section P m e ] Analytical кгейг“' Control i - ¡ Q f Radiopharmaceuticals Proceedings of a Panel Vienna, • ° 7-11 July 1969

INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1970

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS The following States are Members of the International Atomic Energy Agency:

AFGHANISTAN GREECE .NORWAY ALBANIA GUATEMALA PAKISTAN ALGERIA HAITI PANAMA ARGENTINA HOLY SEE PÂRAGUAY AUSTRALIA HUNGARY PERU AUSTRIA ICELAND PHILIPPINES BELGIUM INDIA POLAND BOLIVIA INDONESIA PORTUGAL BRAZIL IRAN ROMANIA BULGARIA IRAQ' SAUDI ARABIA BURMA IRELAND SENEGAL BYELORUSSIAN SOVIET ISRAEL SIERRA LEONE SOCIALIST REPUBLIC ITALY SINGAPORE CAMBODIA IVORY COAST SOUTH AFRICA CAMEROON JAMAICA SPAIN CANADA JAPAN SUDAN CEYLON JORDAN SWEDEN CHILE KENYA SWITZERLAND CHINA KOREA, REPUBLIC OF SYRIAN ARAB REPUBLIC COLOMBIA KUWAIT THAILAND CONGO, DEMOCRATIC LEBANON TUNISIA REPUBLIC OF LIBERIA TURKEY COSTA RICA LIBYAN ARAB REPUBliC UGANDA CUBA LIECHTENSTEIN UKRAINIAN'SOVIET SOCIALIST CYPRUS LUXEMBOURG REPUBLIC CZECHOSLOVAK SOCIALIST MADAGASCAR UNION OF SOVIET SOCIALIST REPUBLIC MALAYSIA REPUBLICS DENMARK MALI UNITED ARAB REPUBLIC DOMINICAN REPUBLIC MEXICO UNITED KINGDOM OF GREAT ECUADOR MONACO BRITAIN AND NORTHERN EL SALVADOR MOROCCO IRELAND ETHIOPIA NETHERLANDS UNITED STATES OF AMERICA FINLAND NEW ZEALAND URUGUAY FRANCE NICARAGUA VENEZUELA GABON NIGER VIET-NAM GERMANY, FEDERAL REPUBLIC OF NIGERIA YUGOSLAVIA GHANA *• ZAMBIA

The Agency’s Statute was approved on 23 October 1956 by the Conference on the Statute of the IAEA held at United Nations Headquarters, New York; it entered into force on 29 July 1957. The Headquarters of the Agency are situated in Vienna. Its principal objective is "to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world".

Printed by the IAEA in Austria July 1970 PANEL PROCEEDINGS SERIES

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS

PROCEEDINGS OF A PANEL ON ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS ORGANIZED BY THE INTERNATIONAL ATOMIC ENERGY AGENCY AND HELD IN VIENNA, 7-11 JULY, 1969

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1970 ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS IAEA, VIENNA, 1970 STI/PUB/253 FOREWORD

Many countries are now producing radioisotopes and labelled compounds, and it is a common experience that the bulk of such materials is for medical use. Any chemical, whether radioactive or not, which is intended for ad­ ministration to patients must be regarded as being a drug'and thus should be subject to rigorous and stringent quality control. To ensure that the necessary information is made available, the Inter­ national Atomic Energy Agency convened in Vienna on 7 - 11 July 1969 a panel of experts on the Analytical Control of Radiopharmaceuticals.- The panel was attended by 25 participants from 14 Member States and three inter­ national organizations. The panel participants represented producers and users of radiopharmaceuticals, developed and developing countries,and a variety of professions. Since the matter is of interest to the International Atomic Energy Agency and to the World Health Organization, both have collaborated in the evalua­ tion of the analytical methods used for the control of radiopharmaceuticals. The World Health Organization was among the international organizations represented at the panel meeting. It is hoped that the present book, containing the papers presented at the panel, will be found useful to the nuclear centres of both developed and developing countries which are producing radiopharmaceuticals, and will create a widespread awareness of the need for careful quality control. It is also hoped that the publication will facilitate the incorporation of the neces­ sary specifications and regulations in the -various national and international pharmacopoeia.

CONTENTS

Purity criteria and general specifications of radiopharmaceuticals (IAEA PL-336/1)...... 1 Y . Cohen Quality control of radiopharmaceuticals and its organizational aspects (IAEA-PL-336/2) ...... 31 V.K. I y a and N.G.S. Gopal Analytical control of radiopharmaceuticals by the Spanish Nuclear Energy Board (JEN) (IA EA -PL-336/3)...... 51 M. del Val Cob, D.V. Rebollo Garrido and F. Casas Medina Methods of testing radiopharmaceuticals used by the Argentine National Atomic Energy Commission (IAEA-PL-336/4)...... 61 A.E.A.- Mitta and R. Radicella Analytical control of radiopharmaceuticals in Norway (IAEA-PL-336/5)...... ; ...... 63 E.Steinnes Analytical control of radiopharmaceuticals in the Department of Chemistry, Reactor Centre, Seibersdorf (IAEA-PL-336/6). .. . 69 H. Sorantin Quality control and chemical analysis of radiopharmaceuticals at a small research centre (IAEA-PL-336/7)...... 83 Y . S. Kim Role of the hospital radiopharmacy (IAEA-PL-336/8)...... 99 J'. L . Quinn III Testing procedures for individual batches of radiopharmaceuticals (IA E A -P L -336/9)...... , ...... I ll K . Frühauf Limits of accuracy in the determination of purity by thin-layer and ' paper chromatography (IAEA-PL-336/10)...... 115 С . E . Me llis h Determination of inorganic radioiodides in 131I~labelled compounds -by means of thin-layer chromatography (IAEA-PL-336/11). . . . 127 J . Alvarez, N.G.B. de Salas, P. Raban and A.E.A. Mitta Self-decomposition of some 131I-labelled radiopharmaceuticals (IAEA-PL-336/12)...... 131 I. Galatzeanu and G.B. Cook Radiochemical purity and stability of some radiopharmaceuticals (IA EA -PL-336/13)...... 153 J . С i f к a Degradation of 131I-hippuran, 131I-lipiodol, 131I-Rose Bengal and 198 Au colloidal gold (IA EA -PL-336/14)...... 181 F . Casas Medina, D.V. Rebollo Garrido and M. del Val Cob Possible artifacts in the chromatographical determination of radio­ chemical purity of 35S- and 75Se-labelled methionine (IA EA -PL-336/15)...... 189 I. Galatzeanu Radiometric titration of inactive and radio-pharmaceuticals (IAEA-PL-336 /16)...... 201 J. Tölgyessy and T. Braun Summary of the activities of COMECON with regard to the control of radioactive medical products (IA EA -PL-336/17)...... 213 D. Ostrovski Conclusions and Recommendations of the P an el...... 217 List of Participants...... 221 IAEA-PL-336/1

PURITY CRITERIA AND GENERAL SPECIFICATIONS OF RADIOPHARMACEUTICALS

Y . COHEN CEA Centre d'Etudes de Saclay, Gif-sur-Yvette, France

Abstract

PURITY CRITERIA AND GENERAL SPECIFICATIONS OF RADIOPHARMACEUTICALS. A limiting definition of radiopharmaceuticals is given. They are defined as compounds (radionuclides or labelled compounds) intended for administration to human beings for therapeutic or diagnostic purposes. Sealed sources are not considered as radiopharmaceuticals. The useful purity criteria, i.e. radionuclide, radiochemical and chemical purity, are distinguished and explained. The general methods of quality control are briefly reviewed and their limitations indicated. Various radiopharmaceuticals for different uses are described, including solutions for oral administration and parenteral injection, capsules and isotope generators. The purity criteria applied will differ, depending on the accuracy of the available production methods and the intended m edical use. Specifications laid down by the World Health Organization (WHO) and national pharmacopoeias may vary slightly from country to country without any adverse effect on the users and without the intrinsic quality of the radiopharmaceutical products in question being affected.

1. PRESENT POSITION

The quality control of radiopharmaceutical substances raises a number of interrelated problems which are frequently the responsibility of different authorities and administrative bodies. These problems may be classified under the following headings: technical, medical, administrative, legal and international. The technical reasons for quality control are connected with the desire of producers to ensure that their products are able to satisfy market requirements. Hospital services which are users of radiopharmaceuticals see in quality control a guarantee that these products are harmless for admini­ stration to their patients and an assurance that the therapeutic results will not give rise to undesirable side effects and that the diagnostic indications will not be impaired by errors attributable to poor quality of the products. The administrative departments of health ministries responsible for pharmaceutical substances insist that radiopharmaceutical products should be subject to control and should satisfy the purity criteria laid down by the pharmacopoeia commissions with a view to protecting public health in general. A concept of legal liability enters at this point and in case of culpable negligence the judicial consequences may be serious for the parties concerned. International organizations are concerned with the question in several respects, since radiopharmaceutical products manufactured in one country are often used in several other countries and are transported by sea, air or rail under international transport regulations. Certain countries have adopted the regulations proposed by international organizations for the handling of radioactive substances, the protection of personnel and the

1 2 COHEN protection of the population at large. The provisions of the International (WHO) Pharmacopoeia have often been adopted in national pharmacopoeias. Radiopharmaceutical substances are governed by these provisions inasmuch as they are both radioactive substances and pharmaceutical products. The question is to determine whether they belong primarily to the one or the other category:

(i) As radioactive substances, they come under the regulations issued for the protection of operational personnel and the general popu­ lation; (ii) As pharmaceutical products, they have to meet the criteria for good therapeutic or diagnostic agents.

We have now to consider the scientific and technical aspects of the problem:

(1) What characteristics require to be controlled? (2) What are the best methods for analysing these radiopharmaceutical substances?

The criteria should not be laid down in abstract terms since the temptation to control all aspects and to require an ideal standard of purity must be resisted. We are helped in this by the inescapable fact that the radionuclides used in medicine have short lifetimes. If all aspects were to be controlled, the elements would cease to exist before our trials were concluded. The demand for absolute purity is an ideal which has soon to be abandoned owing to the lack of precision in existing analytical methods. In any case, is absolute purity essential? I do not think so, since it is an ideal which goes far beyond the real requirements. Radiopharmaceutical products may be inadequate from the strictly pharmaceutical point of view but yet give satisfaction in terms of nuclear quality. Cliggett and Brown [1] and Vetter [2] have recently drawn attention to products which were for various reasons defective: incorrect shipment, erroneous assay date, wrong volume, incorrect supplier dosage, incon­ sistent label. To my mind, these causes are strictly "pharmaceutical". Among them two are directly connected with radioactivity: uncertainty in the decay scheme and radioactive contamination. We shall concern ourselves here with nuclear quality and disregard any inadequacies due to faulty pharmaceutical preparation of the radioactive compounds. '

2. DEFINITION OF THE RADIOPHARMACEUTICAL PRODUCT

The definition of a radiopharmaceutical product seems to have been generally accepted by various authors: Cohen [3], Kristens en [4], Gopal and Iya [5], Wolf and Tubis [6], Balaban [7]: it is a radioactive compound intended to be introduced into the human organism for therapeutic or diagnostic purposes. In its aim this definition corresponds to that given in the International Pharmacopoeia and the national pharmacopoeias. How­ ever, this definition raises a difficulty in that it implies that sealed sources IAEA-PL-336/1 3 are radiopharmaceutical products since they also are intended to be intro­ duced into the human organism for therapeutic purposes, whereas the experts appear to agree that since traditional radium sources have not been accepted as medical agents, radioactive substitution products such as colloid gold (198Au) seeds and wires, yttrium (90 Y) oxide pellets, 60Co needles, 192Ir wires and needles are not radiopharmaceutical substances. These radionuclide needles, wires, seeds and pellets are medical aids for use in radiotherapy. The definition of the radiopharmaceutical product could therefore be improved by excluding sealed sources and agreeing that radiopharma­ ceuticals are radioactive preparations, which are administered to man for therapeutic or diagnostic purposes and are not contained in sealed sources and which therefore undergo metabolic changes in the organism. Even this definition does not avoid the difficulty raised by the existence of ceramic seeds which contain a radioactive substance and are adminis­ tered in suspension in a liquid. These seeds undergo metabolic changes since they are transported by the blood, but they constitute separate m icro­ scopic sealed sources which are perfectly insoluble and do not give off radioactivity. We can see already that the definition of radiopharmaceutical sub­ stances is by no means perfect. What are.these radiopharmaceutical substances? They may be classified either on the basis of their method of preparation — and this is the concern primarily of the radioisotope specialist — or as a function of their method of administration, which is more in accordance with the traditional pharmaceutical classification. We shall therefore treat these two types of classification separately.

3. CLASSIFICATION BASED ON THE RELEVANT NUCLEAR CHEMISTRY OPERATIONS

Five groups of preparations can be distinguished: 3.1. Radioactive preparations obtained by irradiation of a target followed by solution of this target. Sodium-24, potassium-42 andbromine-82 belong to this category. 3. 2. Radioactive preparations obtained by chemical separation of a radionuclide from an irradiated target. Such chemical separation may involve distillation, oxidation-reduction, adsorption-desorption on an ion- exchange resin or precipitation followed by re-dissolving. This is the most common case and we may mention the examples of iodine-131, phosphorus-32, arsenic-76, chrome-51, copper-64, iron-55, sulphur-35, etc. 3. 3. Radioactive preparations obtained by labelling synthesized o r­ ganic molecules or an organic molecule of biological (animal or plant) origin with a radionuclide. This category includes a large number of 131I-labelled molecules used in medicine: Cholografin, diiodotyrosine, diodone, Glomerfil, Hippuran, Hypaque, iodo-antipyrin, iodophenyl- indandione, monoiodotyrosine, Renografin, Rose Bengal, thyroxine, tr i­ iodothyronine, uracil, uridin, Urokon, Vasurix and human fibrinogen, human serum albumin, insulin, oleic acid, triolein, Lipiodol, etc. Some of these molecules have aiso been labelled with 1251: e.g. cytosine, de- oxyuridin, mono- and diiodotyrosine, diodone, Hippuran, antipyrin. 4 COHEN

Renografin, Rose Bengal, thyroxine, triiodothyronine, uracil, uridine and human fibrinogen, human serum albumin, insulin, oleic acid, triolein, etc. Seleniomethionine (75Se), , Neohydrin (197Hg and 203Hg), mercurihydroxy- propane (197Hg and 203Hg), cyanocobalamin (57Co, 58 Co or 60Co) also belong to this category. 3. 4. Radioactive colloidal preparations are produced by the precipi­ tation of metals, metalloids or salts, such as colloid gold-198, .yttrium-90 or chrome phosphate (32P) in the form of particles of diameter less than 100 nm in a stable suspension. A derived form is represented by suspen­ sions or aggregates of diameter exceeding 100 nm and of a biological nature: e. g. denatured albumin. 3. 5. Isotope generators producing short-lived radioactive emitters when required. These preparations are the most complicated since they involve the irradiation of a target, the separation of the parent radioactive element, its adsorption on a carrier (ion-exchange resin, inert adsorbent powder, etc. ) and elution of the daughter radionuclide which is alone of interest from the point of view of the user. The preparations available at the present time include gallium-68 generators from germanium-68, strontium-87 m from yttrium-8 7, technetium-99 m from molybdenum-99, rhodium-103 m from palladium-103, barium-137 from caesium-137, barium-140 from lanthanum-140, iodine-132 from tellurium-132 and indium-113 m from tin-113.

The concept of generators has led commercial firms to propose the use of kits by means of which radiopharmaceuticals could be produced extemporaneously.

4. CLASSIFICATION AS A FUNCTION OF THE PHARMACEUTICAL FORM

Classification by pharmaceutical form depends on the method by which the labelled radionuclide or molecule is introduced into the human organ­ ism. Two principal methods of administration are used, oral and parent­ eral, and these govern which of the four pharmaceutical forms is preferred.

4. 1. Solutions for oral administration

A large number of radionuclides are prepared in solution for oral administration. They are supplied in solution in penicillin-type bottles from which the required volume can be removed in one or more opera­ tions. This involves radioactive contamination of glassware, pipettes, and of the lips and oral cavity of the patient. Removal of the solution in more than one operation can give rise to errors in volume measurement and to bacterial or chemical contamination. The aim should always be to use single-dose bottles thus avoiding these disadvantages. The products are prepared in the form of aqueous, dilute alcoholic or oily solutions.

4. 2. Gelatin capsules

The capsule form has the advantage of avoiding dosage errors since each capsule contains a specific amount of the ra'dionuclide in question. After being swallowed the capsules disintegrate in the stomach and give IAEA-PL-336/1 5

off radioactivity only after travelling a considerable distance along the’ alimentary tract (in the stomach or first part of the small intestine). The disadvantage of this form is the possible development of faults in the capsules —for example, failure of the capsule to dissolve or incomplete solution in the digestive tract or combination of the radionuclide with the capsule components (e. g. gelatin, antiseptic, colouring agent or material presumed to be inert and designed to impart rigidity to the capsule).

4. 3. Solutions for injection

As in the case of solutions for oral administration, these are supplied in penicillin-type ampoules or bottles containing one or more doses. They are sterile, generally isotonic and apyrogenic. They have to be removed under aseptic conditions in a sterile syringe prior to injection. They run the risk of becoming contaminated with bacterial microorganisms if suf­ ficient care is not taken in removing them from the bottle. The solutions are aqueous or dilute alcoholic and sometimes incorporate a bactericidal preservative.

4. 4. Solutions in single-dose syringes

This modern pharmaceutical form, used originally in first-aid kits for the army medical service, has been extended to cover radioactive substances also, since it has the advantage of reducing to a minimum handling by medical personnel with the consequent risk of occupational irradiation and prevents the occurrence of bacterial contamination, pro­ vided that all aseptic precautions are taken at the production stage.

4. 5. A final possibility is the preparation in bottles of lyophilized products which can be dissolved extemporaneously with an appropriate solvent. These belong to the category of pharmaceutical forms 1 or 3.

5. PURITY CRITERIA FOR RADIOPHARMACEUTICAL PRODUCTS

The purity criteria will depend on the chemical processing undertaken during preparation of the radioactive product and on the pharmaceutical form in which the product is supplied. It is obvious that the purity of a sodium iodide (131I) solution without carrier which contains only sodium carbonate and sodium iodide in very small quantities will be greater than that of a gelatin capsule containing sodium iodide (131I), since the latter will contain impurities due to the gelatin colouring agent, antiseptic and reducing agent which is added to preserve the dry state of the iodine in iodide form. We shall therefore make a distinction between the criteria of nuclear purity and chemical purity. The criteria of nuclear purity are known. They include the following elements:

5. 1. Radionuclidic purity, or radioactive purity, or radioisotopic purity, is the ratio, expressed as a percentage, of the activity of the radionuclide in question to the total activity of the source. 6 COHEN

5. 2.' Radiochemical purity is the ratio, expressed as a percentage, of the radioactivity of the radionuclide in question, present in the source in the given chemical form, to the total radioactivity of this radionuclide present in the source.

The chemical purity criteria also apply to the carrier.

5. 3. Chemical purity is the ratio, expressed as a percentage, of the mass of material present in the given chemical form to the total mass of material contained in the source.

5. 4. The isotopic carrier is made up of stable isotopes of the radionuclide in question added to the radioactive preparation and present in the same chemical form as the radionuclide.

The combination of the concepts of nuclear purity and chemical purity gives rise to the definition of specific radioactivity and to the radioactive concentration of the solutions.

5. 5. The specific radioactivity is the ratio of the radioactivity of the radio­ nuclide in question to the total mass of the element or of the chemical form in question.

5. 6. The radioactive concentration of the solutions is the ratio of the radioactivity of the radionuclide in question to the volume of solution in which it is dissolved.

5. 7. Observations on the definitions

5. 7. 1. The definition of radionuclide purity adopted above implies that any radionuclide present in the solution other than the required one is an im­ purity. Such contamination is due to the presence of impurities in the target which give rise to other than the required radionuclide or to the occurrence of parasitic nuclear reactions, occurring at the same time as the principal nuclear reaction; for example, an np or na reaction occurring at the same time as an n-у reaction. This may be the result of an incom­ plete separation of the radionuclide from its target during chemical pro­ cessing. In the separation of phosphorus-32, for example, sulphur-35 originating with the irradiated sulphur may also be carried over. Where the nuclear reaction gives rise to several radionuclides (198Au and 199Au), the 199Au is considered as an impurity in the 198Au but it will not be possible to separate the two. The physical characteristics of the 199Au, and also its half-life and beta-particle and gamma-ray emission, do not differ greatly from those of 198Au and the presence of 199Au does not present any inherent difficulty. The tolerance limits can therefore be flexible. In any case, the appearance of 199Au depends on the neutron irradiation conditions in the reactor. A distinction has therefore to be made in regard to radionuclidic purity between the presence of several radionuclides of the same element and the presence of radionuclides of different elements. The importance of these impurities depends on their biological properites: traces of potassium-42 in sodium-24 are very much more troublesome than the presence of rubidium-86 in potassium-42. IAEA-PL-336/1 7

Radionuclidic purity is essential if satisfactory proportionation (cali­ bration) is to be obtained, since calibration of the source and m easure­ ment of the radioactive concentration will depend on the absence of para­ sitic radiation which can interfere with the principal radiation. Determination of the radionuclidic purity involves identification of the particular radionuclide. Radionuclidic purity is not a stationary phenomenon. It depends on the respective half-lives of the radioactive impurities and the principal radionuclide. It becomes of greater importance if the impurities have half-lives longer than that of the principal radionuclide since the proportion of impurity will then tend to increase with time. On the other hand a short-lived impurity will disappear if a certain period elapses be­ tween irradiation of the target and medical utilization of the radiopharma­ ceutical substance. . This is the case with the preparation of mercury-203 where one waits for the m ercury-197 to decay. It is necessary therefore to specify the date on which the radionuclidic purity determination has been carried out. F o r solutions obtained from isotope generators the criterion of radionuclidic purity is of primary importance and the solutions should not contain the parent radionuclide, the radioactive half-life of which is always longer than that of the daughter product. The presence of the parent radionuclide as an impurity is proof of a poor quality generator which must be discarded. On occasion the radionuclidic purity is checked in conjunction with the radiochemical purity. '

5. 7. 2. Radiochemical purity is a relatively simple concept for mineral molecules: the presence of sodium iodate in a solution of sodium iodide constitutes an impurity. In the case of labelled .organic molecules the phenomenon is more complex. Mention may be made by way of example of Rose Bengal labelled with iodine-131 which is the compound tetraiodo- tetrachlorofluorescein. Not only does sodium iodide in a Rose Bengal solution constitute a radiochemical impurity but the presence of insufficient­ ly iodized tetrachlorofluorescein molecules (mono-, di-, or tri-iodo- tetrachlorofluorescein) also constitutes an impurity. Furthermore, if the molecule is insufficiently chlorinated, the molecules of mono-, di-, or tri-chlorofluorescein, even if tetra-iodized, will constitute radio­ chemical impurities. Sources of impurities may be present in commercial non-radioactive products which are considered satisfactory for normal pharmaceutical or chemical uses, but they would render such products completely unsatisfactory from the point of view of nuclear purity. Radiochemical purity depends also on the stability of the labelled molecule as a function of time, temperature, exposure to light and internal irradiation. A radiochemical purity which is adequate at the time of preparation of the radiopharmaceutical product may alter with time, giving rise to the formation of new chemical compounds due to radiolysis or radiosynthesis leading to the displacement of the radionuclide within the molecular structure. The strictness with which the tolerance limits have to be applied to these radiochemical impurities depends on the intended medical application of the radiopharmaceutical product. F o r some radioactive compounds the presence of 5% radiochemical impurity is acceptable, whereas others have to be rejected if the radiochemical impurity content exceeds 1%. This is со

TABLE 1.a VARIATION IN DISTRIBUTION OF COMMERCIAL SELENOMETHIONINE t75Se) AND ITS OXIDATION PRODUCTS (Fixation in the rat at T = 1 h)

Oxida­ Distribution in the rat Chemical analysis Serial No. pH ЛТЬ tion №g) period Amount deposited Oxides Acids SeM Impurity Liver Pancreas Kidney Lungs Blood

c SeM 3. 6 305 1. 05 5.89 2. 88 88. 71 1.42 1.34 2.1 1. 83 0.45 0.27 CEN 30 ' ^ SeM oxid. 16 h 2. 79 7. 30 3. 51 80.81 5.52 1.93 2. 3. 0.8 0.54 c SeM 6 330 0.18 0.66 1.05 95.6 2. 70 1. 55 2. 32 1. 32 0.33 0.28 Exptal 16 h \ 1.84 1.19 1. 74 92.2 3. 05 1. 94 4.42 1. 54 0.47 0. 50 series 1. 84 2.35 1. 95 0.59 0.50 of 9. 5 ^ SeM oxid. 7. 5 263 32 h 0.78 8. 73 5. 44 83 2.09 1. 79 3. 59 1.92 0.30 0.60 COHEN c SeM 6 3Ó8 2.11 7. 68 4. 93 63.9 21.29 1.5 1.33 1. 09 0.39 0. 30 Exptal series of 30.5 ^ SeM oxid. 5 392 40 h 3. 03 26. 55 12. 17 45. 3 12.48 1.67 1. 32 1. 64 0.63 0.60

c SeM 5.3 291 1.42 3. 79 2.03 88.9 3.82 1.65 2.2 1. 60 0.57 0.68 CEN 39 ^ SeM oxid. 5 420 36 h 0. 85 4.46 2. 68 88. 6 3. 34 1. 46 3. 1. 80 0. 55 0. 62 c SeM 4. 9 292 1. 5 12. 0 8.4 74. 5 3. 8 1. 65 2. 1. 67 0.48 0. 59, CEN 40 ^ SeM oxid. 7 384 35 h 2.0 * 5.0 4.3 66. 0 22. 6 1. 79 2. 94 1. 89 0.43 0.45 c SeM 5.1 283 0.3 2.5 0.5 94.2 2. 0 1. 75 4.13 2. 03 0.46 0.56 CEN 41 ^ SeM oxid. 7.1 373 36 h 0.8 1. 8 1.4 82. 8 11. 0 2.15 4.15 2. 38 0.44 0. 39

With the collaboration of Mme Besnard and Mile Costerousse k ДТ = isotonicity, expressed in milli-osmols. c Selenomethionine ^ Oxidation products of selenomethionine. IAEA-PL-336/1 9

the case with 131I-labelled Hippuran which is unsuitable for certain diag­ nostic purposes if the iodine content not included in the organic molecule exceeds 1%. On the other hand it is open to question whether an excessive radio­ chemical purity is necessary for certain preparations: selenomethionine (75Se) becomes concentrated in the pancreas where it is easily oxidized into methionine selenoxide and methionine selenone which constitute radiochemical impurities. We have been able to show experimentally the difference in the distri­ bution in the animal between commercial selenomethionine (75Se) prepara­ tions and their oxidation products (see Table I). When specifications for radiopharmaceutical products are drawn up, biological, medical and radiochemical data should therefore be taken into account. 5. 7. 3. The chemical purity of radiopharmaceutical preparations refers to the absence of substances not included in the specified formula for the preparation. Its application does not extend beyond the conventional case of pharmaceutical substances, except in so far as it relates to the radio­ nuclide. Chemical impurities which are regarded as negligible in a con­ ventional preparation may combine with the radionuclide to render it in­ soluble or'to facilitate its adsorption on vessel walls. We meet the same situation again, for example, with preparations of phosphorus-32. To . prevent this phenomenon the addition of a carrier is recommended, pro­ vided that such addition does not alter the biological characteristics of the preparation. 5. 7. 4. In point of fact, although the addition of a carrier, as described in the previous paragraph, may prevent the chemical phenomena which occur at high dilutions, such additions sometimes have the disadvantage that the radionuclide loses its essential characteristic of a tracer which will not modify the steady state of the organism. This is particularly noticeable with iodine-131 which should not be diluted with iodine carrier beyond a fairly low limit. No more than 100 ц g iodine should be administered in the form of a tracer. 5. 7. 5. The specific radioactivity and radioactive concentration of the solutions are obtained by means of calculations based on radioactivity measurements and chemical or volumetric measurements. These factors are important where the reactor flux is insufficient to obtain compounds of high specific activity or where the target has to be enriched with a specific stable element (chromium-50, iron-58, etc.). Preparations of insuffici­ ently high specific activity would involve the need to administer to patients fairly large quantities of chemical components which might be toxic. Too highly diluted solutions would mean the injection of excessive volumes which could lead to a lack of precision in the initial pulse in the case of functional explorations of a dynamic type.

6. METHODS OF PURITY CONTROL

The organization of purity control checks on radiopharmaceutical preparations requires the establishment of a complex circuit of operations, ’ since, in view of the shortness of the half-life, examination of the product TABLE II. CHROMATOGRAPHIC SEPARATION OF RADIOCHEMICAL IMPURITIES IN ORGANIC RADIOPHARMACEUTICALS • (a) 58Co Labelled Molecules

Duration Rf of Radiochemical Rf of thè Labelled molecule Support Solvent Proportions (h) molecule impurity impurity

Cyanocobalamin 58Co Whatman No. 4 paper Sec. butanol: 100:1:50:0.25 0. 25 Cobalt ion acetic acid: water: 5ft KCN

Whatman No. 4 paper Sec. butanol: 100:1:50:0.25 0.30 Cobalt ion aqueous NH3: water: 5ft KCN Silica gel G Water 0.22 Silica gel G Glacial acetic 5:5:20:70 0 acid-.acetone: • methanol: benzene Silica gel G Sec. butanol: 100:0.1:24:2:30 aqueous NH3: 3. 5U NaCN: water

(b) 125I or 131I-Labelled Molecules

Hippuran Silica gel G Acetic acid: 3 :9 7 0. 75 Iodide 1 water

Rose bengal Whatman No. 1 paper 2ft ammonium 1 Í 0 .0 5 Iodide • 0. 8 citrate, pH 7 0 .1 with ammonia

Rose bengal Whatman No. 1 2 5 ft ethanol 1:1 2 0 .6 Iodide + Xj 0. 8 and 5ft ammonia • *. x2 0. 3 + X3 0 .1

Rose bengal •Thin layer of Merck Isopropanol and 6 0 :0 .5 1 0 .4 2 X 0 activated silica and ammonia Diiodotetra- 0. 25 gel G (d = 0. 925) chlorofluorescein iodide 0 . 65

Rose bengal Thin layer of Merck Chloroform and 9:1 * 0 ,8 8 Iodide 0 activated silica acetic acid + X] 0 . 31 gel G * хг 0 . 74 + X3 0 . 78 + x4 0 .8 2 TABLE Il.(cont)

(b ) 125I o r 131I-Labelled Molecules (cont. )

Duration Rf of Radiochemical Rj- of the Labelled molecule Support Solvent Proportions (h) molecule impurity impurity

Rose bengal Whatman AE 30 0. 25M sodium 1 0 Iodide 0. 6 paper citrate, pH 8.2

Rose bengal Whatman DE 20 0, 25M sodium 1 Iodide 0.4 paper citrate Thyroxine Whatman AE 30 0. 25M sodium 1 0 - 0.1 Iodide 0. 6 paper citrate, pH 8. 2. IAEA-PL-336/1 Thyroxine Whatman DE 20 0. 25M sodium 1 0 Iodide 0.4 paper citrate Thyroxine Thin layer of Merck Isopropanol, 35:30:25:20 1 0. 3 Monoiodo- 0. 22 activated silica ethyl acetate, thyrosine gel G acetone, and 3, 5-diodo- 0. 42 ammonia thyronine (d = 0.925) 3, 3, tri­ 0. 38 iodothyronine Iodide 0. 05

(c) 197Hg or 203Hg-T_,abel] ed M olecules

'

Neohydrin Ethanol-phosphate 0. 50 - 0. 58 Mercuric ion 0. 04 buffer pH 7. 4 0. 82 - 0. 88 Neohydrin n-butanol: methanol: 5:7:3:1 Mercuric ion aqueous ammonia (d = 0. 88) water

ч TABLE III. CHROMATOGRAPHIC SEPARATION OF RADIOCHEMICAL IMPURITIES IN INORGANIC RADIOPHARMACEUTICALS

Support and Duration Radiochemical Rf of Radioisotope Solvent Proportions Rf of conditions (h) radioisotope impurity impurity

згр-sodium APC Isopropanol, water, 75:25:5:0.3 16 0. 7 Pyrophosphate 0.45 orthophosphate trichloro-acetic acid, Meta phosphate 0 ammonium hydroxide APC Water trichloracetic acid, 170:25:17:325 1 - 2 ammonium hydroxide, acetone DPC t-butylalcohol, water, 40:20:5 4 formic acid TLC Ethanol, water, tri­ 80:10:5:0.3 0.5 0.9 Pyrophosphate 0.6 chloroacetic acid, Polyphosphate <0.5 ammonium hydroxide 5,Cr-chromium Ethanol, HCl(d = 1.19) 50:25:125 2. 5 0. 7 - 0, 8 Chromium 0 chloride distilled water complex 51Cr-sodium APC Water, ethanol, and 125:50:25 2. 5 0. 9 Trivalent 0 chroma te ammonia chromium (d = 0. 925) 59Fe ferric APC Butanol, water, acetic 50:30:10:5 6 0. 7 Ferrous ion 0.25 chloride acid, and ethyl aceto- acetate

APC - Ascending paper chromatography TLC - Thin-layer chromatography W - Whatman No. 1 S - Silicagel. TABLE IV. ELECTROPHORETIC SEPARATION OF RADIOCHEMICAL IMPURITIES IN ORGANIC AND INORGANIC RADIOPHARMACEUTICALS

Rf of Radiochemical Labelled molecule Paper Electrolyte Rf of impurity molecule impurity

Sodium phosphate (32P) 0 .1M lactic acid

Sodium chiomate (51Cr) Glass fibre paper 0 .05M 1 Trivalent 0 Na2 HP04 pH 9. 6 chromium Sodium chloride (51Cr) Glass fibre paper Na2 HP04 pH 9. 6 0 Hexavalent 1 chromium

Vitamin B12 (58Co) Whatman No. 540 0.3N acetic acid 0 1 Sodium iodide (13II) Whatman No. 540 0. 01N sodium Iodate hydroxide

Whatman No. 540 0. 05M Na2 HP04 - 1 Iodate 0.7 NaHjP04 pH 6. 5 Hippuran (134) Whatman No. 540 0. 05M Na2 HPO4 - 0. 31 ' Iodide 1 NaH2P04 pH 6. 5

Rose Bengal (131I) Whatman No. AE 30 0. 025M or No. DE 20 Sodium citrate 0 Iodide 1 Rose Bengal (m I) Whatman No. 540 0. 05M Na2 HP04 - 0 Iodide 1 NaH2P04

Rose Bengal (131I) Whatman No.l 0. 025M sodium 0 Iodide 1 citrate

Human serum Whatman No. 1 (1) Iodide albumin 131I Iodide 0. 07 Human serum Whatman No, 540 0 .05M Na2 HP04 - 0 Iodide 1 albumin 13,I NaH2PO„ pH 6. 5 Thyroxine (131I) Whatman No.l 0 .025M sodium 0 Iodide 1 or No. AE 30 citrate or No. DE 30

Neohydrine Whatman No. 540 0 .1M NaCl 0. 52 Mercuric ion 1 (l97Hg or 203Hg)

Colloidal gold Whatman No. 540 0. 075M Na2Sj03 0 Sodium chloro- (19* Au) aurate

(1) - Barbitone sodium: 5 g; sodium acetate: 3. 25 g; sodium octoate: 4 g; hydrochloric acid; 34. 2 ml water: to 1000 ml 14 COHEN must either be carried out very rapidly or deferred until after dispatch of the preparations. The first case represents a true check of the pharma­ ceutical product, whereas in the-second'case it is the production conditions (good manufacturing processing) that are being checked. In any case certain control checks must be carried out before dispatch, viz..: (a) Identification of the radionuclides; (b) Measurement of the radioactivity; (c) Verification of the radionuclide purity; (d) Verification of the radioactive concentration; (e) Verification of the chemical purity. The remaining control checks can be deferred. We presented at the Oak Ridge Symposium in 1965 a diagram of the required radiopharmaceutical control checks which we still consider to be valid (Fig. 1). Figure 2 shows the control card which is prepared in triplicate for each preparation. (All Figures appear in Annex I).

6 . 1. The methods of radionuclide identification include, for the case of beta emitters, plotting a curve for absorption in matter and a decay curve, and also using a gamma spectrometer to verify the absence of gamma emitters. In the case of gamma emitters, plotting the gamma emission spectrum can generally be used for detecting the presence of other gamma emitters but not that of beta emitters. The gamma-ray spectrum can be plotted point by point by using a manual single-channel gamma spectrometer or on a more sophisticated, 400-channel gamma spectrometer. We give in the subsequent figures the characteristics spectra of the radionuclides most frequently used in medicine (Figs 3-11, Annex I) .1

6 . 2. Verification of the radiochemical purity is by the electrophoresis and chromatography methods described in the Manual of Radioisotope P ro ­ duction published by the IAEA2 (see Tables II, III, IV).

6 . 3. The chemical purity can be estimated with sufficient accuracy and speed by determining the metal and metalloid content by means of radiation emission spectrometry. With this method the following elements can be measured in a single operation: Al, As, B, Be, Ca, Cd, Cr, F e, Mg, Mn, Ni, P, Pb, Si, Te, Zn.

7. RADIOPHARMACEUTICAL SPECIFICATIONS

Radiopharmaceutical specifications are drawn up by mutual agreement between producers of radionuclides, analytical control laboratories and hospital physician users. They cover:

1 These spectra were obtained with an SA 40 В analyser produced by the INTERTECHNIQUE firm in France. 2 INTERNATIONAL ATOMIC ENERGY AGENCY, Manual of Radioisotope Production, TRS No. 63, IAEA, Vienna (1966). IAEA-PL-336/1 15

(a) Radioactive concentration; (b) Radionuclidic purity; (c) Chemical purity; (d) Specific radioactivity; (e) Radiochemical purity; and the purely biological criteria:

(a) Sterility; (b) Pyrogenicity; (c) Isotonicity; (d) Biological affinity.

By way of example we reproduce the French specifications 3 [8 ] for solutions of sodium iodide, injectable sodium chromate, thyroxine and bromo-mercurihydroxypropane solution (Figs 12-15, Annex I). The drawing up of these specifications is the responsibility of inter­ national health organizations (WHO) or national bodies within the fram e­ work of national pharmacopoeias.

8 . CONCLUSION

It has been our intention to contribute a few thoughts to the question of the purity control of radiopharmaceutical compounds. Certain control checks appear to us essential and should be carried out prior to distri­ bution of the radiopharmaceutical compounds: these relate to radionuclidic purity, chemical purity and radiation concentration. Others such as radio­ chemical purity, salt content, isotonicity, etc. can be carried out subse­ quently. The ideal arrangement would be for all control checks to be carried out prior to delivery of the radiopharmaceutical substances. We have also set out the results of our experience with the control of radio­ pharmaceutical substances based on daily contact with such problems over a period of 15 years. We earnestly hope that this work will prove of general use.

3 The French specifications have been adopted in agreement with the CEN (Belgium) and the SORIN (Italy). COHEN

ANNEX I

CONTROLES DE PRODUCTION

contrôle contrôle mesure spectroscopique spectrométrique gamma ß-ö solutions en stock

_ pureté — pureté _ concentration chimique radionuciéidique radioactive

CONTROLES RADIOPHARMACEUTIQUES

contrôle contrôle contrôle forme chimique biologique physique

radio—

pharma pureté stérilité pureté 1 1 ceutique radiochimique apyrogénéïté entraîneur concentration atoxicité radioactive tampon affinité radioactivité concentration spécifique ionique totale

FIG. 1. Chart of radiopharmaceutical control checks.

DÉPARTEMENT DES RADIOÉLÉMENTS CONTROLE DE

OpéroHon n° du Laboratoire de Contrôle Pharmaceutique A N A L Y S E N»

Concentration radiooctive ;

Pureté radioactive : Stérilité : Pureté radiochimique Absence de toxicité : Concentration chimique : Apyragénéité : Mesure du pH : Affinité biologique : par résistivité Î par cryométrie OBSERVATIONS - CONCLUSION

Opérateur Dote Visa

A envoyer ou service de Préparation des Radioéléments

FIG.2, Control card. IAEA-PL.-336/1- 17

82 Br

FIG. 3. Gamma-ray spectra obtained with the SA 40 B INTERTECHNIQUE 400-channel spectrometer. Spectrum of bromine-82. - •• COHEN

FIG.,4. Spectrum of ctiromium-51.' IAEA-PL-336/1 ae Poste: Date:

181 Kev I

FIG. 5. 4 Spectrum o f iron-59. I 20 COHEN / 7 .

¡

О

FIG. 6. Spectrum of iodine-131. IAEA-PL-336/1 21

78Kev » 197 Hg

FIG. 7. Spectrum o f m ercury-197. COHEN

198 Au

4ÎÎ Kev I EA N*7 CE.A.

FIG. 8. Spectrum of gold-198. IAEA-PL-336/Í 23 24- ÇOHEN ■lAE А-PL-336/1- 25

9 9 mTc COHEN

4 ; * ' ■ ■*' / ■ . ’ ■ ' lODURE

DE SODIUM ■ ' ; • ; ; . / . ’ •- ■* ' . мпа »ntralneur Г ” .* 1 ’ solution пав injectßblt io d e 1 3 1

.Solution aqueuse .stérilisée'd'iodure de'sodium • Na131l, dè pH 7 à 10, répondant aux spécifications suivantes/.

Concentration radioactive, mesurée à 5 % près : - ' - ". pouvant aller jusqu'à.100 mCi/ml.

P u re té radioactive : teneur en ’ VJ > 99.%. (Spectre y caractéristique de l'iode 4 31).' .• : , .

' .Pureté radiochimique: teneur en iódúres > 98 %, v-

Activité spécifique : supérieure, à 20 Ci/rrig: ' '

Composition de la solution conforme aux indications de la Pharmacopée française'(1965)...... 'Radioiodure de sodium ' Quantité impondérable . ‘ NaîCOs anhydre - 0,26 mg" J NaHCCb anhydre 1,68 mg Na2Sî03 anhydre ' •• ' - 1,50 mg • Eau distillée . -.Q.S.P.* 1 ''ml;....-

Absence d’impüretés chimiques gênantes.,' ■ «

FIG..12. CEA, CEN and SORIN radiopharmaceutical specifications.Tor sodium'iodide solùtion.(131 IAEA-PL-336/1 27

C H R O M A T E

D E S O D I U M

solution injec tibia • • c h r o m e 5 / RÉF. . Cr-61-S-1(B ÉT F)

; -Solution neutre, isotonique, stérile et apyrogène de chromate de sodium Na2 6'CrO< répondant aux spécifications suivantes : • •

C o n c e n tra tio n ra d io a c tiv e , mesurée à 5 % près : • .1 à_20 mCi/ml.

• Pureté radioactive : teneur en 61C r>99% . (Spectre y caractéristique du chrome 51 ). • ,

Pureté >radiochimique : teneur en chromate > 98 %. '

Activité spécifique : supérieure à 50 mCi/mg.

Affinité .pour'les hématies-: rendement de marquage in vitro des hématies de Lapin : supérieur à 75 %. ■ .

S té rilité .

Apyrogénéité.

Isotonicité.

FIG. 13. CEA, CEN and SORIN radiopharm aceutical'specifications for in jectable sodium chromate solution (5ICr). 28 COHEN

THYROXINE

MARQUÉE A L IO0E 126

solution injectable iode- 125

Solution stérile et apyrogène de L-thyroxine mar­ quée à l'iode 125, dans' le propylèneglycol. à , 50 % , de p H .voisin de 6 , répondant aux spéci­ fications suivantes :

Concentration radioactive, mesurée à 5 % près : voisine de 250 (iCi/ml. -, >

Pureté radioactive tèneur en ,26| < 2 %. (Spectre y caractéristique de l'iode 125).

P u re té radiochimique : teneur en iode organique > 98 %.

Activité spécifique : 5 à 15 mCi/mg.

S té rilité .

Apyrogénéité-

FIG. 14. CEA,. CEN and SORIN radiopharmaceutical specifications for injectable thyroxine solution (125I). IAEA-RL-336/l

solution injocttàlo

RÉF. : Hg-MM-2 (F) Solution aqueuse, de pH 9 à 10, isotonique et stérile de bromomercurihydroxypropane (BMHP) à la concentration de 6 m g /m l.

Concentration radioactive, mesurée à 5 % près : 0,3 mCi/ml en moyenne.

P ureté ra d io a c tiv e : teneur en ,9,Hg > 99 %. (Spectre y caractéristique du mercure 197).

Pureté radiochim ique : teneur en mercure organique > 98 ^

Activité spécifique : 50 à 100 ji Ci/mg de BMHP.

Affinité pour les hématies et absence d'effet hémo- iy s a n t in vitro.

S té rilité .

Isotonicité.

FIG. 15. CEA, CEN and SORIN radiopharmaceutical specifications for.bromomercuri-hydroxypropane solution ( l97Hg). 30 COHEN

■ R E F ERE N C E S

[1] CLIGETT, P.A ., BROWN, J. M ., J. nucl. Med. 9 (1968) 236. [2] VETTER, H., I. nucl. Med. 10 (1969) 147. ■ - [ 3] COHEN, Y ., in Radioactive Pharmaceuticals Symp. Oak Ridge, 1-4 November 1965, USAEC (1966) 67. [4] KRISTENSEN, K ., Acta Radiológica Suppl.-254 (1966) 131. • [5 ] GOPAL, N. G. S. , IYA, V. K'. , J. Scient. Indust. Res. 26 (1967) 153. [6 ] WOLF, W. , TUBIS, M ., J. Pharm'. Sei. 56 (1967) 1. .• [7] BALABAN,- A. T ., in Proc.2nd Interamerican Conf.- Radiochemistry, Mexico City 22-25 April 1968 (in press). [8 ] CEA, CEN, SORIN, Spécifications radiopharmaceutiques (février 1969). [9 ] INTERNATIONAL ATOMIC ENERGY AGENCY-, Radioisotopes, Production and Control, IAEA, Vienna, - in press.. 1 LAEA-PL-336/2

QUALITY CONTROL OF ., ... RADIOPHARMACEUTICALS AND ITS ORGANIZATIONAL ASPECTS

V.K. IYA, N.G.S. G OPAL / - ' Isotope Division, • ■ ' V , ‘ Bhabha Atomic Research Centre, . ' Trombay, Bombay/,- India

Abstract ■/ '/ , . ...

. QUALITY ^CONTROL OF-RADIOPHARMÂCELÎTICALS AND ITS ORGANIZATIONAL ASPECTS.. • . Methods for-.the.quality control of starting materials in the production of radiopharmaceuticals, as well as'for thè. control of the finite products,. are reviewed. The various types of purity criteria are.defined. As an example forthe organization and-running of a radiopharmaceutical control laboratory, the situation in. the' Bhabha Atomic Research .Céntre, Trombay, is presented.

The term' radiopharmaceutical; as it is accepted today covers over a hundred simpleand/'complex-ràdioactive chemicals used for in-vivo diagnostic and therapeutic applications, and for in-vitro clinical diagnostic tests;.- While a majority/of. tiiese products ar.e formulated as neutral,, .basic or,acidic iaqueous solutions there are a few hydroalcoholic and oil solutions, ,'as-wellá's colloids and suspensions. Some are distri- ■ buted as .adsorbates oruinert anhydrous supports'in gelatin capsules for ease of administration.. Quite a few radiopharmaceuticals are obtained as eluates from generator columns. , 1 ■ - ; v . • Pharmaceuticals, whether they áre to be distributed as solids or simple solutions ,orJ multi-ingredient solutions, are subjected to various ^pe'cific controls, such as ¿identification tests, colour examination, melting point, boiling point,] solubility, specific rotation, heavy metal and arsenic content, chemical assay, tests for undue, specific, or abnormal toxicity, and-tests for pyrogens and sterility. Some.of.these tests need not be carried out .on oral products, while some are to be carried out on products of biosynthetic and biological origin, and on certain products which may be contaminated with a.toxic impurity. .The controls for radiopharmaceutickls, howevèr, ' differ in some respects, from the above-mentioned criteria. For instance, examination of colour is not often a meaningful test as radiation has a darkening- effect.on. containers as well as the radioactive products. -Determination-.of melting point and-solubility are not practicable-since only-very, small quantities;of the organic labelled compounds are involved. Instead o.f these, other, indirect methods of characterization.are therefore required to assess the purity of the product. . , .. ■ It is well known .that some compounds and ionic species of a few elements,-when present .in-solutions at low concentrations, undergo pronounced hydroly.tic'and;phptochemical reactions, combine with certain impurities to fpfm. colloids,-, or are,lost from 'solutiön on account of

31 32 IYA and GOPAL

adsorption on to the walls of the container.. Besides these factors, in solutions of radionuclides and their labelled compounds, another important phenomenon known as radiation decomposition contributes, to the. complexity of the changes. ' ' ' ' .Sometimes the presence of certain impurities, which would not be objectionable in conventional pharmaceuticals, might be .totally undesirable in radio pharmaceuticals. These considerations, coupled -with_ the fact that a number of radionuclides and their compounds.of medical importance are used on human beings, necessitate á careful control of. the quality of the products and sometimes of .the raw m aterials also.:

1. CONTROL OF THE RAW MATERIALS

It would be difficult to carry out quality control on all-raw'm aterials, - target materials for irradiation and reágent chemicals used in’the various processing'stages. This problem is solved to some-extent by^the use of raw m aterials of the highest grade of purity.available^- A few of them are, however, subjected to certain specific controls,-, which are.carried out on a representative sample obtained from a bulk packet of'the ráw m aterials in question. Tellurium, the target element.employed for iodine-131 production, is specifically treated for natural iodine content.' it: is isolated by a series- of processes involving dissolution of tellurium-in chromic sulphuric acid mixture, reduction with oxalic acid and_ steàm distillation HI. The' steam distillate is trapped in dilute sodium bisulphite solution. The iodide in the latter is examined amperometrically [2] 'or spectrqphptomëtrically [3].'" The iodine content should be less than 1 ppm., ' - Potassium chloride, which is the target-for sulphur-3 5 production,’’ is previously assayed for its sulphate content,’-'which'should be less-than' 1 ppm. The sulphate is isolated by chromatography on an alumiria ’■ column [4] and determined by spectrophbtomètry [5]. ' - ■ The chromic ion content of potassium chromate is determined by repeated co-precipitation with-Al(OH)3'in an alkaline-cycle [ 6 ] and finally, determined by spectrophotometry with diphenylcarbazide [7] and should be less than a few ppm. •' '• Rose Bengal, to cite ah example of a raw material employed for ' - labelling by isotopic exchange, is tested for tetra-chloro-.tetraiodo- ‘ . fluorescein content by chromatographic techniques [ 8 ]. It is also tested for its apyrogéneity and frèedom from undue toxicity.' : ' ” •" Certain target m aterials are subjected, to neutron activation analysis before they are employed for regular production. This method of analysis is often more sensitive than chemical or-spectrogräph’ic .tests, and may be the only helpful method of examination of c'ertáin target elements-. Thus, ' cobalt impurity ( 0 . 1 ppm) in.nickel-is detected by .neutron irradiation and ' subsequent gamma spectrometry of the 60mCo (10-m),radionuclide.[8 d ].’ Water, which is the vehicle, in most of the radiopharmaceutical in- . jections, solutions of buffers for adjustment of pH, saline solution for adjustment of isotonicity, and solutions'of gelatin utilized for preparaticm of colloids — to quote à few of'the important items is examined for sterility and absence of pyrogens and undue-toxicity. ' IAEA-PL-336/2 33

2. CONTROL ON RADIOPHARMACEUTICALS

The controls carried out on radiopharmaceuticals differ in some respects from those carried out on conventional pharmaceuticals and fall into five groups — physical, chemical, radiochemical, biological and consignment controls. Some aspects of these controls have been dis­ cussed elsewhere [9-11]. Some special features of the radionuclidic, chemical and radiochemical purity and possibly biological controls, and some aspects of the organization and running of a radiopharmaceutical control laboratory are discussed in this paper.

3. RADIONUCLIDIC PURITY

There still appears to be a lack of unanimity concerning some of the terminology used for quality control of radiopharmaceuticals. The term radionuclidic purity is undoubtedly to be preferred to the terms radio­ active or radioisotopic purity. For example, if cobalt-60 and iron-59 are present in cobalt-58, the term radioisotopic impurity can be applied to cobalt-60 but not to iron-59. This difficulty and ambiguity does not arise when the term radionuclidic purity is used. In some recent litera­ ture, however, the phrase "radiochemical purity" has been mistakenly used for radionuclidic purity. Radionuclidic impurities arise when nuclear side reactions proceed concurrently with the main nuclear reaction. Traces of impurities in targets may have very high cross-section and thus produce significant amounts of radionuclidic impurities, which may or may not be isotopic with the radionuclide of interest. We shall consider a few examples.

C ase I

3 5 С 1(п ,т)3 6 С1, 3 5C l(n, p)3 5 S, 3 5 C l(n ,a ) 3 2 P , 4 1 K (n, y)42K

These reactions are met within the production of 35 S from KC1. The non­ isotopic radionuclidic impurities like 3 2 P , 3 6 C1 and 42K may easily be re­ moved by chemical purification, and therefore do not pose much of a p roblem .

C ase II

5 4 F e ( n , 7 ) 5 5 F e , 5 8 Fe'(n, 7 )59F e .

1 9 6 Hg(n, 7 )197 Hg, 202 Hg(n, 7) 203Hg

These represent cases of isotopic radionuclidic impurities, which can be considerably minimized by using enriched targets or sometimes, avoided by employing alternative nuclear reactions. The amount of MFe and 58 Fe in natural .iron are 5.82 and 0.33%, respectively. Irradiation of such a target for four weeks produces 55Fe and 59Fe activities in a ratio 3:1. The ratio drops to 1:100 when the iron target contains 80% 58F e . 34 IYA and GOPAL

N aturally o cu rrin g m e rc u ry , containing 0.146% 196Hg and 29.8%) 302 Hg yields, on irradiation for a week, ia,Hg containing about 2% 2 0 3 Hg. W ith a ta rg e t containing 4% 196 Hg the 203Hg contam ination drops to 0 .1 . In the two cases considered above, the contaminants are long-lived. Radionuclides containing short-lived contaminants can, however, be allowed to decay for a suitable time and then employed. 82 B r produced by irradiation of natural bromine for 36 h contains 82Br : ( 80mB r + 80B r ) activities in the ratio of'1:5, but 36 h after pile shut-down, the ratio drops to 1 0 0 : 1 , which would be satisfactory for use.

Case III

197Au(n, y) 198Au(n, 7 ) 199Au

This is a case where isotopic radionuclidic impurities are produced by a secondary burn-up of another radionuclide. The thermal neutron activation cross-sections of 197Au and 198Au are, respectively, 99 and 26 000 b. Hence 198Au employed for medical application usually contains 19 9 Au, the actual quantity of which depends on the neutron flux and energy of the neutrons. For identical quantities of gold irradiated for the same period, an increase in the neutron flux from. 1 0 13 - 1 0 14 (n/cm 2 sec) results in an 85-fold increase in the production of 199Au [12]. Thé secondary reaction can be greatly reduced by shielding the gold target with a thin sheet of cadmium foil [13]. Thus, 198Au practically free from 199Au can be produced, but attended by a diminished production rate of 19 8 Au. From the above considerations it can be seen that radionuclidic im ­ purities are sometimes unavoidable. In some cases they can be tolerated, as for example 199Au in 198Au, since the half-lives and emissions are not very different. The same cannot be said of the contaminants 203Hg and 60Co in 197Hg and 58 Co, respectively, as can be seen from the radiation dosimetry data given in Table I. Radionuclides are distinguished from each other by their half-lives, the types of radiation emitted, and the energy of the radiation. Radio­ nuclidic purity is established by examination of one or more of these characteristics. Determination of the half-life is not always a practicable method for determining the radionuclidic purity of radiopharmaceuticals before their release for use. On the other hand, an examination of their three main types of radiations, beta particles, positrons, X-rays and gamma photons serves as a quicker means of establishing the identity and purity of radio­ n u clid es. Beta particles are detected by Geiger-Müller or proportional counters. Their energy is calculated from an analysis of an absorption curve, which is constructed by plotting the count-rates versus the various absorber thicknesses (mg/cm2), which are interposed between the source and the counter. The absorber is usually beryllium or aluminium. The purity of the radionuclide is considered as satisfactory if the value of the energy so determined falls within +15% of the reported values [15]. Liquid-scintillation spectrometry can be employed for determining or checking the radionuclidic purity of some radionuclides as, for example, in an estimation of up to 2% iron-59 [16] and as a qualitative check for IAEA-PL-336/2 3 5

TABLE I. BETA DOSE DELIVERED TO TISSUES BY SOME RADIONUCLIDES

Dose delivered H alf-life Radionuclide Eß T (MeV) P rads/g of tissue Delivery duration by 1 цС i (d)

198Au 2 .6 9 d 0 .3 2 32 2 .7

lwAu 3 :1 5 d 0 .1 3 15 3 .2

5¥ e . 2 .9 4 yr 0.0 0 6 14 45

59Fe 4 5 .0 d 0.1 1 8 196 45

197Hg. 2 .7 d 0 .0 7 12 48

203Hg 48 d 0 .1 0 177 48

58Co 72 d 0 .0 3 5 93 . 72

60Co 5 .2 yr 0.0 9 3 342 72

The beta dose delivered is calculated from formula [ 14] :

Dß | t= 7 3 .8 C EBT p ( l - 2 ' t/TP) rad/g where С = дСл/g

Eg= Me V/disintegration

Tp = h a lf-life in days

t = dose delivery duration

traces of weak beta emitters in strong beta emitters (e.g. 35S in 32P solution) and as a qualitative means for determination of hard beta contami­ nants in weak beta emitters [17]. Gamma photons are usually detected by means of a scintillation counting assembly consisting of a crystal of sodium iodide and a photomultiplier. The energy of the gamma photons is determined by a multichannel spectro­ meter coupled to the scintillation counter. The energies of the gamma photons by which a few important radionuclides may be identified are: 51 Cr 0.323 MeV; 59Fe 1.10 and 1.29 MeV; 131I 0.36 MeV; 198Au 0.412 MeV etc. The shape of the spectra obtained is compared with standard spectra [18]. The presence of a pure beta emitter mixed with a gamma emitter goes undetected by mere Nal-scintillation gamma spectrometry. Also, a mixture of gamma emitters of closely related energies is unlikely to be differentiated by gamma spectrometry alone. In some cases, for example 95Zr in 95Nb, 36 IYA and GOPAL

FIG .l. Gamma spectrum of 95 Zr - 95Nb with Ge(Li) detector.

FIG.2. Gamma spectrum of 95 Zr - 9sNb with Nal(Tl) detector. it is possible to differentiate the gamma photons by means of a lithium- drifted germanium detector (Figs 1 and 2). For many other cases radio­ chemical separations will have.to be made. Sometimes paper chromato- graphic-or electrophoretic separations with added carriers can be carried' out and the zones are cut out for characterization of their radiations. These operations are time-consuming and cannot always be performed on a radio­ pharmaceutical batch before its release for use. They can only be carried out periodically. To a considerable extent reliance must be placed on the much stressed "good manufacturing practices". Absence of long-lived radionuclidic impurities is ascertained by an examination of the radio­ pharmaceutical solutions after the lapse of several half-lives. For example, IAEA-PL-336/2 37 the 60Co contamination in batches of 58Co may in this way be shown to be less than 0 . 0 1 %. An examination of the half-life, beta absorption curve, and the gamma spectrum is thus helpful in identifying the radionuclide as well as a s­ certaining its purity, which is an important criterion for all radio - pharmaceuticals. The radionuclidic purity is generally greater than 99%. In the few exceptional cases cited already, leaving'aside the inevitable contaminant radionuclide, the purity of the main radionuclide is usually greater than 99%. The percentages of the two should, however, be clearly stated before being supplied to the user.

4. CHEMICAL PURITY

The target materials employed for radioisotope production and the reagent chemicals employed in the various stages of processing are of the highest grade of purity available. The production procedures involve techniques such as distillation, solvent extraction, selective precipitation and ion-exchange, all of which by their nature purify the desired product. Processing is 'carried out in closed units and on a small scale. Hence, radiopharmaceutical solutions are of a very high order of purity. Emission spectrography is a convenient method for simultaneously deter­ mining several elements present in a small aliquot of a sample. Though it is an invaluable technique, one encounters several problems in employing it to analyse radioactive solutions. In addition to the considerable radi­ ation hazard involved in handling highly radioactive solutions, the contain­ ment of the radioactive vapours in the sparking chamber requires elaborate arrangements. In the absence of such a facility, one can adopt the well- known limit tests given in the pharmacopoeias for a semi-quantitative determination of heavy metals as hydroxide or sulphide precipitable matter at pH 7-9 [9]. Polarographie techniques are also useful in estimating heavy metals in solutions [19]. A toxic impurity such as tellurium in iodine-131 solution is semi-quantitatively estimated by spot test [20]. Traces of arsenic in most of the radiopharmaceutical preparations are estimated spectrophotom etrically [21]. Sulphate in phosphoric acid solutions, may be determined by precipi­ tating with benzidine which is spectrophotometrically estimated by dia- zotizing with thymol (Amax 500 m/u) [5]. The cyanide concentration of vitamin B-12 solutions is routinely assayed by reaction with chloramine-T, pyridine-barbituric acid reagent (\max 580 тпц) [22]. . Methods used for determining the carrier concentrations in other radiopharmaceuticals are mentioned in an earlier publication [ 1 0 ].

5. RADIOCHEMICAL PURITY

This aspect of purity is unique to.radiochemicals. If a radionuclide is in a chemical form other than that stated or expected, then it is said to be radiochemically impure. Reasonable amounts of non-radioactive impurities are often relatively unimportant, while radiochemical impuri­ ties would not be considered so. Radiochemical impurities must be kept to a minimum as they can falsify the conclusions derived from the use of 38 IYA and GOPAL a labelled compound or give rise to faulty diagnosis and act as a source of unwanted radiological dose to certain specific organs of the body. The form in which radiochemical impurities are commonly manifested in some well-known radiopharmaceuticals are:

(i) The labelling agent accompanies the labelled compound (e.g. Nal in radioiodinated compounds); (ii) Chemically related impurities are also labelled along with the compound of interest (e.g. the mono- and di-oleins in triolein); (iii) Compounds used as preservatives get inadvertently labelled and constitute a major radiochemical impurity (e.g. beta naphthol in Na 131I - gelatin capsules undergoing iodination [25]). (iv) A radionuclide in a definite valency state appears in different molecular forms caused by overheating (e.g. pyro-, poly- and meta- phosphates in orthophosphate-32P).

Radiochemical impurities may arise as a result of chemical instability of the compound or as a result of radiation decomposition during storage. To some extent, the conditions of storage also affect the radiochemical purity. In general, many labelled organic compounds with very high specific activities are prone to decomposition. Measures to safeguard the radiochemical purity of compounds are maintenance of optimum pH, use of moderate specific activity, careful sterilization, addition of stabilizing agents, protection from light and storage at low temperatures. Methods for determining radiochemical purity are based on various physico-chemical techniques such as paper chromatography, thin-layer chromatography, paper electrophoresis, ion-exchange chromatography, polyacrylamide gel electrophoresis, gel filtration, reverse radioisotope dilution analysis, solvent extraction and dialysis. Polyphosphates, which appear in orthophosphoric acid as a result of overheating, are determined by paper chromatography on acid-washed Whatman 540 using isopropanol:trichloracetic acid:ammonia:water (75:5:0.3:25) [23]. The duration of chromatography is 18-20 h and the Rf values of the various species are meta (0), pyro (0.40-0.45), and ortho (0.7-0.8 ). It is essential to add a few micrograms of each of the carriers to the radioactive phosphoric acid solution prior to chromatography. Radioactive sodium iodide solution is examined for iodate impurity by paper electrophoresis in 0.05 M Na2 HPQj on Whatman No.540 at 8 V/cm over 2^ h [10]. A few micrograms of each of the two carriers is added to the solution before electrophoresis. The distance of anodic migration is 0.7 that of iodide. Under the same experimental conditions a host of other organic radioiodine-labelled compounds are examined for radiochemical purity. Chromatography on ion-exchange cellulose papers affords another convenient method of separating free iodide from a number of radio­ pharmaceuticals [24, 25]. The radiochemical purity of radioiodine-labelled hippuran is examined by paper electrophoresis in phosphate buffer as mentioned under radio­ iodide or by paper chromatography in the solvent benzene-glacial acetic acid-water (2:2:1) [26] and n-butanol-glacial acetic acid-water (4:1:1) [27]. The brownish colouring matter that develops as the solution of the labelled product ages, is separated by filtration through DEAE-cellulose (OH ) and elution with normal saline. The brown substance is strongly adsorbed IAEA-PL-336/2 39 in the top layer of the column and is eluted only by concentrated sodium chloride solution or much more easily by dilute sodium hydroxide solution. The brown zone has negligible radioactivity [17]. Methods for other iodine- labelled compounds are also available [28]. Vitamin B-12 labelled with 57Co or 58Co is examined by ion-exchange chromatography using DEAE-cellulose (OH ) and CM-cellulose (H+)29 which separate red acids and aquocobalamin respectively from B-12 activity. Reverse isotope dilution analysis, solvent extraction and paper chromatography [30] are also employed for this analysis. During storage the decrease in the radiochemical purity of cyanocobalamin-57Co/58 Co is due to a partial conversion to hydroxy and aquocobalamin form as evidenced by increased activity retention on the CMC column [17]. The radiochemical purity of the organomercurial neohydrin, and mercuri-hydroxypropane labelled with l97Hg or 203 Hg is verified by paper electrophoresis on Whatman No.540 and O.lMNaCl, pH 5-6 at 8 V/cm for 2-2| h [10], and by paper chromatography on Whatman No.l with 0.01M ammonia. Carrier m ercuric nitrate and organomercurial carriers are added to the solutions prior to application on electrophoresis or chromatographic paper strips [17]. The radiochemical purity of serum albumin labelled with 51Cr, 95Nb, 125I, and 131I is verified by paper electrophoresis, by gel filtration through Sephadex-G-150 or 200 and by polyacrylamide gel electrophoresis which gives a rapid and clean separation of the various protein fractions [17]. Dialysis through a cellulose'membrane (48 Á core diam.) and gel filtration through Sephadex-G 15 or 25 [17], paper chromatography on Whatman No.540 using the solvent system acetone; conc. HC1 (d= 1.19): water (70:10:20) [31] and paper electrophoresis on Whatman No.540 in 0.075M sodium thiosulphate as electrolyte [32] are employed for deter­ mining the radio-chemical purity of gold colloid-198Au.

6 . BIOLOGICAL CONTROLS

The routine biological tests carried out on the radiopharmaceuticals at Trombay are those for sterility, pyrogens, and undue toxicity, in-vitro labelling tests on som e products, and m icrobiological assay. The sterility test is carried out according to BP procedures [33]. In the case of most products the test is started immediately after the preparation of the product so that at least a 24-h observation is made before its despatch. In the case of a few injectables o_f high radioactive concentration, because of the high radiation dose to the container, the test is carried out after allowing a suitable decay period. The sterilizing condition prevailing during an autoclaving operation is checked with spores of Bacillus stearothermophillus [34] . Environmental control tests and total sterility tests [3 5] are done in cubicles and boxes where aseptic manipulations are carried out. Pyrogen tests are made on all injectable products. The quantity of activity injected varies between 1-60 times the human dose (proportional to body weight) [ 1 1 ]. An undue toxicity test is carried out on all orals and injectables and on some primary radioisotopes employed for the purpose of labelling (e.g. 57Co, 58Co) as soon as they are ready. This is based on the method prescribed in the BP for phenol-sulphon-phthalein [36]. An aliquot (which 40 IYA and GOPAL is several times the human dose proportional to the body weight) of the stock solution is injected into each of five mice, each weighing 20 g. The products are released for human use only if all the mice are alive and normal. Both pyrogen and undue toxicity tests are invariably complete before the release of the products of radionuclides, that are not too short-lived.

7. ORGANIZATION AND ADMINISTRATION OF A RADIOPHARMA­ CEUTICAL CONTROL LABORATORY

It is important that the Quality Control Section be quite independent of the production organization. In the Isotope Division at Trombay, this has been taken into account and the head of the Quality Control Section reports directly to the head of the Isotope Division. The Quality Control Section is not only concerned with the analysis of the final products, but also of raw materials procured for the production of all radiopharmaceuticals. The section consists of four groups which look after the physical, chemical, and biological controls on every individual batch of the various radiopharmaceuticals produced, and also consignment controls on every individual outgoing consignment. As soon as a radiopharmaceutical is ready aliquots of it are sent to the Quality Control Section in separate vials for physical, chemical, and radiochemical controls, and for pyrogen, sterility and undue toxicity tests. Besides these samples, another aliquot of the product is dispensed and retained by the Quality Control Section for any future reference, especially if complaints from customers are to be investigated. The details of the product given for analysis are entered in an analysis request slip (Appendix I). The physical control group carries out radioactive standardization (by primary as well as secondary methods) radionuclide identification, certi­ fication of the radionuclidic purity, and periodical studies on particle size of colloids. The equipment necessary for the routine physical controls of radiopharmaceuticals is given in Appendix II. The chemical control group carries out controls such as determi­ nation of pH, acidity and toxicity, limit tests, estimation by volumetric, spectrophotometric and electroanalytical techniques, and determination of radiochemical purity by various physicochemical techniques. The equipment and facilities required for carrying out the various controls are listed in Appendix III. The biological control group carries out pyrogen and sterility tests (on all injectables) and undue toxicity test (on orals and injectables) verification of the in-vitro labelling properties of some products, microbiological assays etc. Data on pyrogen and sterility tests are entered in laboratory data sheets shown in Appendices IV and V. The basic facilities and equip­ ment for biological controls are lis,ted in Appendix VI. The consignment controls and packing inspection are carried out by a separate group. Data regarding the consignments going out to a customer are entered in a "consignment history sheet" (Appendix VII) by the dispensing group of the production section. These documents, along with the consignments, are handed over to the Quality Control Section. Before despatching them to the customers each and every consignment IAEA-PL-336/2 41 is examined for the following criteria: (i) clarity; (ii) a gross check on the radioactivity by direct measurements on an ionization chamber where- ever possible or with a gamma scintillation counter in the case of ampoules ofvitaminBi2 57Co/58 Co; (iii) the volume of the solution in the container; (iv) any potential leak of the radioactive material; (v) ascertaining whether the product complies with controls such as radiochemical purity, undue toxicity tests, etc. wherever possible. The corresponding entries are made in the consignment history sheet, A packing note (Appendix VIII) containing all the important particulars about the product is then prepared. The consignments are transferred to suitable packing containers which bear the label and the packing note. Adoption of these measures has been very helpful in preventing faulty consignments reaching customers. Any complaints from customers are jointly attended to by the Sales Department and the Quality Control Section.

8 . RECORDS MAINTAINED

An inventory of the raw materials employed in the production of the radiopharmaceuticals is prepared by the Production Section and a copy deposited with the Quality Control Section. The identification numbers of the materials in the inventory are always mentioned in the production schedules. Separate analysis records are maintained for each radio­ pharmaceutical. Details of the analysis carried out on each and every batch of a radiopharmaceutical are recorded in the log-books and the analysis request slips coming from the Production Sections are documented along with these records. As and when the results of the various controls are ready, the Production Sections are provisionally informed in order to facilitate their dispensing programs. Soon after completion of all controls a composite analysis report (Appendix IX) is prepared and given to the Production Section. Duplicates of these reports are maintained in the Quality Control Section. The packing note, which is supplied to the customer along with the consignment, gives all pertinent details of the product. 42 I YA and GOPAL

APPENDIX I

BHABHA ATOMIC RESEARCH CENTRE Isotope Division

Quality Control Analysis Request Slip

(a) Description of the product. (b) Date of processing: (c) Batch No. (d) Code;

Location Activity of the S. No. Description of the sample Volume Date and tim e with tim e sample

1 Batch Control ml

2 Physical Control m l

3 Chem ical and Radio­ ml chemical controls

4 Undue toxicity test m l

5 Sterility test ml

6 Pyrogen test ml

Mode of sterilization:

Please arrange for the above-mentioned sample to be analysed at an early date.

Processed by:

Head, Special Medical Preparations Sec.

Head, Quality Control Section IAEA-PL-336/2 43

APPENDIX II

BASIC EQUIPMENT FOR ROUTINE PHYSICAL CONTROL OF RADIO­ PHARMACEUTICALS

1. End-window gas-flow proportional counting assembly with associated electronics. 2 . 2 -pi gas-flow proportional counting assembly with associated electronics. 3. Automatic absorber changer fitted with end-window G-M Counter or end-window gas-flow proportional counter with facilities for counting for preset counts or present time, and and digital print-out of counts per minute. 4. Gamma-scintillation spectrometer with 3 in.X 3 in. Nal(Tl) well-type detector assembly. Well dimension,! in. diam X l j in. deep, approximately. 5. A multichannel analyser with standard 3 in.X 3 in. Nal(Tl) integral assembly having resolution better than 10% (for energy 0.67 MeV) for qualitative and quantitative gamma spectrometry with digital printing and x-y plotting facilities. 6 . Liquid-scintillation counting assembly with associated electronics. 7. Calibrated gamma ionization chamber for beta and gamma emitters. 8 . A semi-micro balance (accuracy ±0.01 mg) for source preparations. 9. A beta-gamma box for preparation of sources for counting. 10. A beta-gamma box for storing calibrated solutions of radionuclides required for routine standardization.

APPENDIX III

FACILITIES AND EQUIPMENT FOR ROUTINE CHEMICAL AND RADIO­ CHEMICAL CONTROLS IN RADIOPHARMACEUTICALS

1. Beta-gamma boxes with adequate air change for handling low levels of activity with facilities such as centrifuge, water bath, small drying oven, infra-red heater etc. 2. Glove-cum-tong boxes with shielding for high levels of activity, one exclusively meant for handling 131I. The boxes must be provided with facilities given under Item 1. 3. Micrometer syringes for pipetting radioactive solutions, e.g. Agla syringes and Metrohm micro-hand burettes. 4. pH meter with combined microelectrode assembly. 5. Magnetic stirrers with hot plates. 6 . Ultra-violet spectrophotometer. 7. Polarograph. 8 . Coulometric titrator. 9. Equipment for paper and thin-layer chromatography. 10. Equipment for paper electrophoresis. 11. Equipment for column chromatography with a fraction collector. 44 IYA and GOPAL

12. Ultra-violet lamp assembly. 13. A radiochro'matogram scanner for paper and thin-layer chromatograms 14. A separate cubicle for carrying out chromatographic and electrophoreti analyses, and fume hoods for air-drying strips, and for spraying strips, and for spraying reagents on to strips. 15. A dark-room for autoradiography.

a p p e n d ix IV

BHABHA ATOMIC RESEARCH CENTRE ISOTOPE DIVISION

STERILITY TEST REPORT

Sample: Batch No.:

General Remarks: Date:

Observation on following dates

Media

Thioglycollate Fluid Medium

Conclusion: The sample ------^------— with B. P. test for sterility. does not comply

Analyst:

Head, Quality Control Section IAEA-PL-336/2 45

APPENDIX V

BHABHA ATOMIC RESEARCH CENTRE ISOTOPE DIVISION

PYROGEN TEST REPORT

Sample: - Batch No.: General Remarks: Date of Test:

Weight Kg Kg Kg Kg Kg

Rabbits Nos. 1 2 3 4 5

Body Temp, Tim e Tem p. (day previous 1) to the test) 2)

Body Tem p, 1) (day of test) 2)

Mean Initial Temp.

ml. injected

Temp, at +i hr.

Temp, at +1 hr.

Temp, at +l|hrs.

Temp. at+2hrs.

Tem p, at +2£ hrs.

Tem p, at +3 hrs.

Maximum Temp.

Rise of Temp.

Conclusion: The sample ------^------— withB.P, test for limit of pyrogens. r does not comply r/ ° Analyst:

Head, Quality Control Sec. 46 IYA and GOP AL

APPENDIX VI

FACILITIES AND EQUIPMENT FOR ROUTINE BIOLOGICAL CONTROLS ON RADIOPHARMACEUTICALS

1. Animal house for keeping rabbits and mice. 2. Special racks and cages with facilities for continual flushing with water and for removal of excreta. 3. Separate constant temperature room for carrying out pyrogen and undue toxicity tests. 4. Fume hoods for pyrogen and undue toxicity tests. 5. Temperature measuring device with thermistor probe; the measuring device is preferably kept in the adjoining room. 6 . A beta-gamma box for diluting stock solutions for pyrogen and undue toxicity tests. 7. A separate cubicle with an ante-room and with filtered air supply for carrying out sterility tests. One or two asecptic glove boxes with ultra-violet lamp fixtures, and electric bunsens. 8 . A separate room for microbiological assay. 9. A separate room for media preparation incubators, autoclave, ovens, shakers, weighing balances and other equipment. 10. Spectrocolorimeter. IAEA-PL-336/2 47

APPENDIX VII

ISOTOPE DIVISION Consignment History Sheet

1. PRODUCTION DETAILS IDA No. 213 Cons. No. 4328

a) Application Customer Client x Isotope; *®Co C lient.x Activity 75 /Ci on 3 1 .3 . 69 Code: COM-2

Remarks

(b) Stock solution Stock soln.No. 5*Co Vit B-12/40 Control No. 58Co Vit B-12/40/C

Assayed activity 25 дс/ml date 5.10.68 T im e

Activity at 5 дс/ml date 25.3.69 T im e dispensing.

(c) Dispensing 1. Vol. of stock solution in ml 2 x 10 ml

2.

3.

4 .

Total volume: 2 x 10 ml

Chemical form Cynocobalamin pH 4 .1

Container Vial

Activity required 100 цс date 25.3.69 Tim e

Activity dispensed 100 дс date 25.3.69 Tim e

Physical Appearance: Clear to the naked eye.

Remarks: For oral use only. Specific activity: 34 ßCi/ßg

Dispensed b y :...... Approved b y :...... •

Health Physicist Certificate: No contamination outside the vial.

2. QUALITY CONTROL DETAILS:

Activity on date date 5.10.68 Time of checking_____ 2 x 252 jiCi______

Activity on reqd. date 31.3.69 Tim e - date 2 x 45 /iCi

Volume 2x10 ml pH 4 .1 .

Test for sterility: _

Pyrogen test: -

Undue toxicity test: Passes

LABEL 2 X 4 5 /iCi in 2 x 10 m l on 3 1 .3 .6 9 .

Remarks: Specific activity: 32 jiCi/pg.

Checked b y : ______(Head, Quality Control Section) 48 IYA and GOPAL

APPENDIX VIII

ISOTOPE PACKING NOTE DIVISION BH A BH A A T O M IC R E S E A R C H C EN T RE/T RO M B A Y/BO M B A Y 74 ( AS)/ IN D IA

CABLE: ISOTOPES, BOMBAY TEL: 521401 & 523260 TELEX: ATOMERG, BOMBAY 355

Order No. Date Quantity Ordered

■Our Ref. Packing Note No. IDA/ Consignment No.

• Invoice to

Despatch Particulars Outer Surface is free from radioactive contamination.

Surface dose rate • mr/hr

Health Physicist

Cons. Date & Code D escription W t./Vol. Activity No. T im e

COM -2 Vitamin B-12 Co-58 solution 4328 2 X 10 ml 2 X 45 iiCi 3 1 .3 .6 9 (High specific activity) NON-INJECTABLE

Specific activity 32 fiCi/fig B-12.

Remarks:

To be stored below 10°C.

Date: for Head, Isotope Division IAEA-PL-336/2 49

APPENDIX IX

BHABHA'ATOMIC RESEARCH CENTRE Isotope Division

Quality Control Section

Report on Analysis of Cynocobalamin stock solution 57 Co/ 58 Co

S . No. Date Initials

1 Stock solution No:

Date of preparation:

2 pH

3 Radioactive concentration

4 Radionuclide identification

5 Radiochem ical purity

6 Cyanide content

7 Specific activity

8 Undue toxicity Test i • .

9 Sterility Test

10 Pyrogen Test •

Head, Quality Control Section 50 IYA and GOPAL

REFERENCES

[■1] IYA, V .K ., MURTHY, T . S . , BALASU BRAMANIAN, K .R ., RAMACHANDRA, M ., NAIR, V. C ., AEET/Radiochem/50, Bhabha Atomic Research Centre, Bombay (1964). [2] LAITINEN, Anal. Chem. 28 (1956) 666. 1 [3] CUSTER, J.J. , NATELSON, S. , Anal. Chem. 21 (1949) 1005. [4 ] FRITZ, J . S . , YAMAMURA, S . S . , RICHARD, M .J ., Anal. Chem. 29 (1957) 158. [5] ALLPORT, N .L., KEYSER, J.W ., Colorimetric Analysis, 1, 2nd Edn, Chapman and Hall Ltd. (1957) 326. [6 ] GOPAL, N .G .S ., In Press. [7] CHARLOT, G ., Les Méthodes de la Chimie Analytique, 4th Edn, Masson et Co. Paris (1961) 705. [8] TAYLOR, K.B., Nature 185 (1960) 243. [8a] GUINN, V .P., in Production and Use of Short-lived Radioisotopes from Reactors, (Proc. Seminar, Vienna, 1962) IAEA, Vienna (1963) 3 SM-32/27. 1 [9] IYA, V.K. et a l., AEET/Radiochem/43 (1963). [10] GOPAL, N .G.S., IYA, V.K., J. scient, industr. Res. 26 No.4 (1967) 153-158. [11] GOPAL, N .G.S., PATEL, K.M ., "Radiopharmaceuticals and their biological control" 19th Session Indian Pharmaceutical Congr., Hyderabad, 25-27 Dec. 1967. [1 2 ] FRIEDLANDER, G ., KENNEDY, J. W. , MILLER, J .M ., "Nuclear and Radiochem istry". [13] BAKER, P .S ., "Reactor produced radionuclides", "Radioactive Pharmaceuticals" (ANDREWS, G .A . et a l . , Eds), USAEC/Div. T ech . Inf. (April, 1966) 134. [14] SILVER, B ., Radioactive Isotopes in Medicine and Biology - Medicine, LEA and Febiger, Phil. (1962) p.215. [15] OVERMAN, R .T., CLARK, H.M ., Radioisotope Techniques, McGraw Hill Book Co., Inc. (1960) 221. [1 6 ] GOPAL, N .G .S ., HETHERINGTON, E .L ., Unpublished results. [17] GOPAL, N.G.S., Unpublished results. [18] HEATH, R.L., Scintillation spectrometry gamma-ray spectrum catalog, US Atomic Energy Commission Report ID 016408. [19] MEITES, L ., Polarographie Techniques, Interscience, N.Y. (1955) 247-307. [20] FEIGL, F ., Spot Tests in Inorganic Analysis, Elsevier Publishing Co. (1958) 350. [2 1 ] GRAWFORD, A ., PALMER, J .G ., WOOD, J.H ., M icrochim . Acta 2 ( 1958) 277. [2 2 ] ASM US.-E. , GARSCHAGEN,. H. , Z . Anal. Chem ie 138 (1953) 404, 414. [2 3 ] EBEL, J .P ., Mikrochim. Acta 6 (1954) 679. [24] GOPAL, N .G.S., Int. J. appl. Radiat. Isotopes 17 (1966) 75-77.. [25] GOPAL, N .G .S ., FRAPART, P., Int. J. appl. Radiat. Isotopes 17 (1966)129-32. [26] The United States Pharmacopeia, 17th Revision, p.619. [27] MAGNUSSON, G. , Nature 195 (1962) 591. [28] COHEN, Y ., "Chemical and radiochemical purity of radioactive pharmaceuticals", Radioactive Pharmaceuticals (ANDREWS, G.A. et al., Eds), USAEC/Div. Tech. Inf. (April, 1966) Ch.4. [29] GOPAL, N .G.S., IYA, V.K. , MAJALI, M., PATEL, K.M ., Indian J. Pharmacy 28 No. 6.(1966) 1 5 1-56. [30] British Pharmacopoeia, 1963, Addendum 1964, p .19. [31] MAJUMDAR, A. K., CHAKRABURTHY, M. M. , Anal. Chim. Acta 19 ( 1958) 129. [3 2 ] BEEN, U ., HOYE, A ., J. Chromat. 17 (1965) 631. [33] British Pharmacopoeia, 1963, Addendum 1964, pp.83-85. [3 4 ] HARRIS, M ., Pharm aceutical Microbiology, Bailliers, Tindall and C o x ., London (1964) 216. [35] KENNETH, E. Avis, "Parenteral preparations", Remington's Pharmaceutical Science, XIII”, (MARTIN, E. W. etal.,Eds), Mack Publishing Co. Easton(1965) 508-509. [36] British Pharmacopoeia(1963) 593. IAEA-PL-336/3

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS BY THE SPANISH NUCLEAR ENERGY BOARD (JEN) .

M. del VAL COB, D.V. REBOLLO GARRIDO, F. CASAS MEDINA, Junta de Energía Nuclear, Madrid, Spain

Abstract

ANALYTICAL CONTROL OF PHARMACEUTICALS BY THE SPANISH NUCLEAR ENERGY BOARD (JEN). Radiopharmaceuticals currently produced in the Junta de Energia Nuclear, Mádrid, are enumerated and their control is described. Several suggestions are made on the possible role of the IAEA in the field of radiopharmaceuticals.

I. INTRODUCTION

The production and control of radiopharmaceuticals in Spain, first begun in 1960, is carried out by the Isotopes Section of the Nuclear Chemistry Division, which is part of the Chemistry and Isotopes Depart­ ment of the Nuclear Energy Board (JEN). The process starts with radio­ isotopes produced within JEN by the Radiochemistry Section, which in turn are checked for chemical purity by the section mentioned and the Spectro­ scopy Section, both of which come under the Chemistry and Isotopes Department. At present the following radiopharmaceuticals are produced and analytically controlled:

32P Sodium phosphate (injectable) Sodium phosphate (non-injectable) Colloidal chromium, phosphate, 600-2000 A (injectable), Colloidal chromium phosphate, 300-700 A (injectable) Colloidal chromium phosphate, 100-300 Â (injectable) Colloidal zirconium phosphate, 250-350 Â (injectable)

51C Chromium chloride (injectable) Sodium chromate (injectable)

59Fe Iron chloride Iron citrate (injectable)

64Cu Copper acetate (injectable)

82Br Oleic' acid (non-injectable) Triolein (non-injectable) Olive oil (non-injectable)

51 52 del VAL COB et al.

131I Sodium iodide (injectable) Sodium iodide (non-injectable) Rose Bengal (injectable) Hippuran (injectable) Diiodrast (injectable) Oleic acid (injectable) ( Triolein (non-injectable) Olive oil (non-injectable) Lipiodol (injectable) Human serum albumin (injectable) Macroaggregates of colloidal human serum albumin, 10-50 ium (injectable) Microaggregates of colloidal human serum albumin 1 0 - 2 0 ;um (injectable) Gamma globulin (injectable) Fibrinogen (injectable)

198Au Colloidal gold, 200-300 Â (injectable) Colloidal gold, 70-90 A (injectable) Colloidal gold, 40-50 A (injectable)

203 Hg Neohydrin (injectable)

These radiopharmaceuticals come in two forms — injectable and non- injectable. In either case it is required that they combine a series of physical, chemical and biological properties which enables them to be administered to,humans, and it is necessáry to control these properties prior to their distribution. The control is threefold and takes the form of: J 1. Physical checks, which ensure the requisite radioactive concen­ tration, radionuclidic purity, colloidal particle size, pH value of the solution, etc. 2. Chemical checks, which ensure the requisite chemical and radio­ chemical purity, constitution and concentration of the solution, etc. 3. Biological checks, which ensure the requisite pharmacological characteristics of the radiopharmaceutical, such as, for example, sterility, isotonicity, absence of pyr.ogens and atoxicity, as well as other biological characteristics, such as biological affinity and distribution throughout the animal organism, etc.

The need to specify these characteristics and provide for checks has . been expressed in a number of countries, as a result of which these specifications have been published, usually together with the list o'f radio­ isotopes, the specifications which must be met by the radiopharmaceuticals and which have to some degree been incorporated in the relevant pharmacopoeias. Being aware of this need, the Argentine Nuclear Energy Commission (CNEA) and the Spanish Nuclear Energy Board have begun collaboration in order to establish the specifications with which radiopharmaceuticals must strictly comply, together with the procedures or standards which should be applied on a routine basis to test whether those specifications have been met. IAEA-PL-336/3 53

II. SPECIFICATIONS AND STANDARDS FOR RADIOPHARMACEUTICALS

Reference [l] lists the specifications and standards applied to the radiopharmaceuticals manufactured by both countries, with the appropriate definitions at the end. Chemical purity is determined by emission spectroscopy. The speci­ fication or criterion for the chemical purity selected is such that a radio­ pharmaceutical cannot be considered suitable for human application unless its chemical purity is above 98%. In many instances, and whenever possible, a purity greater than 99.9% is provided for. At the same time, the experience we have gained has led us to lower the purity limit to 98% in certain cases, such as for colloidal phosphates, since the chemical treat­ ment needed to produce the radiopharmaceutical is more complex. Radioactive purity, or rather radionuclidic purity, is determined by the techniques listed in Ref.[l] for each radiopharmaceutical individually, and is always specified as 99.9%. Radiochemical purity is likewise determined by a variety of methods for each radiopharmaceutical. The methods aré listed in Ref. [1]. Generally speaking, a purity greater than 98% is specified, although the use of products containing as much as 5% impurity is permitted.

III. POSSIBLE CAUSES OF DETERIORATION IN RADIOPHARMACEUTICALS. STORAGE CONDITIONS AND THE CONCEPT OF DECOMPOSITION TIME

Once the specifications to be met by each individual radiopharmaceutical have been established and have been checked by suitable methods or com­ pared with suitable standards, those specifications must be complied with, especially at the time when the products are administered to the patient. Ideally, therefore, the methods of checking should be applied immediately before administration of the radiopharmaceutical to human subjects. If this is not feasible, a decomposition time has to be fixed for each of the products, i.e. the period over which the primary specifi­ cations, as verified by the laboratory at the time of issue of the product, are preserved. The decomposition time of the radiopharmaceutical depends.not only on its basic characteristics but also on the mode of storage. It is there­ fore advisable to recommend or specify a suitable storage method to be followed by the users that will guarantee the decomposition time fixed by the production laboratory. Reference [1] gives the recommendations on storage established for Spanish radiopharmaceuticals under the heading "Observations". To establish the storage time it is first necessary to realize the main causes of deterioration for each radiopharmacëutical individually. Generally speaking the causes are as follows:

(1) Deterioration owing to radiolytic processes; (2) Deterioration owing to external physical agents, such as light or heat; (3) Non-fulfillment of the specifications due to radioactive decay, the drop of the specific activity below a threshold value, and so on. 54 del VAL COB et al.

In the first two cases a study should be made by varying the radio­ active concentration, specific activity and intensity of the external physical agents, as the case may be, so as to determine the effects of these variables a priori and then establish the decomposition time for given storage conditions. Reference [2] contains a study on the deterioration of 131I-Rose Bengal, 131I-Hippuran and 19sAu-colloidal gold, with the passage of time and with variation in certain external physical agents. In the third instance, the decomposition time must be specified as a function of the radioactive decay, provided the latter is the most important factor, in such a way that the radiopharmaceutical is used at a suitable level of specific activity. In this respect an important case to consider is the administration of colloidal gold-198 with such low specific activity that the injected solution produces physiological disorders due to the chemical excess of gelatin. An excess of Hippuran or Neohydrin may also produce a diuretic effect. In all the cases, in addition to the indication of the decomposition time, the shipment of a radiopharmaceutical must be accompanied by information on how it should be stored: for example, labels indicating that it should be kept in a refrigerator without light and minimum specific activity.

IV. ADMINISTRATION OF A LABORATORY FOR ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS. CHARACTERISTICS OF SHIPMENTS

The radiopharmaceutical control laboratory should be clearly dif­ ferentiated within a radiopharmaceutical production and distribution de­ partment. It must operate independently and control all the batches produced in accordance with standards similar to those described in Ref. [1], and the judgement it makes must be the final word concerning whether the radiopharmaceuticals produced within that department are acceptable for distribution. . As far as the control group is concerned, we consider that it should be made up of at least the following technical specialists:

One pharmacist One chemist , One laboratory assistant; should make use of the following equipment and techniques:

Electrophoresis (of all types) Chromatography (of all types) Equipment for measuring radioactivity (ionization chamber) Gamma spectrometry equipment Radioactivity analyser for electrophoretic diagrams and chromatograms Conductometer pH meter Spectrophotometer Densitometer Cryoscopy/ebullioscopy equipment Autoclave IAEA-PL-336/3 55

Complete bacteriological equipment for inoculating in culture media and for incubation Autoradiographic equipment Pyrogen detectors and detection techniques Pharmacological equipment and techniques for determining the. toxicity and selectivity of-radio pharmaceuticals in relation to different organs; and should have available suitably equipped laboratories as well as a dark­ room in which the above-mentioned equipment and techniques can be properly used. There should also be services for analytical emission spectroscopy, provided by an instrument analysis laboratory. In most cases control of each batch can and-must be carried out prior to its preparation and distribution. In some special instances and on the basis of facts emerging from previous research, because of urgent require­ ments (related primarily.to half-life), the control can be carried out concurrently with distribution; in this case the user can be advised by some rapid form of communication prior to administration of the pharma­ ceutical, that it should not be used and be given an explanation for the action taken. Normally, the tests for sterility and absence of pyrogens are carried out at set periods and in our particular çase provide information on the acceptability of the working conditions. However, they do not represent a special control for each batch. Shipments suitably labelled to give all the basic data of the pharma­ ceutical (activity, volume, date, pH, sterility and isotonicity) should also be accompanied by a data sheet with the rest of the specification.. A sample data sheet is shown in Appendix I. It is also the practice of the JEN to send out a technical bulletin on each new radiopharmaceutical that appears on the market for general distribution as well as to 'acquaint medical personnel with its characteristics. The data sheet should accompany every shipment of radio pharmaceuticals. It is advisable to send, imaddition, certificates relating to the inspections made, for example, photocopies of autoradiograms etc. Irrespective of the technical data supplied in the data sheet, every shipment of radiopharmàeeuticals should be accompanied by the appropri­ ate administrative documents, transport and packaging instructions, labels, and so on, in compliance with the IAEA transportation regulations. All this documentation is entered in the records of the control group.

V. CONCLUSIONS AND POINTS TO BE BORNE IN MIND

We have described the specifications and standards that the JEN applies for the control of radiopharmaceuticals. We have considered the principal causes of deterioration in radio­ pharmaceuticals and the methods employed by the JEN to establish their decomposition times. We have summarized the basic organic characteristics of an ana­ lytical control laboratory for pharmaceuticals and the basic data that should be recorded by the laboratory and supplied to the users. We con­ sider it appropriate to make the following suggestions: 56 del VAL COB et al.

(1) The International Atomic Energy Agency should publish radiopharma­ ceutical specifications and standards. In a few exceptional cases the national pharmacopoeias deal with radiopharmaceuticals, and in certain countries where the official documents have included references to the matter,, they cover a very limited number of such products. Such inter­ national specifications and standards would be of great benefit to nuclear energy organizations and serve as guidelines for the inclusion of radio­ pharmaceuticals in the national pharmacopoeias in a standardized form, especially for developing countries intending to produce radiopharmaceuticals.

(2) The specifications, criteria or lower limits for purity as applied to ■radionuclidic, chemical and radiochemical purity must be formulated with the greatest possible scientific rigour. In the majority of cases these limits are established by practice, analogy or the sensitivity of the control equipment employed.in each laboratory. An example that can be given is the percentage of free 131I permitted in the respective radiopharmaceuticals to be administered for diagnosis or therapy to normal persons, children or pregnant women (5% free 131I is permitted in Rose Bengal to be ad­ ministered to normal persons).

(3) The concept of decomposition time should be accurately defined and systematic research should be begun on the decomposition of radio- pharmaceuticals, beginning with those that are most commonly used.

(4) The lower limit for specific activity that can be administered in each case has not yet been clearly defined. Problems of tolerance and secondary effects make it advisable to fix this limit with the greatest possible scien­ tific accuracy.'

(5) Optimum storage conditions should be fixed and decisions taken with regard to the labels and additional information supplied to the user. Tem­ perature and light are fundamental factors.

(6 ) A table should be drawn up showing incompatibility in the administration of radiopharmaceuticals in relation to the general condition of the patient.

(7) In many cases the selectivity of a radiopharmaceutical with respect to an organ is not entirely clear. This is a matter of interest and should be given due attention. IAEA-PL-336/3 57

APPENDIX I

Technical Bulletin

JUNTA DE ENERGIA NUCLEAR DIRECCION DE QUIMICA E ISOTOPOS MADRID-3 ESPAÑA-SPAIN

TELEFONO: 24412 0013231 CORREO: AVDA. COMPLUTENSE APARTADO DE CORREOS: 3.055 CABLES Y TELEGRAMAS: JENISOTOPOS. MADRID ISOTOPOS

INFORMACION TECNICA TECHNICAL BULLETIN

PROOUCTO Product

REFERENCIA PERIODICIDAD PREPARACION Code n.° Availability

METODO DE PREPARACION Preparation method

DATOS TECNICOS Technical Data

Radiactivo Estable Compuesto marcado Radlofármaco Radioactiva □ SUW« □ O ffe4toph«rmeeevtfc*l O Actividad especifica Riqueza isotópica Specific activity I(oto pie enrichment

Pureza radioquímica Pureza química Radiochemical purity Chemical puHty

Concentración química Concentración radiactiva Chemical concentration Radioactiva concentration

Estéril Libre de pirógenos leotónlco Principal macrocomp. I I Starlle □ O laotonlo □ Main meerocemp. I I METODOS DE CONTROL Quality control methods 58 del VAL COB et al.

Technical Bulletin (cont.)

FORMA DE ENVASADO Y ALMACENAMIENTO. Packaging and Storage

OBSERVACIONES Additional remarks

PRODUCTOS ANALOGOS DISPONIBLES Related materials available

PRECIO Price

BIBLIOGRAFIA References

II IAEA-PL-336/3 59

APPENDIX II

D a t a S h e e t

JUNTA DE ENERGIA NUCLEAR DIRECCION DE QUIMICA E ISOTOPOS MADRID-3 ESPARA • SPAM

TELEFONO: 24412 0013231 CORREO. AVOA. COMPLUTENSE APARTADO OE CORREOS: 1055 CABLES Y TELEGRAMAS: JENtSOTOPOS. M A D ttD ISOTOPOS

HOJA DE CARACTERISTICAS

PRODUCTO Product

REFERENCIA LOTE FECHA PREPARACION Code n.° 8atch Preparation dato

CARACTERISTICAS Specifications

Radiactivo Establo Isótopo Г Radioactive □ Stable □ Isotope t Forma química Clwnlcal form

Actividad específica Riqueza isotópica 8poclfle »eil »It* liotoplo tnHchminl

Concentración radiactiva | Concentración química Radioactiva concentration Chemical concentration

Actividad total | i Fecha I I Hora I Total activity I I 0(1» I I Tima I

Cantidad total | i Volumen Total quantity Volum*

Pureza química f j Pureza radlolsotópica | | Pureza radioquímica d Z I

Estéril I------i Isotónico i I Libre de pirógenos I Startle I I loo tonta I I Pyrogen free |

M ETO DO S D E CO N T R O L Vease información técnica n.s FORMA DE ENVASADO

FECHA DE LA ULTIMA DETERMINACION: Date of the last assay

PREPARADO POR FECHA: Preparation made by Date 60 del VAL COB et al.

REFERENCES

[1 ] DEL VAL COB, M ., REBOLLO GARRIDO, D. V ., CASAS MEDINA, F . , Especificaciones y Normas de Radiofármacos (Specifications and standards for radiopharmaceuticals), CNEA (Argentina) and JEN (Spain), JEN, Madrid (1969). [2 ] CASAS MEDINA, F ., REBOLLO GARRIDO, D. V ., DEL VAL СОВ, M ., Estudios de degradación de Hippuran - I31l, Oro Coloidal — l98Au y Rosa Bengala - 131I (Studies on the disintegration of 131I- Hippuran,!98Au-colloidal gold and 131I-Rose Bengal), JEN internal rep. IS-1218/I-1, Madrid (1969), IAEA-PL-336/4

METHODS OF TESTING RADIOPHARMACEUTICALS USED BY THE ARGENTINE NATIONAL ATOMIC ENERGY COMMISSION (CNEA)

A .E .A . MITTA AND R. RADICELLA CNEA, Buenos Aires, Argentina

Short contribution

INTRODUCTION

In the CNEA some short-lived isotopes are produced and labelled molecules are prepared from different radioisotopes. In addition, im­ ported products are made up into lots. Tests are made on the raw ma­ terials and on the finished products before the latter are sent to the user. The production and testing activities are carried out in laboratories under the Commission's Directorate of Energy and Directorate of Research. Packing and distribution are arranged by the Directorate of Energy, which also takes care of the marketing of these products.

CHEMICAL TESTS

Radiochemical tests

Paper chromatography Thin-layer chromatography Paper electrophoresis Thin-layer electrophoresis Autoradiography - radioscanning

Identification tests for biological materials

Electrophoresis in polyacrylamide gel Electrophoresis in cellulose acetate Immuno-electrophoresis Radial immuno-diffusion

Quantitative tests for chemical and biological materials

Spectrophotometry

Determination of hormone damage

' Chromato-electrophoresis Reaction with specific antiserum and precipitation with Carbon-Dextran

61 62 M ITTA and RADICELLA

PHYSICAL TESTS

Measurement of particles

Haematocytometry and microscopy Spectrophotometry and electron-microscopy

BIOLOGICAL TESTS

Pyrogens Sterility Toxicity Distribution of organs Immunological characterization

RADIOACTIVE PURITY TEST AND ACTIVITY DETERMINATION

The radioisotopes are calibrated with conventional instruments and techniques. The instruments used are: Liquid scintillation equipment; well-type ionization chambers and sample-holders with the appropriate current measuring instruments; Nal(Tl) scintillation counters, flat and well-type, with single-channel and multi-channel spectrometers; thin-window Geiger- Müller counters; and gas-flow 4-7Г proportional counters. Some of the tests mentioned above are performed in the central testing laboratory and others in the production laboratory, as in the case of hor­ mones, proteins and colloidal gold. A new manual on radiopharmaceutical testing and a manual on radio­ active testing are being prepared simultaneously, and are expected to be ■ ready by the end of 1969. IAEA-PL-336/5

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS IN NORWAY

E. STEINNES Institutt for Atomenergi, Isotope Laboratories, Kjeller, Norway

Abstract

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS IN NORWAY. A review is presented of the radiopharmaceutical control unit in Norway. The equipment and methods are described. Some particular problems which apply to small producers of radiopharmaceuticals are emphasized. The co-operation among Scandinavian countries in preparing monographs for the Pharmacopoeia Nórdica is outlined.

1. INTRODUCTION

A major purpose of this Panel is to put forward proposals on how a laboratory for quality control in a smaller nuclear centre producing radio­ pharmaceuticals, should function. In several nuclear centres of this cate­ gory, the production of radiochemicals for medical application has already been in progress for a number of years. Information on the experience gained in some of these laboratories should certainly be of interest when recommendations on the organization of new analytical control laboratories are to be worked out. In our Institute radiopharmaceuticals have been produced regularly in the Isotope Laboratories for more than fifteen years. The steadily in­ creasing range of products has necessitated a rather extensive develop­ ment of the analytical control routine in order to meet the actual quality requirements. At present about 4000 orders for radiopharmaceuticals are dispatched each year. A major part of the production is for the domestic market, but a considerable part of the materials is also exported. The reactor JE E P II, with a maximum thermal neutron flux of about 2 X 1 0 13 n/cm2 sec, is used for the irradiations. Besides being the only producer of radiopharmaceuticals in Norway, the Isotope Laboratories also take care of the import and distribution of radioactive substances from other countries. The personnel engaged in the production of radiochemicals for medical use are mainly persons with a pharmaceutical education. The various steps in the production process are always supervised by one of the pharmacists, who is personally responsible for the quality of the final product.

2. ORGANIZATION OF THE PRODUCT CONTROL

The Isotope Laboratories have a management and four operating sec­ tions, namely a production section, an analytical section, a section for laboratory service and neutron and gamma irradiations, and a section

63 64 STEINNES dealing with industrial applications of radiation and radioactive tracers. The quality control of the radiopharmaceuticalproducts is provided by the two first mentioned sections. The pharmaceutical personnel of the production section take care of the biological control, while the analytical section is responsible for radioactivity calibration and the various tests necessary to establish the radionuclidic, radiochemical and chemical purity of each product. The analytical section also performs the necessary testing of the chemicals to be used in the various productions. In addi­ tion to the activities concerning the various aspects of product control, the analytical section also provides for the standardization of radioisotope solutions and preparation of calibrated sources. Furthermore, a fairly extensive activity is being carried out in activation analysis, dealing with about 3000 samples a year.

3. R ADIOANA LYTIC A L AND CHEMICAL CONTROL

3.1. Gamma-spectrometry

The 7 -spectra of all products are recorded by means of a 400-channel pulse-height analyser equipped with a 3 in. X 3 in. Nal(Tl) crystal. The primary purpose of this measurement is to identify the actual radionuclide, but in many cases the test is also sensitive for the detection of expected radionuclidic impurities. In some cases impurity concentrations as low as 10" 3 % can be detected. The y-spectrometric measurements are es­ pecially important in cases where isotopic y -emitting impurities are likely to occur, such as 203Hg in a 197Hg preparation, 60Co in a 58 Co preparation etc. In such cases, y -spectrom etry is the only rapid means of establishing the level of impurity. In some cases y-spectrometry has also proved use- •ful for the study of unidentified spots on chromatograms.

3.2. В eta-emitting impurities

Impurities of beta-emitters in products based on a purely beta- emitting nuclide, in most cases 35S or 32P, are estimated by conventional- methods using aluminium absorbers. This test is performed on every production of the actual radiochemicals.

3.3. Radiochemical purity

The radiochemical purity of the products is in most cases checked by ascending paper or thin-layer chromatography. If for some reàson the result is needed very rapidly, a method based on high-voltage electro­ phoresis is used. In such cases a result can be obtained in about 30 min, which is substantially shorter than for most chromatographic methods. When a new production is to be introduced, several chromatographic or electrophoretic procedures are investigated, the most suitable of which is selected for routine control. Each method is tested using carriers for probable impurities and establishing Rf values for these as well as for the major component'. Two radiochromatogram scanners, the first equipped with a window-less GM-tube and the second with a thin Nal scintillation crystal, are used for determining radioactivity distribution IAEA-PL-336/5 65 on the paper strips or thin^-layer plates. Some of the procedures routinely used in the laboratory have been described in the literature [1 , 2 ]. In a few cases where no suitable chromatographic procedures have been found, methods based on other techniques such as, for example, reversed isotope dilution, have been used to determine radiochemical purity.

3.4. Chemical purity

Identity tests on all chemicals used in the production are carried out according to procedures described in Pharmacopoea Nórdica. For a number of organic compounds which have not so far been described in the Pharmacopoea, infra-red spectrometry has been employed. Sub­ stances to be irradiated are tested by emission spectroscopy once a new bottle has been opened. In some cases, neutron activation analysis has been used to determine certain impurities in new bottles of chemicals, such as Na in K 2 C03. All production batches are tested for certain toxic elements such as As, Pb, Te etc. by emission spectroscopy.

3.5. Various tests

The radioactive decay is controlled occasionally for most preparations, especially for those prepared according to methods recently developed. Specific activity and dry-matter content are determined regularly for the various products. In a few cases every batch is tested. For the speci­ fic activity determination, spectrophotometric methods, in most cases in the visible region, are used. For proteins, u.v. spectrophotometry has been found useful. Investigation of the rate of decomposition is performed regularly on a number of products such as 131I-human serum albumin, 1311-Hippuran and 203Hg-chlormerodrin. The radioactive decomposition products are identi­ fied and determined by chromatographic methods. In some cases the particle size distribution of the product is important. In the case of 131I-labelled human serum albumin m acro-aggregates, the particle size is estimated by means of a projection microscope. In the case of colloidal gold, the particle size of the preparations is checked by electron microscopy in another research institute.

3.6. Other analytical activities

During the development of new methods for radioactive labelling of organic molecules, an extensive analytical activity is necessary in order to establish a procedure yielding a product of satisfactory purity. Here again chromatographic methods.have proved to be of great importance. Infra-red spectrophotometry has in some cases proved to be a useful means of detecting chemical impurities in the organic starting materials. In some cases more fundamental studies on radiochemical decomposi­ tion have been performed for certain products. The influence of specific activity and time of storage on the breakdown of 131I-labelled L-thyroxine' was carried out recently [3]. Similarly, work is being done on the effect of radiation protective agents on certain radiochemicals. 66 STEINNES v 4. BIOLOGICAL CONTROL

4.1. Sterility

Whenever possible the products for injection are sterilized after sealing in the final container by autoclaving at 120°C for 30 min. Radio­ chemicals that cannot be exposed to autoclaving due to thermal instability are prepared under aseptic conditions and sterilized by passing through a bacterial filter. A suitable amount of a bacteriostatic agent, like benzyl- alcohol, is added to the solution. A sterility test is performed on every solution prepared in this manner.

4.2. Pyrogen testing

Testing for the presence of pyrogens is done regularly on the distilled water used for the preparation of pharmaceuticals for injection. In some cases certain chemicals used are also tested. This is done by the National Institute of Public Health, where a central laboratory for pyrogen testing is situated.

4.3. Toxicity

Every production batch of materials for injection is tested for toxicity by injection of white mice.

5. SOME GENERAL COMMENTS ON THE QUALITY CONTROL ROUTINE

When the number and character of the quality tests to be applied on a certain radiopharmaceutical product is to be decided, there are at least two important factors which must be considered. Generally the most im­ portant point is the u ser1 s requirements for the product to be efficient in the actual type of application. On the other hand, some analytical techniques require the use of expensive instruments or are rather time- consuming, which frequently means a substantial addition to the cost of the product, especially if the production is relatively low. Therefore, for a small producer of radiopharmaceuticals it might sometimes be necessary to omit a certain control test, or to apply the test on a limited number of batches, although the inspection of every production would be desirable. In-some cases, however, such a problem can be overcome by leaving the test to a more specialized analytical laboratory. The point discussed here could be emphasized by examples from the author1 s laboratory: Concerning the test for pyrogens, there is no question that this control is of considerable importance. However, as the test is rather difficult and expensive we find it convenient to have it performed in a central laboratory doing this work routinely. We analyse all samples produced in our laboratories by emission 'spectroscopy. We are able to do this because, in the Materials Depart­ ment of our Institute, there already exists suitable equipment for this type of analysis,'mainly intended for other types of investigations. If IAEA-PL-336/5 67

this possibility did not exist we would probably have used conventional chemical tests for the routine detection of certain toxic element in our products, while for analyses of more extensive character, the consulta­ tion of an external laboratory would have been necessary. The need for equipment to be used in the analytical product control will naturally vary from laboratory to laboratory, depending on the size of the production and the selection of products. At our stage of develop­ ment, apart from conventional counting and laboratory equipment, we would consider the following instruments to be necessary for a suitable product control: A radiochromatogram scanner with a beta-detector, a 400-channel gamma-spectrometer with a scintillation detector and a spectrophotometer. In addition,, we have found some other equipment to be very useful — an infra-red spectrophotometer, an apparatus for high-voltage electrophoresis, a radiochromatogram scanner equipped with a proper Nal(Tl) detector, and a fast scaler/tim er for use in the recording beta absorption curves.

6 . PREPARATION OF MONOGRAPHS FOR THE PHARMACOPOEA NORDICA

In a number of countries, Norway included, radiopharmaceuticals are now classified as drugs, and several products have consequently been incorporated in various pharmacopoeas. In Scandinavia, the work on preparation of monographs for radiopharmaceuticals to be included in the Pharmacopoea Nórdica, started in 1965, and has proceeded con­ tinuously. Our laboratory has participated in the preparation of a con­ siderable number of monographs, including identification criteria and purity requirements with corresponding analytical methods of testing. The monographs prepared for the Pharmacopoea Nórdica are in general similar to those found in other pharmacopoeas for corresponding preparations, but in two respects there might be some difference. First, it should be possible to perform the quality tests in a relatively short time, i.e. before the product is distributed for sale. The half-life of a nuclide is hence generally not acceptable as a purity or identity criterion. Secondly, a more quantitative point of view has characterized some of the requirements. As an example it can be mentioned that, for products based on 7 - emitting nuclides, the y-spectrometric measurement, besides being an identification test, is also accepted as a test of purity. This is expressed in the following manner: The ratio between the number of im­ pulses (Nj), registered under the main photopeak and the total number of impulses (N2) registered in the spectrum should not deviate from the corresponding ratio for a reference solution of the same nuclide by more than

provided that sample and reference are measured under the same con­ ditions, and that the number of counts registered in the highest channel exceeds 50 000. 68 STEINNES

7. CONCLUSION

In our laboratories we follow the principle that, as far as possible, radiopharmaceuticals should be produced and analysed in the same manner as other drugs. The methods used for the chemical and biological control of radiopharmaceuticals should therefore be similar to those used for other pharmaceuticals. The special quality requirements necessary for radiopharmaceuticals, compared with other drugs, are essentially those associated with the radioactive properties of these substances.

REFERENCES

[1] BEEN, U., H0YE, A ., J. Chromatog. П (1965) 631. [2] H0YE, A ., J. Chromatog. 28 (1967) 367. [3] H0YE, Ä ., Acta Chem. scand. (in press). LAEA-PL-33 6/6

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS IN THE DEPARTMENT OF CHEMISTRY, REACTOR CENTRE, SEIBERSDORF

H. SORANTIN Österreichische Studiengesellschaft fur Atomenergie, Vienna, Austria

Abstract

ANALYTICAL CONTROL OF RADIOPHARMACEUTICALS IN THE DEPARTMENT OF CHEMISTRY, REACTOR CENTRE, SEIBERSDORF. Control procedures for target materials and processed radiopharmaceuticals are.described, with emphasis on the use of radioactivation analysis.

1. GENERAL ASPECTS

Production and control of radiopharmaceuticals in the Department of Chemistry were started at the beginning of 1961, shortly after the inaugura­ tion of the first nuclear reactor in Austria. Emphasis is laid on the production of short-lived radionuclides, whereas longer living radionuclides are not produced routinely, but only for special purposes or in special chemical forms. The main reason for this limitation is the fact that the demand for longer living radionuclides is rather small in Austria so they cannot be prepared at economical cost in the centre compared with the prices offered by the distribution organizations of foreign producers. Concerning purchased preparations the activity of our centre is therefore confined to the control of specifications and purity.

2. RADIONUCLIDES

i 8p ; I8 p sodium alumínate 24Na: chloride (injectable) 42K: chloride (injectable) 46Sc: chloride (injectable) 47Ca: chloride (injectable) ®Cr: chloride (injectable) sodium chromate (injectable) 64Cu: chloride 85Sr: chloride (injectable) 86Rb: chloride (injectable) 87mSr: chloride (injectable) 90Y: seeds 99тте: 99'Mo-"mTe generator adsorbed on Fe 2 0 3 12®I: 'sodium iodide 130I: sodium iodide

69 70 SORANTIN

131I: in alcoholic solution sodium iodide (injectable) oleic acid ricinoleic acid triolein olive oil rape oil I32j. 132Te-I-generator adsorbed on CaSiOj 133I: sodium iodide (injectable) 140Ba: chloride 198Au: metal needles or wire

3. CONTROLLING PROCEDURES The direct application of these radionuclides in human medicine for tracing and localization examinations requires a rigorous control. The criteria are radionuclidic, radiochemical and chemical purity. In the case of injectable solutions, sterility and absence of pyrogens are also necessary. The term "radionuclidic purity' 1 excludes the presence of other radio­ nuclides, which can be formed during irradiation from impurities in the targets or by side reactions. Radionuclidic impurities are usually indentified after chemical separation by radioactivity measurements. As shown later, direct information is also possible by using 7 -spectrometry with solid-state detectors. "Radiochemical purity" refers to the chemical form of the produced radionuclide, whereas "chemical purity" confirms the absence of impurities, especially toxic ones.

3.1. Controlling of target-materials, fillers etc.

• To ensure pure products, stress is laid on the checking of target substances. Although generally analytical grade reagents are used, which are obtained in m ost cases with certificates stating the amount of trace elements, they are examined batchwise by neutron activation analysis because often nuclides with large cross-sections are not listed. Irradia­ tion times are the same as that used in the sample preparation. Attention is also paid to the amount of trace elements in plastics used for the encapsulation of target materials during short time irradiations. Extensive studies have shown that high-pressure polyethylenes are usually purer than varieties which are synthetized at low pressures or by the help of catalysts [1, 2]. Even quartz ampules that were analysed after irradiation showed traces 24Na, 56Mn, 64Cu and 97Zr (Fig. 1). Fillers for chromatographic columns, e.g. Al20 3,were also checked by 7 -spectrom etry and revealed differences in the amount of 24Na. Great attention is also paid to the quality of distilled water, including the content of pyrogenic substances.

3.2. Control of processed radionuclides

Essential differences exist in the control of long-lived compared with short-lived radionuclides. The first group is prepared by dissolution of the target, chemical separations, 7 -spectrometric measurements, radio­ chemical analysis, pyrogen and atoxicity tests, chemical analysis of the final solution, activity calibration and dispensing. 1Л О t£) о 3 _

5 0 5 ñ S b & о M CD S5 o M in СО in I c G.. mt qat vial quartz Empty .l. IG F Irradiation tim e: 3 h at 1 2 x l 0 13 n/cm2 sec sec h 13 n/cm2 17 0 l x 2 1 e: at tim h 3 Cooling e: tim Irradiation onig i : 0 in. m 20 e: tim Counting oln i : 6 h 56 sec n/cm2 1013x 3 e: at tim h 2 Cooling e: tim Irradiation IAEA-PL-336/6 71 TABLE I. IMPURITIES IN ALKALI SALTS

Li2C 0 3 NazC 0 3 NaHC03 NaCl NaCl KzC 0 3 KC1 KC1 analyt. grade analyt. grade analyt. grade analyt. grade ultra pure analyt. grade analyt. grade ultra pure m m m №) №) W m (%)

Content 9 9 .5 99 99.5 9 9 .5 99 99.5

H20 in sol. 0 .0 1 0.0 0 7 0.02 0 .0 0 5 0 .0 0 5 0.01

Cl 0 .0 0 2 0.001 0 .0 0 3 '

S. as S 0 4 0 .0 0 3 0 .0 0 5 0.003 0 .0 0 2 0 .0 0 3 0 .0 0 3 '

P and Si 0 .0 0 2 0 .0 0 5 0.008 0 .0 0 5 0.01 0 .0 0 2

N 0 .0 0 1 0 .0 0 5 0.001 0 .0 0 1 0 .0 0 1 0 .0 0 1

Pb 0 .0 0 0 5 0 .0 0 2 0.001 0 .0 0 0 5 5 X 1 0 -6 0 .0 0 1 0 .0 0 0 5 5 x 1 0 -6

Fe 0 .0 0 0 3 0.0 0 0 5 0.001 0 .0 0 0 3 5 X 10“6 0 .0 0 0 5 0 .0 0 0 3 1 x 1 0 -6 SORANTIN

A1 0 .0 0 1 0.0 0 1 1 x lO -6 0 .0 0 1 0 .0 0 1 1 x 10~6

Ca 0 .0 0 1 0.0 0 5 0.005 0 .0 0 2 5 X 10-5 0 .0 0 2 5 x l 0 - 4

Mg 0.0 0 5 0.005 0 .0 0 1 1 XIO-5 0 .0 0 0 5 5 X 1 0 -6

К 0 .0 3 0.005 0 .0 1 l x l O ' 3

Br 0 .0 1

Ba 5 x 10“4 5 x 1 0 -4 Cu 5 X 1 0 -6 1 x 1 0 -6 Co 1 X 1 0 -6 1X10“6 Ni 1 XlO -6 1 xlO -6 Zn 1 x 10-6 1 Xl0"6

Mn 5 x 1 0 "6 5 x l0 - 6 T e 1 x lO -6 Sr 3 X 1 0 -4 3 x 1 0 -4 Li 4X 10“5 4X10-5 Na 0 .0 2 0 .0 2 0 .0 2 4X10-3 IAEA-PL -336/6 73

By contrast the production line of short-lived nuclides starts with the determination o£ radioactivity. Secondly, the radionuclidic purity is checked by 7 -spectrom etry with Ge(Li) detectors. Samples fulfilling the required specifications are immediately handed over to the biological section for pyrogen and toxicity tests, while other aliquots are given to the chemical and radiochemical laboratories for special tests. A short summary of the controlling procedures, applied individually to the produced radiopharmaceuticals, is given in the following pages.

1 8 F : L i 2C 0 3 p. A. is used as target material and is analysed batchwise by neutron activation. Besides 18Fe, 24Na could also be detected in short time irradiations. In other samples traces of radioactive tellurium and strontium could be found by Ge(Li) 7 -spectrometry (Fig. 2).

18F is separated by adsorption on chromatographic alumina and elution with diluted NaOH, all traces of Te and Sr remain on the column. 24Na comes off before 18F, each eluant is examined by 7 -spectrom etry. Aliquots of 24Na-free solutions are given pyrogen tests. The Li-content is determined by flame photometry and the amount of Alby titration with titriplex III and dithizone [3]. Aliquots are examined after precipitation of 18F with calcium phosphate for tritium in a Tricarb scintillation counter.

24Na: NaHC03 or Na2 C 0 3 is used as target. The purity of analytical grade reagents is usually sufficient to be taken as target materials for the prepara­ tion of short-lived nuclides and there is no need for the use of the more expensive "super pure chemicals" listed in Table I. If the content of the different trace elements (Table I) is related to 1 g sodium, the chemical purities are nearly the same. Analytical grade of NaCl (Table I) can also be irradiated, if there is time enough to await the decay of 38C1. The radionuclidic purity was checked by 7 -spectrometry and showed that, in the case of well-thermalized neutrons, no 32P or 36C1 is found. Samples are dissolved in a calculated amount of hydrochloric acid and the weight of total solids determined after evaporation. The pH value is checked before filling, sterilizing and packing. In our laboratory carrier- free 24Na is also produced by irradiation of MgO or Mg (OH) 2 [4].

42K: In all targets, sodium is assayed quantitatively by neutron-activation analysis. Yields of about 200 ppm can be accepted as tolerance level. The As content is also determined after volatilization of AsH3, absorption in silver-diethyl carbamate and measuring the absorption at 538 nm. The relative standard deviation in the 5-/ug range is ± 2. 5% [5]. The As present should not exceed 5 /Jg/g potassium. Control of cyanide-ion is now being established by potentiometric titration using element specific membrane electrodes. Amounts of up to 5 ppm can be tolerated. Dissolution procedures are the same as those described previously.

47 Ca: This is produced from enriched 46 Ca, which is mostly supplied with a certificate stating the impurities. It is advisable to test the radionuclidic purity by pre-irradiation. 24Na is nearly always found when the 46Ca is 74 SORANTIN shipped in glass containers. Preparation is sim ilar to that for 24Na and 42K, but the final solution in also controlled by emission spectrography.

6 1 Cr: For the preparation of injectable isotonic chromium (Ill)-chloride or sodium chromate solutions, chromium or enriched 50Cr in form of metal powder,are used as targets. Chemical purity is checked by emission spectrography, lead and iron are determined by spectrophotometry. By activation analysis the radionuclides 28A1, 52V, 56Mn, 64Cu, 54Mn can be detected, but except for 54Mn they are short-lived and do not influence the radionuclidic purity [6 ].

The irradiated chromium is weighed, dissolved in the calculated amount of hydrochloric acid, the solution heated and filtered in a calibrated flask. Aliquots of the stock solution are analyzed for radioactivity and total amount of solids. Inactive impurities are determined by emission spectroscopy and radioactive impurities by 7 -spectrometry. Part of the solution is converted to Na2 C r04. Aliquots of both solutions are tested for pyrogens and toxicity. For carrier-free 51Cr preparations K 2 C r 0 4 is used as starting material and impurities e.g. Cl, Na, Fe etc. are detected by pre-irradiations. In the preparation cycle after activation, trivalent chromium is isolated by ion exchange or chromatography and dissolved in HC1 or oxidized in alkaline solution to Na2 C r 0 4 . The chromium content of the final solution is measured colorim etrically against standard solutions. The chemical state of the chromium is deter­ mined by extraction techniques or by paper chromatography, the potassium determined as tetraphenyl borate, the amount of total solids by evaporation and the composition by emission spectrography. Chromate solutions are also checked for H2 0 2 by spot tests with KMn0 4 in sulphuric acid. Finally the radionuclidic purity is controlled by 7 -spectrometry and freedom of pyrogenic and toxic substances verified by biological tests.

59Fe: Chemical purity is also checked by activation analysis. In analytical grade iron metal, 28A1, 52V, 58Mn, 84Cu could be identified by 7 -spectrometry using Nal(Tl) detectors. By using a Ge(Li) solid-state detector 7 -rays from 60Co, 76A s, 110mAg, 122 Sb and 187W are discernible. 65Zn was present in greater amounts. A quantitative determination gave 50 ppm for Zn, 20 for copper, 100 for Mn and 2 ppm for arsenic.

Fe (IH)-oxide titration standards were also examined and showed nearly the same impurities [ 6 ]. After irradiation of the target, 59Fe is separated fro m 54м П) 51 Cr, by chemical separation procedures such as extraction. The processed solution containing 59Fe and 55Fe is checked for iron content, acidity, specific activity, content of solids, radionuclidic purity and delivered as F e (III)-chloride in 1NHC1. The valence states are examined by paper chromatography with butanol-12NHC1 mixtures [7]. The ratio of 55Fe to 59Fe is determined by liquid scintillation counting [8 ].

6 4 Cu: The CuO target is examined by pre-irradiation for impurities giving short-lived radionuclides, especially 38C1. It is also analysed batchwise for its arsenic and sulphide content by chemical methods. 64Cu is dis­ solved in HC1+H 2 0 2 and processed in a sim ilar manner to that for 5 1 C r C l3 . IAEA-PL-336/6 75

Counts

FIG.3. y -spectrum of enriched 86Sr Irradiation time: 4 h at 2 x l0 * 3 n/cm2 sec Cooling time: 3 d Counting time: 10 h

86Rb: has been prepared on request as chloride in an injectable form. Traces of the shorter-lived 24Na and 42K were detectable, but did not result in radionuclidic purity. Isotonicity was controlled and freedom ofpyrogenic and toxic substances established by biological tests.

87mSr: Enriched 86Sr is used as target. No impurities can be detected by Ge(Li)—^-spectrometry shortly after the end of irradiation, but after the decay of 87mSr the impurities 24Na, 140 L a and 187W (F ig . 3) appeared in the spectrum in addition to the expected 85S r .

90 Y: This is irradiated in form of seeds; absence of у-emitting radionuclides is established, then calibrated, sterilized and packed.

99M o - 99mTc-generators: 99Mo is separated from uranium fission product solutions and sorbed on A12 0 3 o r F e 2 Os columns. The radionuclidic purity of 99Mo and 99m Tc eluates is checked by -/-spectrometry and half- life determinations of the 99mTe [9]. Longer-lived impurities should not be present. These are sought by means of Ge(Li) y-spectrometry. As can be seen from Fig. 4, traces .of 65 Zn, 124Sb, 134Cs and a fte r exten sion of the spectrum (Fig. 4) 95Z r, 95Nb can be clearly identified in old generators.

126I: This is prepared for double-labelling purposes by (n, 2n) reaction of 1271, elementary iodine being used as target material. The chemical purity was tested by pre-irradiation and showed (Fig. 5) that even in 76 SORANTIN 2090 2090 Sb 124

FIG,4 . Control spectrum of " m M o-Tc Counting time: 100 min

FIG. 5. y -spectrum of elementary iodine and Pbl2 Irradiation time: 7 h at 2 .1 x l0 13 n/cm2 sec Cooling time: 24 h ------Pbl ------= Iodine IAEA-PL-336/6 77 analytical grade, sublimed iodine, bromine is present [10]. Radionuclidic purity is reached by selective extraction of iodine after oxidation with NaNC>2 in sulphuric acid. The other tests regarding.oxidation states etc. are performed for.131I (see below).

131I: This ispreparedby irradiation of telluric acid and precipitation as described in previous work [ 1 1 ]. The chemical purity of the target material is therefore more critical compared with methods which separate 131I by volatilization. Selenium is determined by 7 -spectrometry after precipitation with SO2 from hydrochloric acid [12]. Additional elements determined are copper, arsenic and chloride. After activation, telluric acid is precipitated with alcohol and the rest of telluric acid removed by adsorption on an A12 0 3 colum n.

Na131I: The alcoholic solution is evaporated after addition of NaOH. The total amount of solids is determined and the residue redis solved in bi­ distilled water. The chemical composition is determined by paper chromato­ graphy and autoradiography [13] or precipitation methods and radioactivity measurements [14].

The iodine content is assayed quantitatively by the reaction

A s3+ + 2 Ce4+ ------A s5++Ce3+ [15,16] which proceeds very slowly but is catalyzed by iodine.. The resulting reaction rate is directly proportional to the amount of iodine present. By means of the modification introduced by Spitzy and Knapp [17] the extinction is directly measured against the time and the iodine content thus evaluated. The final solutions are analysed by emission spectroscopy for inactive and by 7 -spectrometry for radioactive contaminants. After toxicity and pyrogen tests the solutions are ready for dispensing.

131I-labelled oleic acid, ricinoleic acid, triolein, olive oil, rape oil are all prepared by direct labelling, a procedure whereby 131I is extracted by the oil to be labelled and involves an additional purification step [18]. The radiochemical purity of the labelled oils is checked by paper electrophoresis and autoradiography. Fixed 131I remains at the starting point, whereas free iodine migrates in the electrical field and gives easily visible traces on the developed radiogram.

130I is often used for double labelling of substances with radioactive iodine. The isotopic composition depends on the kind of target m aterial and has already been discussed in previous work [18]. We isolate 129I from old fission-product solutions. By activation in a thermal flux of 10 13 n/cm 2 s e c , after 25 h a specific activity of 8 m C i 130l/m g (127I + 129I) is obtainable after allowing for the decay of 128l. The radionuclidic purity is reduced by the presence of 1261 originating from the 121I content as a result of (n, 2 n) r e a c tio n s . The final solutions are examined by emission- and 7 -spectroscopy and the iodine content determined by titration. Absence of pyrogenic substances is also established. 78 SORANTIN

FIG.6. Spectrum of 1331 fraction —;------= 32 h after irradiation ------' 51.5 h after irradiation ——------= 72 h after irradiation

133I: The simultaneous application of several iodine isotopes at different times before biopsy allows dynamic examinations of the iodine metabolism in a single tissue sample.

1331 can be used as additional tracer and is prepared by irradiation of uranium as shown in Fig. 6 . A chemically pure iodine fraction can easily be obtained by three extraction and back-extraction cycles. The time of separation, however, influences the isotopic composition greatly. By cal­ culation it is found that a cooling of 40 h [19] gives the maximum "nuclidic purity" (Fig. 6 ). All further tests are performed as already described fo r 131I.

140Ba: 140Ba has been requested for bone scintigraphy. Because of the toxicity of barium compounds it must be prepared in a carrier-free condition. It is therefore precipitated with PbS0 4 as a non-isotopic carrier, dissolved in EDTA solution and purified by ionic exchange [20]. The final ВаС1г solution is tested by 7 -spectrometry for its nuclidic purity and the solids determined by evaporation and emission spectroscopy. .

198Au: Activated gold needles are used for the treatment of tumours. The chemical purity is tested by pre-irradiations by which silver, a very common contaminant, is easily detected. The content should be as low as possible. IAEA-PL-336/6 79

österreichische Studiengesellschaft für Atomenergie Ges. m. b. H. Telefon 573649/327 Wien Vllf, lenougasse 10 Telegrammodrejse AUSTRATOM

SGAE - Bestellungsformular f. Radionuklide

Nome, Adr., Tel., Telegr. des Name, Adr.. Tel., Telegr. der für die Name, Adr., Tel., Telegr, der Stelle, Bestellers (Firmenstempel): Bestellung verantwortlichen Person: an die geliefert werden toll:

Datum d. gewünschten Liefertermins: Gewünschter Abholtermin (nur f. Selbstabholer):

Rodionuklid: Cod.:

Chem. Form : Gewicht (Vol.):

Gesamtakt.: Spez. Akt.:

Verwendung: med. j Nichtgewünschtes streichen!

Kurze Beschreibung d. beabsichtigten Verwendung (dient nur zur Information des Herstellers):

Besondere Bemerkungen (z.B. evt. Wiederholung d. Lieferung an bestimmten Tagen): *

Ort: ...... Unterschrift d. verantwort). Bestellen

FIG.7. Special order form. because the long-lived liOmAg (253 d) is formed. To maintain radionuclidic purity neutron fluxes should not be greater than 1 0 12 n/cm 2 and the needles not kept continuously in the reactor. This avoids conversion to 199Au [21]. After activation the pins are sterilized, the activity measured before dispatch and supplied with a 7 -sp e c tru m .

4. ORGANIZATION

In production and control of radiopharmaceuticals three sections, namely administration, production, and quality control, and to a partial extent the reactor, biology-health physics department and transport- division, are mainly engaged. Consumers are asked to send their orders at least one week in advance to the administration section. Special forms (Fig. 7) should be used for this purpose and are supplied by our company. The demand for radionuclides fluctuates widely with respect to types and amounts. Special programs must therefore be set up nearly every week by the production group, and the targets delivered to the reactor. The processed radionuclides are handed to the control group which carries out the necessary analyses and gives samples to the biology section for pyrogen and atoxicity tests. All radiopharmaceuticals which fulfil the requested specifications are labelled and packed. The surface dose-rates of containers are measured by the health-physics department and marked on a double slip, one part of 80 SORANTIN which is given, together with a certificate stating date, radionuclide, radioactivity, volume or specific activity, sterility, atoxicity, isotonicity etc., to the customer. Transport is carried out by company-owned cars to Vienna city, the railway station or Vienna airfield. The transport slip has to be signed at the place of destination. After return it is given to the payment office. In the case of short-lived radiopharmaceuticals, however, some controls, especially biological ones, must be carried out concurrently with radiochemical examinations and even during the time of transport. The certificate states that the user will be informed as soon as the results are available.

5. RECORDS AND STORES MAINTAINED

One certificate of the processed radiopharmaceutical is kept by the control group, and the number of the target and irradiation order are also liste d . Residues of short-lived radionuclides are not stored longer than 2 -3 weeks. Yet before discarding they are examined by 7 -spectrometry. The spectra are a valuable help in recognizing trace nuclides and are attached to the corresponding certificate.

6 . CONCLUSION AND RECOMMENDATIONS

Control methods and problems arising when a variety of radiochemicals have to be produced in different amounts and at different times have been briefly discussed. Although we do not export too many radiopharmaceuticals we feel that the IAEA should publish specifications for radiopharmaceuticals. We also recommend discussion of the term "carrier free". Often solutions marked "carrier free" contain small amounts of carriers to minimize adsorption on the glass of the container. 1Э1 1-produced from irradiated 235u has been often sold "carrier free" although 127I and 129I are also present as fission products. I hope I have shown some of the problems which arise with the produc­ tion of short-lived radionuclides for medical use. We believe that in this connection 7 -spectrometry with solid-state detectors is a powerful tool for establishing radionuclidic purity, but it simultaneously raises the question concerning to what lim its control procedures should be aimed at.

REFERENCES

[1] SORANTIN, M ., PATEK, P ., Z. analyt. Chem. 211 (1965). 99. [2] WOGROLLY, E ., SORANTIN, H ., Allg. Prakt. Chem. 20 (1969) 49. [3] Kqmplexometrische Bestimmungsmethoden mit Titriplex, E. Merck, AG, Darmstadt (1966) 19. [4] SGAE-Rep. CH-3B (1963). [5] FRIES, G ., Spurenanalyse, E. Merck AG, Darmstadt (1966) 17. [6] MICHA ILOV, M ., SORANTIN, H ., SGAERep., in preparation. [7] BIGHI, C ., Ann. Chim. (Roma) 45 (1955) 532. IAEA-PL-336/6 81

[8] DERN, R .T ., Liquid Scintillation Counting, (Bell, C .G ., Hayes, F.N ., Eds), Pergamon Press (1958) 205. [9] BULBULIAN, S ., SORANTIN, H ., Kerntechnik 8 (1966) 118. [10] SORANTIN, H ., unpublished work. [11] SORANTIN, H ., BILDSTEIN, H .„ J. inorg. nucl. Chem. 27 (1965) 521. [12] LEDICOTTE, G .W ., NAS-NS 3038 (1961) 14. [13] GETOFF, N .. PARKER, W ., Atompraxis 9 (1963) 175. [14] MÜNZE, R ., Kernenergie 3 (1960) 518. [15] SANDELL, E .B ., KOLTHOFF, I.M ., J. Am. Chem. Soc. 56 (1934) 1426. [16] SANDELL, E .B ., KOLTHOFF, I.M ., Mikrochimica Acta 1 (1937) 9. [17] SPITZY, H ., KNAPP, G ., in preparation. [18] DUFTSCHMID, K .E ., SORANTIN, H ., Mh. Chem. 97 (5) (1966) 1332. [19] SORANTIN, H. , in Proc. 2nd Int. Conf. Methods of Preparing and Storing Labelled Compounds, EURATOM, Brussels, 28 Nov.-3 D ec.(1966) 201. [20] SORANTIN, H ., in preparation. [21] TIETZE, H ., BILDSTEIN, H ., GETOFF, N ., SORANTIN, H ., PFEIFER, V ., Atompraxis 12 (1966) 509. [22] SORANTirç, H ., Mh. Chem. 96 (1965) 1754; and CEA-TR-A -1959 (1966).

IAEA-PL-336/7

QUALITY CONTROL AND CHEMICAL ANALYSIS OF RADIOPHARMACEUTICALS AT A SMALL RESEARCH CENTRE

Y .S . KIM Atomic Energy Research Institute, Seoul, Korea

Abstract

QUALITY CONTROL AND CHEMICAL ANALYSIS OF RADIOPHARMACEUTICALS AT A SMALL RESEARCH CENTRE. The methods used for the quality control and chemical analysis of radiopharmaceuticals at the Korean Atomic Energy Research Institute are described. Chromatographic techniques (including paper, thin layer and ion exchange) have proved to be simple and effective means for studying radiopharmaceuticals. Results of a study of radioiodinated human serum albumin by electrophoresis and degradation are presented as well as the results of a study of the stability of selected radiopharmaceuticals. Comparative studies of imported and Korean manufactured products are also presented.

1. INTRODUCTION

In Korea radiopharmaceuticals are used at many hospital establishments ’ and a considerable demand exists for such m aterials. Until recently such products were imported and experience has shown that the quality of the imported materials varied considerably and frequently resulted in incon­ sistent clinical data. In 1965 a small laboratory was established at the Korean Atomic Energy Institute for the preparation of radiopharmaceuticals and the determination of-their quality. In this paper the results obtained from operating this laboratory are presented. Although the procedures used for the quality control and chemical analysis described here are largely routine in nature, it is hoped that an account of our experiences will be helpful for other small institutes or organizations faced with sim ilar problems. As will be seen, chromatographic techniques have proved to be the main techniques used in our studies.

2. QUALITY CONTROL

Complete quality control of radiopharmaceuticals involves consideration of many factors and ideally physical, chemical, biological and pharmaco­ logical tests are required prior to shipment. Our experiences with commer­ cially produced m aterials have not always been satisfactory in respect to both radiochemical and radionuclidic purity. M aterials produced in our own laboratory are derived from purified chemicals and reagents since cost is not a dominant consideration. The quality control of such products is simpler and more rigorous than for commercially produced products. In this account of our work only the chemical aspects and not the bacteriological aspects of quality control will be considered.

83 со 4^

TABLE I. RADIOIODO-HIPPURAN IAEA-PL-336/7 85

3. STUDIES ON HIPPURAN (131I)

131I-labelled hippuran (o-iodohippurate) has structure A.

[ C O -N H -C H 2 -C O O N a О <*. It is usually manufactured by exchange of radio-iodine between active sodium iodide and inactive hippuran at elevated temperatures in an aqueous solvent system at pH 5.5. Improved yields and decreased reaction times are claimed to result from the use of hydrogen peroxide as iodination ca ta ly st [1]. Preparation in this way results in the contamination of the product with other compounds such as o-iodobenzoic acid (B), sodium 2,4, 6 -tri-iodohippurate (C) and others.

CH2-C O O N a

(B) (C)

It has also been reported [.2] that storage of radio-hi'ppuran in the presence of excess carrier iodide results in enhanced contamination by the poly-iodinated compound (C). Such contamination is also favoured by preparation at elevated temperatures in the presence of excess carrier iodide. Thus, radio-hippuran should be checked for the presence of free iodide, o-iodobenzoic acid, and polyiodinated compound (C) after preparation, and checked for the absence of the polyiodinated compound (C) after storage. Radio-hippuran was examined for purity by paper chromatography using Whatman No. 1 filter paper as the stationary phase and n-butanol saturated with 5% acetic acid as mobile phase. Paper chromatography under these conditions gives clear separations of activity peaks corres­ ponding to free iodide, o-iodobenzoic acid and o-iodohippurate. No peaks were observed corresponding to the triiodohippurate, or to other impurities. Table I gives the results obtained from a comparative chromatographic study of radio-hippuran produced at the KAERI, and purchased from two other sources. As is seen from the results in Table I, the imported pro­ ducts are in general less pure, which may be somewhat inevitable as a consequence of large-scale production. When commercial products are considered to contain too much free iodide they require purification before use. The product made at the KAERI, however, showed no trace of free iodide, and is considered to be quite suitable for use. Figure 1 shows a typical scan of a chromatogram of a KAERI-produced sample of radio- hippuran. Thin-layer chromatography proved to be insufficiently sensitive to separate radiochemical impurities. Clearly, since decomposition and polyiodination are enhanced by auto­ radiolysis and as this is greatest for products of high specific activity, a constant study of the purity of radio-hippuran prior to administration is necessary to achieve satisfactory clinical results. 86 KIM

R t =0.8

Solvent front Point of application / Iodide Rf=0.3 \ I

FIG. 1. Paper chromatographic analysis of 1311-hippura'n (KAERI). Batch prepared on 6.4 .1 9 6 9 ; Date of analysis - 14.4.1969; Solvent - n-butanol: acetic acid 30°jo (4 : 1); non-bound iodide < 1°¡o.

4. R ADIOIODO - ROSE BENGAL

Rose Bengal has the structure (D).

Cl

I I (D)

There are four sites to be radio-iodinated and unless care is exercised in its preparation, complex mixtures will result. Clearly radiolytic degradation will likewise produce complex mixtures. Figure 2 shows a typical scan of a paper chromatogram of a sample of Rose Bengal ( 131I). The complex nature of the mixture is clearly evident from the numerous peaks. Special care must be exercised when selecting the developing solvent in order to achieve adequate resolution. Figures 3 and 4 show scans of a KAERI preparation [3, 4] of Rose Bengal. Although superior to the sample used to produce Fig. 2, impurities are still present. Mani [ 1] claims that a different mode of preparation produces a superior product. That this claim is true is evidenced by Fig. 5, which shows the scan of a chromatogram obtained from a sample of Rose Bengal prepared according to Mani's directions. IAEA-PL-336/7 87

FIG.2. Paper chromatographic analysis of 131I-Rose Bengal supplied by Company A. ■ Solvent - О.б'Уо aqueous ammonia. Ascending.

FIG. 3. Paper chromatogram of 1311-Rose Bengal batch prepared on 9.4 .1 9 6 9 . Solvent - Butanol saturated with 5°]o acetic acid; major zones - Rf 0. 24, R^ 0. 72.

' Table II gives the results obtained from a comparative chromatographic study of several samples of Rose Bengal. The improved product synthesized at the KAERI exhibited the best purity and dilute ammonia was found to be the best developing solvent. As it is difficult to purify Rose Bengal,' due attention must be paid to its mode of preparation. Purification by repeated recrystallization is often effective. The presence of free iodide ion in a preparation of radio-Rose Bengal results from a complicated autoradiolysis of the material. Free iodide ion is readily detected by any of the developing solvents listed in Figs 3, 4 and 5. In our experience, thin-layer chromatography of Rose Bengal • showed inadequate resolution and too low a sensitivity to be used for its quality control. TABLE II. RADIOIODO - ROSE BENGAL IAEA-PL-336/7 89

FIG. 4. Paper chromatogram of 1311-Rose Bengal batch prepared on 9. 4.1969. Solvent - 0. б^о aqueous ammonia; major zones - Rf 0.27, R f0.45, R f0.62, R f0.85.

0.37

FIG. 5. Paper chromatogramic analysis of 1311-Rose Bengal prepared at KAERI. Solvent - 0. 6°lo aqueous ammonia. Ascending.

5. RADIOIODINATED HUMAN SERUM ALBUMIN (RISA)

There have been many complaints (arising from its clinical use in Korea) about the instability and de-iodination of RISA. A typical specifi­ cation for RISA states that it should contain "less than 3% of non-bound radioiodine. Each gram-molecule of albumin should contain no more than one gram-atom of iodine". The percentage of non-bound radio-iodine in RISA is likely to increase with time. At the KAERI we have studied methods of preparing RISA with a view to reducing the loss of bound iodine. It was found that a chloramin-T 90 KIM p roced u re [5,6] gave preparations of greater stability than those obtained by conventional methods. Degradation studies on RISA prepared in this way showed that the chloramin-T procedure produced more iodine labelling at the site of the histidine residue of the albumin. Figure 6 is a scan of a chromatogram of a hydrolysed RISA. Material prepared via the chloramin-T route exhibited considerably reduced de-iodination during transportation, storage and clinical use. At present the author believes that iodination of the stable site of the protein molecule (i.e. h istid in e and other amino-acid residues) is an effective way of increasing the stability of RISA. We have found paper chromatography of RISA,using 75% methanol- 25% water as developing solvent,to be an effective way of analysing RISA. Samples prepared at the KAERI were compared in this way with samples purchased from two other sources. All showed the presence of free iodide ion. The site of iodination in the albumin molecule must, of course, be checked by means of a degradative study.

DISTANCE cm X0.5 FIG. 6. .Paper-partition chromatography of the hydrolysed product of radioiodinated human serum albumin. Developing solvent - 75°Jo methanol-water solution.

As a result of radiolysis, preparations of RISA frequently contain denatured proteins which obscure clinical observations. Control of de­ natured proteins is best done by means of dialysis. Ion-exchange techniques serve for the elimination of inorganic ions such as iodide while the identi­ fication of the denatured protein is best achieved by means of electro­ p h o re sis.

6 . M ER C U R IA LS

At this institute the mercury compounds Neohydrin a'ndl-bromo- mercury-2-hydroxy propane (BMHP-203Hg) have been prepared. The quality of the preparations was checked using paper chromatography and Fig. 7 shows a typical result with an impurity at the spotting point. Although only limited studies have been carried out at this institute, these suggest that the purity of the product is significantly affected by the method of pre­ p aration . Because of the high toxicity of the m ercuric ion, the most important tests for the quality control of mercurials are the biological tests. They will not be discussed in this paper. IAEA-PL-336/7 91

FIG. 7. Paper chromatogram of MBHP 203 Hg. Solvent - Benzene: HOAc: H2 0(2 : 2 : 1); Front - 23 cm; - 0.60.

(c)R , =0093

A

FIG. 8. Radio-paper-partition chromatogram of the eluate of и зт 1п-”со\/’ (i) The final pH of the eluate was adjusted to: (a) 1. 5 (b) 1. 0 (с) 0 (d) 2 (e) 6 (f) 14. (ii) Filter paper - Whatman No. 1 (in) Developing solvent - 75% CH3 OH

7. PREPARATIONS OF '113mIn COLLOIDS

The eluate from a nsmjn generator is generally used for preparing a colloid employed in liver scanning. Such an application requires a uni­ formly sized colloid. The particle size of the colloid depends on the column characteristics, the pH of the elution medium, the elution time and the final pH of the eluate. Uniform size of colloidal preparations used for liver scanning can be achieved by elimination of inorganic ions by passing the eluate through an ion-exchange resin,[7]. In a series of experiments, the pH of the eluate from a ll3m[n generator was varied and the resultant solutions examined by paper chromatography. The results are shown in Fig. 8 . These results show that at low pH inorganic ions are present and that the R{ values of the ionic species varied with decreasing pH. It is known, however, that best results are obtained by elution of "cows" at a pH of 1.5. The eluate from a generator (initial pH = 1.5) was passed through a cation-exchange resin and the resultant solution examined by'paper chromatography. The results are given in Fig. 9. By adjustment of the pH of the eluate obtained from the cation-exchange column to a value of 3.5, solutions are obtained which should give clear and selective liver scans [ 8 ]. This procedure, however, results in a considerable loss of radioactivity. Experiments are now in progress to improve the recovery of radioactive m aterial from the ion-exchange column. Although this pro­ cedure for preparing solutions of 113mIn for liver scanning greatly reduces the available amount of radioactivity, a clear clinical observation is guaranteed and the technique is recommended when it is necessary to obtain results from a particular scanning observation. 92 KIM

anion exchanger

I33mjn '-cow-'eiuatepHI.S

cation exchanger

FIG. 9. Separation of the components of the eluate by an ion-exchange resin.

FIG. 10. Triolein-1311. Developing solvent - petroleum ether : acetone (5 : 1); stationary phase - thin-layer plate (Eastman 6060). o - o : I . Co. x - x : D. Co.

8 . 99mTc PREPARATIONS

At the KAERI we have used paper-partition chromatography to check the quality of various eluates from a -99mTc generator: 99mTc-pertechnetate, 99mTc colloid and 99mTc albumin have been checked for radiochemical purity úsing 80% methanol-2 0 % water as developing solvent. IAEA-PL-336/7 93

Thin-layer chromatography is also suitable for the examination of these preparations and has the advantage of reduced developing time with conse­ quent reduced loss of radioactivity. An Eastman Kodak thin-layer sheet 6060 (silica gel) gave good resolution in the case of pertechnetate and c o llo id a l- 99mT c .

FIG. 11. Oleic acid-13iI. Developing solvent-petroleum ether: acetone (5 : 1). o - o : I . Co. X - X : D. Co.

9. MISCELLANEOUS RADIOPHARMACEUTICALS

We have also examined at the KAERI triolein 131I and oleic acid 131I. At present these m aterials have only been prepared on a laboratory scale and their quality control has, therefore, been limited to laboratory ex­ periments. Tables III and IV give the results obtained from comparative studies on commercial samples of triolein-131I and oleic acid- 131I. F o r t r io le in - 131I, the results obtained from examination of a product prepared at the KAERI are also included. The commercial products are usually contaminated by various glycerides or fats, the presence of which is readily demonstrated by thin-layer chromatography (see Figs 10 and 11). C are is required when using these radiopharmaceuticals and ideally their purity should be checked before use. Studies at the KAERI are now directed towards examining the quality of thyroxin and thyronine.

10. STABILITY TESTS

Commercial products are usually of high specific activity which, in general, will result in enhanced decomposition during storage. At the KAERI, we have been concerned with the stability during storage of pro­ ducts obtained commercially and those locally manufactured. Tables V and VI show the results obtained from the analysis of hippuran and Rose Bengal preparations derived from various sources as a function of their storage time. Although the stability of a radiopharmaceutical TABLE III. RADIOIODINATED TRIOLEIN

Details of Method of Developing Rf values of Activity in Name of Remarks supplier solvent analysis time (h) active zones the zones (°]o )

KAERI Petroleum -ether : acetone PPCa 3 Zone A: 0.05 < 3 Zone A may be due to iodide Zone B: 0. 86 > 97 Zone В is due to triolein ( 5 : 1 ) TLCb 1 Zone A: 0. 05 < 5 Zone A is due to iodide Zone B: 0. 73 > 95

Company I petroleum-ether : acetone PPC 3 Zone A: 0. 86 > 99 Organically bound iodide

(5 : 1) TLC 1 Zone A: 0.13 13 Zone B: 0. 53 20 Zone C: 0.73' 67

Company D Petroleum-ether : acetone PPC 3 Zone A: 0. 86 > 99

(5 :1 ) TLC 1 Zone A: 0.73 > 99

KAERI CHC13 : ether PPC 3 Zone A: 0.05 < 3 Zone B: 0. 82 > 97 (5 : 1)

Company I CHC13 : ether (5 : 1) PPC 3 Zone A: 0. 81 > 99

Company D CHClg : ether (5 : 1) PPC 3 Zone A: 0.84 > 99

a PPC. : Paper-partition chromatography

^ TLC. : Thin-layer chromatography TABLE IV. RADIOIODINATED OLEIC ACID

Name of Details of Method of Developing Rf values of Activity in Remarks supplier solvent analysis time (h) active zones the zones (°¡o)

Petroleum-ether : acetone PPCa 3 Zone A: 0. 82 > 99 KAERI (5 : 1) TLCb 1 Zone A: 0.41 69 Zone B: 0. 55 29

Zone C: 0. 92 2 IAEA-PL-336/7

CHCL : ether PPC 3 Zone A: 0. 83 > 99 (5 : 1) Company D Petroleum-ether: acetone PPC 3 Zone A: 0. 79 > 99

(5 : 1) TLC 1 Zone A: 0.40 40 Zone B: 0. 52 20 Zone C: 0.90 40

CHC13 : ether (5 : 1) PPC 3 . Zone A: 0. 91 > 99

a PPC. : Paper-partition chromatography b TLC. : Thin-layer chromatography

CD СП CO TABLE V.. STABILITY OF 1311 HIPPURANa CT>

Sample Date of preparation Date of analysis Non-bound 1311 1311 as organically (°Jo) bound1311 C7o)

KAERI Batch 28.4.69 14. 5.69 « 1 > 99 2 .6 .6 9 « 1 > 99 10.6.69 « 1 > 99 Sample obtained from 2 9.4 .6 9 14 .5 .6 9 < 4 > 96 company I 2 .6 .6 9 < 4 > 96 10.6.69 < 4 > 96 Sample obtained from 24. 4.69 14. 5.69 < 4 > 96 company D 2 .6 .6 9 < 6 > 94 10.6.69 < 5 > 95

a Stored in a refrigerator.

Я § TABLE VI. STABILITY OF 131I ROSE BENGAL

Sample Date of preparation Date of analysis Non-bound 1311 1311 as organically 1311 as 1311 as (%) bound 1311 (%) 1311-TITCFa labelled (°l°) impurities (%)

KAERI batch 2 8 .4 .6 9 14.5.69 <■ 1 > 99 97 <3 2 .6 .6 9 99 <1 10.6.69 " 99 <1

Sample obtained 2 9 .4 .6 9 14.5.69 7 93 34.4 58.6 from company I 2 .6 .6 9 5 95 48 45 10.6 .6 9 < 1 > 99 ' 50 50

Sample obtained 2 4 .4 .6 9 14.5.69 < 1 > 99 87.6 <12.4 from company D 2 .6 .6 9 " " 99 <1 <1 10.6 .6 9 • 99

Tetra-iodo tetrachlorofluorescein. IAEA-PL-336/7 97 depends on many factors, e. g. specific activity, structure, solvent, impurities etc., it must be borne in mind that considerable decomposition can occur during transport and storage. Multidose vials are particularly lia'ble to contamination by foreign materials and the decomposition products themselves. Products obtained from a reputable manufacturer should be used and for particular appli­ cations we consider it preferable to prepare a small batch in our labora­ tories immediately before use. In our experience preparations made at' our laboratories usually have a longer shelf-life than those obtained commer­ c ia lly .

ACKNOWLEDGEMENTS

The author expresses his sincere thanks for the suggestions and work done by Dr. R. S. Mani, International Atomic Energy Agency expert. Further appreciation is due to M essrs T. Y. Kim and B.K. Rhim, for their excellent laboratory work.

REFERENCES

[1 ] MANI, R. S ., Private communication, April, 1969. [2 ] HOSICK, T. A. et al., J. nucl. Med. ¿(1 9 6 4 ); and HOSICK,T.A . etal., ibid. 6 (1965) 136. [3 ] KIM, Y .S ., KIM, T .Y ., UHM, K. C. » .Korean J. nucl. Med. 2 (1968) 67. [4 ] KIM, Y .S ., KIM, C .D ., KIM, S. O., Korean J. nucl. Med. 1 (1967) 83. [5 ] KIM, Y .S ., KIM, C .D ., J. Korean chem. Soc. (1967) 51. [6 ] KIM, Y. S ., J. nucl. Sei. (Korean Office of Atomic Energy) 7^ (1967) 91. [7] KIM, Y .S ., KIM, T .Y ., J. nucl. Med. (Korea) 3 (1969) 69. [8 ] КО, С. S ., (Radiology Institute, Seoul), Private communication, 1968.

».

IAEA-PL-336/8

ROLE OF THE HOSPITAL RADIOPHARMACY

J .L . QUINN III Department of Radiology, Northwestern University Medical School, and the Chicago Wesley Memorial Hospital, Chicago, 111., United States of Am erica

Abstract $

ROLE OF THE HOSPITAL RADIOPHARMACY. The paper discusses the responsibilities of radiopharmacists in nuclear medicine units. With the advent of radioisotope generators, the role of the hospital radiopharmacy becomes very important. Current practices in the Chicago Wesley Memorial Hospital are reviewed.

INTRODUCTION

The growth of nuclear medicine tracer techniques over the past ten years in the diagnosis and treatment of m an's illnesses has been nothing less than phenomenal. Approximately million diagnostic radionuclide procedures were performed in the United States of America during 1966 [1]. The development of this new clinical discipline has occurred through a most gratifying co-operative effort between physical scientists, biologists and physicians with pharmacists playing an unfortunately minor role. The United States Atomic Energy Commission has stated "Byproduct m aterial shall not be used in humans until its pharmaceutical quality and assay have been established" [2]. Who should assess the pharmaceutical quality of a radioactive product? Should it be the reactor physicist, the radiation chemist, or perhaps the physician who in the end bears the final responsibility for his patient's safety? Have any of these scientists or practitioners had sufficient training in pharmaceutical procedures and practice to guard against patient harm and make such determinations? As a physician I can say that I have not; and my colleagues who are more conversant than I in this area have had to seek out a great deal more in­ dividual training than was available in any of their specialty training programs. In the United States, the hospital pharmacist, with all his knowledge of proper pharmaceutical practices in the manufacture and dis­ pensing of drugs sits as an untapped talent, sadly under-used in the field of hospital nuclear medicine. The pharmaceutical industry has not failed to use these individuals in the preparation of radiopharmaceuticals for sale, but in most instances this talent stops at the shipping room door.

ROLE IN HOSPITALS

The most meaningful justification for the presence of a trained pharma­ cist in a hospital nuclear medicine laboratory is on the basis of need [3]. An adequately trained pharmacist is qualified to evaluate pharmaceutical

99 100 QUINN

products with regard to pharmacological and chemical incompatibility, vehicle or diluent of choice, compound stability, etc. Radiopharmaceuticals should be no exception. In a given institution the functions of the pharma­ cist would depend on the scope of the hospital's nuclear medicine program. In a relatively small community hospital, where only a few diagnostic procedures may be offered, the scope of the radiopharmaceutical service would be limited. -However, at larger institutions where extensive teaching, research, and patient service programs are in operation, the extent of the services may be infinitely greater.

FUNCTIONS OF A HOSPITAL RADIOPHARMACY SERVICE

The major functions of the radiopharmaceutical service can be grouped into five major categories: (1) Formulation, (2) control, (3) research and development, (4) consultation, and (5) inventory and record keeping.

1. Formulation

This would include anything from the aseptic dilution of a radio­ pharmaceutical bulk-shipment to the tagging and preparation of a sterile solution for parenteral administration. Aseptic dilution may seem a rather simple procedure but a number of considerations may be overlooked by personnel without proper pharmaceutical orientation, such as the proper choice of a diluent to avoid inactivation or degradation of a radiopharma­ ceutical. The simple presence of a bacteriostat such as benzyl alcohol may cause unexpected problems [4]. This agent causes vasodilatation in patients and would be detrimental in a radiopharmaceutical intended for regional blood-flow measurement.

2. Quality control

Quality control testing should include both qualitative and quantitative assays for the radionuclide or the radiochemical. It is all too convenient to accept blindly the label on the delivered package. The identity of the radio­ nuclide and assay of the radioactivity as well as the radiochemical purity of such materials is certainly within the area of competence of trained hospital radiopharmacists. Thin-layer or paper chromatography is necessary in compounds known to be unstable, or subject to radiolysis, such as radio- iodinated thyroxine. Radionuclidic purity is very important in the generator systems such as molybdenum-99:technetium-99m, and tin-113:indium-113m because of the threat of parent contamination. It is also vital in the use of •short-lived radionuclides such as iodine-123, where the build-up of longer- lived contaminants could be quite hazardous. The frantic one-day order schedule, which the radiopharmaceutical industry provides, is conducive to packaging errors, and they have occurred. Therefore, it is of considerable importance that the product has some control and analysis in the hospital where it is received for use. The radiopharmacist at a hospital would complement the quality control effort of the manufacturer and only serve to effect better patient care. When radiopharmaceuticals are formulated at the hospital in which ■they are to be used, the services of a radiopharmacist become mandatory. IAEA-PL-336/8 101

3. Research and development

The talents and training of a radiopharmacist are invaluable when clinical research teams require high quality radiopharmaceuticals not commercially available. Physicians and technicians are seldom trained in an appropriate, fashion to perform the task of proper pharmaceutical compounding.

4. Consultation

The hospital pharmacist possesses a great deal of information con­ cerning equipment and supplies available to facilitate drug administration. This type of information may seem quite simple, and perhaps even un­ important to the pharmacist, but it is frequently lacking in the training ■ and experience of nuclear medicine personnel including physicians. Pharmacological incompatibility is an area in which the hospital pharmacist is trained and quite competent. The interference of iodide-containing compounds with the radioiodine uptake and scan is well known. However, many physicians are not cognizant of the wide variety of pharmaceutical products which contain iodine and they would welcome and benefit from the advice of a pharmacist on such m atters.

5. Inventory

Because of his training and experience the hospital pharmacist makes his presence very quickly.felt and appreciated with regard to maintaining an inventory on radioactive pharmaceuticals which may have a very short shelf life and getting optimum use out of expensive tracers through judicious ord erin g and, co-o rd in ated scheduling. An essential role in radiopharmaceutical practice is the proper re­ cording of all appropriate data for future reference. This includes the identity, quality, quantity, and sources of all ingredients present in products used in patients by the nuclear medicine staff.

THE CHICAGO WESLEY MEMORIAL HOSPITAL RADIOPHARMACY

With the increasing use of radionuclide generators in the preparation of technetium-99m and indium-113m compounds for parenteral injection we felt compelled to call upon the services of skilled hospital pharmacists in the formulation and'dispensing of these radiopharmaceuticals. Accordingly, our radiochemist, the hospital pharmacist, and the nuclear medicine physicians formulated a system for the quality control and dispensing of radiopharmaceuticals which has worked quite satisfactorily at our h osp ital [5].

Reagent and/or vehicle preparation

Reagents used in the formulation of radiopharmaceuticals and vehicles used for dilution or elution are prepared in advance and each batch is given a control number from a master log book. Analytical reagent grade materials are used to make up the reagents whenever possible. When a 102 QUINN

Reaqent Amount Lot

Chemical Formula Manufacturen Lot Amount -

Procedure »

Label Date Amount/vial Made by

5 4 4 -5

F IG .l. Reagent manufacturing form(Form 1).

LABORATORY USE ONLY О О z 7) О

NUCLEAR MEDICINE LABORATORY CHICAGO WESLEY MEMORIAL HOSPITAL

FIG .2. Reagent label (Label 1).

batch of a particular reagent is prepared, a reagent manufacturing form is filled out with total identification of all the products included (Fig. 1). The reagents are then sterilized either by autoclaving or membrane filtration (0.45 ium) depending on which method is consistent with reagent stability. They are labelled (Fig. 2(a)) and stored in 10-30 cm 3 volume serum vials. The vials are filled using a Cornwall pipette. Specially hardened Wheaton glass vials are used to minimize silicate formation and radiopharmaceutical adsorption. Each reagent batch has to clear recommended pyrogen and sterility testing [6 ]. The materials which are autoclaved are subjected to no less than a seven-day fluid thioglycollate culture at 30-32°C and those materials sterilized by membrane filtration are subjected both to the thioglycollate and the fluid Sabouraud culturing. The latter is for no less IAEA-PL-336/3 103

LABORATORY RECORD

RADIOACTIVE MATERIALS WORKSHEET

PRESENT DECAY ASSAY ml ml me fj с DESTINATION DATE TIME or Min. mc/ml REMOVED REMAINING REMOVED PATIENT Hrs. pc/m l Days.

DATE MEDIAC READING me m с

FIG.3. Radiopharmaceutical inventory sheet (Form 2). than 10 d at 22-25°C incubation. Only when these materials have passed the sterility and pyrogen tests are they used in the compounding of radiopharma­ ceuticals. The negative testing results are attached to the rear of the manufacturing form and filed for future reference.. No vial is entered more than five times or used for more than one week before it is replaced.

Radiopharmaceuticals

(1) From commercial manufacturers

Upon receipt the radiopharmaceutical is checked to see that the outside and inside labels match and, after solutions are checked for colour and clarity, placed in a dose calibrator where the radioactivity is assessed. A wipe test of the vial is made to see if there is removable contamination as 104 QUINN

Radiopharmaceutical Control Form

Compound 99mTc Sulfide Lot

IsotoDe Source

Reagent Lot Amount

Premix Sulfide

1 N HCl

Phosphate Buffer

0.5 M NaOH

Time m c./m l.

Date m l.

M adebv mc.

5 4 6 -2

FIG.4. " mTc*sulphide manufacturing form (Form 3).

Radiopharmaceutical Control Form

113m Compound In - Fe(OH), Aggregates Lot

Isotope Source

Reagent Lot A m ount

Eluting Solution

FeCI3 - ImgVml.

) N NaOH

20V# Gelatin

IN HCl

Tim e m c./ml.

Date m i­

M ade by me.

5 U -4

FIG. 5. 113mIn aggregates manufacturing form (Form 4). IAEA-PL-336/3 105

Radiophar maceu ical Control Form

Compound Lot

Isotope Source

Reagent Lot A m ount

Time mc./ml.

D ate ’ ‘ m l.

M ade by me.

544-6

FIG. 6. Miscellaneous radiopharmaceutical manufacturing form (Form 5).

a result of leaking vials or poor packaging techniques. The dose calibrator is checked on a daily basis against long-lived standards. An aliquot of the shipment is next subjected to spectrum analysis with a 1024-channel pulse-height analyser. Certain radiopharmaceuticals such as thyroxine, Rose Bengal, and tagged iodinated human serum albumin are then analysed on a strip-chart radiochromatogram. Extra labels supplied by the manu­ facturer are then placed on the radioactive material work-sheet, which is used as an inventory log (Fig. 3).

(2) Hospital produced radiopharmaceuticals

Radiopharmaceuticals which are made in our laboratory are prepared under the direct supervision of a registered hospital pharmacist using production methods developed in co-operation with our radio-chemist. A lot number is assigned from the master control book and a radiopharma­ ceutical control form is filled out for each batch of an individual radio­ pharmaceutical (Figs 4 and 5). This form records all the data pertinent to the compound. The compounds are prepared either in a closed system using sterile reagents and aseptic techniques or in. an qpen system with sterilization of the final product. After preparation the product is assayed for radioactivity. Such radiopharmaceuticals as technetium albumin and technetium sulphur colloid routinely have radiochromatographic analysis before they are used. Every time one of the two generator systems that we use is eluted for injection without further processing, a sim ilar form is filled out (Fig. 6 ). The eluents from the generators are checked for 106 QUINN

radionuclidic purity using chemical and physical methods before being used clinically. If radionuclide impurity is above legal limits, the contaminant is removed or the eluent is not used. Labels are affixed to each vial of compound identifying the radionuclide, the chemical form, the lot number, and the radioassay data (Figs 7 and 8 ). The tin-indium generator is a closed system supplied to the laboratory to deliver sterile and pyrogen- free material after elution. We used the closed technetium-molybdenum system but have recently replaced this with a liquid-liquid extraction system modelled after that at the Argonne Cancer Research Hospital. High specific activity molybdenum-99 in basic solution containing approximately 2 C i/cm 3 is delivered on a weekly basis. This is placed in a separating vessel and sodium pertechnetate-99m is extracted using methyl-ethyl-ketone.

STERILE SOLUTION

TECHNETIUM 99M _____

TIME______DATE CAUTION RADIO _ MC/ML ACTIVE _ TOTAL VOLUME MATERIAL . TOTAL MILLICURIES NUCLEAR MEDICINE CHICAGO WESLEY MEM.H0SP

FIG. 7. Label for technetium-99m compounds (Label 2).

STERILE SOLUTION

INDIUM 113M _____

_ DATE______CAUTION RADIO _ MC/ML ACTIVE MATERIAL _ TOTAL VOLUME _ TOTAL MILUCURIES NUCLEAR MEDICINE CHICAGO WESLEY MEM.H0SP

FIG. 8. Label for indium-113m compounds (Label 3).

It is not possible to complete bacteriological and pyrogen testing of the final radiopharmaceutical form for these two short-lived radionuclides prior to administration. Therefore, post hoc testing must be performed. The testing of all reagents used in formulation prior to their use in pro­ duction minimizes the hazard of contamination. The bacteriological tests are run on pooled samples of all formulated radiopharmaceuticals including generator eluents after they have decayed. The samples which comprise the pool are kept until the results return so that should a pooled sample show growth or pyrogenicity the individual samples can be tested in order to locate the contaminated lot. The control forms, with the bacteriological and pyrogen test results attached, and the batch inventory sheets are kept on file. This allows every compound to be traced from its source to its final destination. The system has worked well in our hands insofar as, since its in­ stitution in May 1967, we have had no bacterial contamination of any injected compound in over 2500 lots of material. IAEA-PL-336/8 107

Radiopharmaceutical dispensing

When a diagnostic nuclear medicine procedure is requested by a physician, the secretarial staff checks with the radiopharmacy to be sure that the proper radiopharmaceutical will be on hand in the required dose at the time the procedure is to be performed. When the patient arrives, the nuclear medicine physician prepares a prescription (Fig. 9) and presents it

NUCLEAR MEDICINE

C H IC A G O WESLEY MEMORIAL HO SPITAL NUCLEAR MEDICINE DOSE CALCULATION SHEET

PATIENT

D A T E ______W EIGHT

PERCHLORATE ’ YES ______N 0

ASSAY LOT NO.______TIME(Date)

. H O U R S ____ MINUTES

ORIGINAL DECAY A SSA Y ______F A C T O R - m Ci jUCl

PRESENT ASSAY m ci >u С i

------|mCiJ(/uCi) ______m l ______(mCi)ÿuCi)

DRAW N BY:

G IV E N BY:

T IM E : ______

RADIOPHARMACEUTICAL

FIG. 9. Radiopharmaceutical presciption and dose calculation form (Form 6).

to the radiopharmacist. The prescription is filled and the dose drawn up in a sterile disposable syringe. The syringe is assayed in the dose calibra­ tor and necessary corrections for minor volumetric errors in drawing up the dose are made. The dispensed dose is logged out on the radiopharma­ ceutical inventory sheet, thereby updating the inventory. The syringe is placed in a lead syringe holder with the prescription dose calculation sheet attached. The dose is then taken to the injection station for administration to the patient after the physician who ordered the m aterial has checked the dose calculation sheet. He injects the radiopharmaceutical using disposable plastic gloves and initials.the dose calculation sheet recording the time of in je ctio n . 1 08 QUINN

TRAINING

The training of a radiopharmacist would include a minimum requirement of being a graduate pharmacist with a strong background in the preparation of injectable products. The necessary training of a pharmacist in health physics, instrumentation, and radiopharmaceutical chemistry can be accomplished in as little as three months with concentrated course work, demonstrations, and closely supervised practical experience at a large nuclear medicine facility. We have had no difficulty in training a hospital pharmacist to practice "safe and proper radiopharmaceutical techniques. It is more difficult to train a chemist or technician in proper pharmaceutical techniques. Good chemical laboratory techniques may bear little or no resemblance to proper pharmaceutical techniques. The basic education and knowledge which a pharmacist obtains should never be underestimated. Their background ,in physics maybe lim ited ; however, other nuclear medicine personnel may have less training in pharmaceutical skills needed to ensure patient safety.

COST

It has been our judgement that the only cost of establishing a first- class radiopharmacy is the salary of the pharmacist himself. All the equipment and record keeping which the pharmacist uses should be available in a formulating and dispensing nuclear medicine laboratory whether a pharmacist is there or not. We have found in our experience that the pharmacist justifies his salary on the basis of savings from proper in­ ventory control, let alone the peace of mind he brings to all of us.

SUMMARY

The radiopharmacist plays a vital role in a nuclear medicine laboratory whether he is at the sophisticated level necessary for a large research- oriented nuclear medicine facility or whether he is the general practice hospital pharmacist, who is responsible for the service at the small com­ munity hospital level where only a few nuclear medicine techniques are available. In nuclear medicine when a problem arise's involving the physical sciences, then the wisdom and judgement of a nuclear physicist or radio­ chemist should be sought. When medical judgement is needed, a physician should be called upon for advice. It is reasonable that, when pharmaceutical judgements concerning radiopharmaceutical products are needed, a trained pharmacist should be used to help solve these problems [7 ]. This does not mean that a pharmacist should be a physicist, a radiation chemist, or anything else, but a practitioner of and contributor to the profession for which he had committed his life's work. A nuclear medicine laboratory in this day and age with the scope of the field broadening daily needs a trained hospital radiopharmacist who is well versed iri the properties and uses of radionuclides currently employed in the diagnosis and treatment of the ills of mankind. IAEA-PL-336/3 109

REFERENCES

[1] Survey of the Use of Radionuclides in Medicine, Preliminary Rep. MORP 68-10, US Dept, of HEW (July 1968). [2] USAEC, A Guide for the Preparation of Applications for the Medical Use of Radioisotopes, US Atomic Energy Commission (March 1965). [3] BRINER, W .H., Radioactive Materials: New Dimensions for Pharmacy, Hospital Topics (June 1965). [4] CHARLTON, J.C ., "Problems of radioactive pharmaceuticals", Radioactive Pharmaceuticals * ■ (ANDREWS, G .A ., KNÏSELEY, R.M ., WAGNER, H .N .,Jr., Eds) USAEC C0NF-651111, Washington (1966) 45-46. [5] CREWS, M .C ., QUINN, J.L ., III, BRYANT, N.. A quality control system for short-lived radio- pharmaceuticals, J.nucl.Med., In Press. [6] The United States Pharmacopeia, Seventeenth Revision, Mach Publishing C o ., Easton, Penn. (1965) 829. [7] BRINER, W .H., Radiopharmacy, the emerging young specialty. Drug Intelligence 2 (1968) 8-13.

IAEA-PL-336/9

TESTING PROCEDURES FOR INDIVIDUAL BATCHES OF RADIOPHARMACEUTICALS

K . FRÜHAUF Farbwerke Höchst AG, Frankfurt/Main-Höchst, Federal Republic of Germany

Abstract

TESTING PROCEDURES FOR INDIVIDUAL BATCHES OF RADIOPHARMACEUTICALS. The paper describes the particle size distribution tests for radioactive gold colloids and albumin particle suspensions. Bioassay is considered essential.

The basic analytical controls for radiopharmaceuticals are the uniformity tests, i.e. radionuclidic and radiochemical purity. While genuine solutions may be sufficiently characterized by these criteria, those preparations that are offered in the form of colloidal solutions or particle suspensions call for additional determinations of mean particle size and particle-size distribution. The examples given below have been taken from the range of diagnostic and therapeutic preparations. The more stringent requirements are set by the diagnostic applications, and here it is especially the group of functional testing procedures, where- quantitative determinations of accumulation or disappearance rate are carried out, that require the pure and uniform product. In the case of gold colloids or albumin particle suspensions the normal radiochemical purity determination by paper chromatography or electrophoresis will result in a value for dissolved activity; the particle- size distribution has to be checked by microscopic methods. Albumin particles intended for intravenous or intra-arte rial administration with a mean particle size of 20 to 30 /um can be counted and measured under the light microscope. While a determination of the number of particles per m illilitre of suspension is not a routine procedure, the size range has to be checked for any individual batch. Our own specifications call for a size range of 5 - 50 /um; the standards of the United States National Institutes of Health require more than 90% of the particles to be between 5 and 70 цт. A large number of very small particles will increase the portion of activity accumulated in the liver; bigger particles may cause embolism. Particles above 50 |im have to be carefully avoided, especially when the .suspension is to be administered intra-arterially. By observing the suspension in a hanging drop under the light m icro­ scope with an ocular m icrom eter, ten different areas with ten particles each can easily be examined in one sample. As long as all the particles fall within the desired lim its, only the total number of particles observed has to be recorded. Otherwise, the smallest and the highest diameter values have to be noted together with the number of particles exceeding the lim its. To supplement this characterization by particle-size distribution albumin particle suspensions are tested for dissolved activity, both in

111 112 FRÜHAUF the form of dissolved labelled albumin and unbound iodide. The total activity dissolved is determined by counting the supernate of an assayed sample after centrifugation of the particles. The supernatant is then submitted to an electrophoretic separation of bound protein and free iodine. The usual findings are 1 - 2% total dissolved activity, about equal parts of which are bound and unbound iodine. Among the radioactive gold colloids there exist special prepara­ tions for the evaluation of liver function. With this technique the dis­ appearance rate of gold activity from the circulation is determined. The disappearance rate as observed in normal individuals is influenced by the particle size. A change in mean particle size will alter the normal rate, while a broad spectrum of particle diameters reduces the sensitivity of the method to small deviations from the normal behaviour. To arrive at reproducible and comparable results, therefore, a narrow and reproducible particle-size distribution is essential. In the 30-nm range an electron microscope has to be used to prepare the necessary micrographs. These can easily be taken in a laboratory not equipped to handle radioactive m aterials, since only minute amounts of the dilute suspension have to be applied as the sample, corresponding to activities well within the licence-free lim its. An entire distribution spectrum need not be taken in every instance. As a routine check with the 30-nm preparation a visual examination for the predominant particle size and the absence of particles below 20 nm and above 40 nm is considered sufficient. Again, a chromatographic determination of ionic gold in the suspension supple­ ments the particle-size determination. After all appropriate physical and chemical tests have been passed, there remain in some cases qualities of a preparation that can only be verified by bioassay. We seriously question the effectiveness of the so- called safety testing procedures required in quite a number of radio­ pharmaceutical standards, which call for injection of several m illilitres of a radiodiagnostic agent into mice or guinea pigs, and observation of these animals over a period of time. We are considering here biological testing procedures which simulate the actual test for which the radio­ diagnostic agent will be used in practice. Through these methods, with the aid of test animals or other biological material, we can guarantee the performance of the preparation, whereas physical and chemical methods of analysis will yield only standards of purity. Two further examples shall serve to illustrate the importance of the bioassay procedures. If the user of a sodium chromate preparation arrives at unsatisfactory results in an erythrocyte labelling procedure, a radiochemical analysis showing the absence of trivalent chromium in the solution will not always convince him of the usefulness of the material. We have evidence for the fact that other impurities besides the lower oxidation states of chromium may interfere with the fixation of chromium activity to red blood cells. A determination of the labelling yield is therefore made after the radiochemical analyses have shown satisfactory results, and a batch of sodium chromate will only be re­ leased for dispensing when under standard incubation and washing con­ ditions more than 90% of the total activity is bound to the cells. . For the final example we return to the aggregated albumin prepara­ tions for lung scanning. The microscopic examination is routinely sup­ plemented by a study of organ distribution in the rat as the test animal. IAEA-PL-336/9 113

Three rats are sacrified 10 min after intravenous injection of the particle suspension. To pass the test more than 90% of the activity- administered has to be found in the lungs, and less than 5% in the liver. Experience gained during the development of the labelled albumin particles revealed the fact that, even with a satisfactory particle-size distribution, which was also documented by a sufficient initial accumula­ tion in the lungs, unsatisfactory lung scans with the beginning of a de­ lineation of the liver may be-obtained, especially when more than the usual ten minutes elapsed between the injection of the m aterial and the beginning of the scan. These findings had to be attributed to the fact that the albumin aggregates disintegrated too fast into sm aller particles in the circulation. Evidence for the suitability of a particle preparation for the lung scan can best be given by a lung scan, and therefore this procedure was maintained when the routine production of the albumin- 131I particles was taken up. Two consecutive scans are performed on an anaesthetized dog after injection of 100 ßCi from a fresh batch. The liver region is intentionally scanned together with the lungs; The first scan is started ten minutes after the injection, and the liver region of the second one will be scanned two hours after the injection. No failure of a product tested in this way has been reported from a hospital, so one may consider this a satisfactory testing method.

IAEA-PL-336/10.

LIMITS OF ACCURACY IN THE DETERMINATION OF PURITY BY 'THIN-LAYER AND PAPER CHROMATOGRAPHY

C.E. MELLISH The Radiochemical Centre, Amersham, Bucks, United Kingdom

Abstract

LIMITS OF ACCURACY IN THE DETERMINATION OF PURITY BY THIN-LAYER AND PAPER CHROMATOGRAPHY. The factors determining the accuracy with which a purity figure can be derived from a paper or thin-layer chromatogram of a radioactive substance are discussed. An approximate expression is derived for the standard deviation of the fractional amount of impurity present due to counting statistics, in terms of the quantities measured on the chromatogram. The examples and graphs presented show in particular the importance of accurate background determination.

INTRODUCTION

The merits of thin-layer and paper chromatography as analytical techniques are that they are quick, easy and cheap. In their application to measurements of radioactivity they also offer the possibility of more accurate results than when they are used for ordinary inactive chemistry. This is because it is'easier to obtain quantitative measurements from a radioactive chromatogram, either by scanning with a counter, or by cutting up pieces and counting them individually, than it is to obtain quantitative results from areas of inactive m aterial spread on the same chromatogram. For these reasons, then, thin-layer and paper chromato­ graphy have come to be accepted very generally as methods of deter­ mining the purity of labelled compounds in the field of radiopharma­ ceuticals and ordinary radiochemicals. A considerable amount of work has been done on the relative ef­ ficiencies of separation of different chromatographic techniques when seeking confidence in either identification of a m aterial or the purity of a material, and this has shown in fact that thin-layer and paper chromato­ graphy are not capable of such high definition or resolution as column chromatography or ion-exchange chromatography. This paper does not deal with the ultimate capacity of chromatographic methods, but with the best way of using these techniques as a routine quality control procedure. It will always be true that the man who is prepared to make a research project out of the investigation of a particular system will be able tö find out more about it than the manufacturer whose analytical work must in the end be limited by economic considerations.

THE PRINCIPLES OF PURITY MEASUREMENTS

Figure 1 is a diagram of a record of a thin-layer or paper chromato­ gram obtained by scanning it with a counter of some kind. The radio­ activity which has been recorded has four components. First, there is

115 .116 MELLISH the activity in the main peak, which we will assume for the time being comes from the pure m aterial that we are trying to prepare. Secondly, there is the radioactivity from the small impurity peak marked-I on the diagram. Thirdly, there is the background. Fourthly, there is the radioactivity which corresponds to the raised area of background between the start position and the impurity peak I. The activity from this part of the chromatogram may be some kind of background effect or due to trailing of pure material from the main peak, or it may be due to im­ purity which is smeared out along the chromatogram rather than presenting us with a single-discrete peak.

FIG. 1. A typical chromatogram scan.

' In analysing this chromatogram, we may be interested in giving a purity figure ("the material is 92% pure"), or we may be interested in a particular impurity, and thus in trying to say that the impurity is less than some fixed percentage of the total activity. In either case the first step in the analysis is to measure the background of the chromatogram, so that we can subtract it from the total counts and obtain the de­ nominator of the two fractions on the diagram. It is immediately clear how important the accurate determination of background is, either to a purity figure or a measurement of impurity content. In the diagram, the determination of background can only be done certainly from the very small amount of trace on the right-hand side of the start point or the left-hand side of the finish point. If the background near the finish point had followed the dotted line in the diagram, it would have been attractive to use this apparently normal background to determine its value. This would have been an unwise procedure as there may be impurity registering in this part of the chromatogram; it only appears IAEA-PL-ЗЗб/10 117 reasonable to use this part of the scan for background determination because the count-rate looks about the same as the background on the other side of the diagram. In the case of a measurement of impurity on the little peak, labelled I, background is shown as a substantial propor­ tion of the total counts recorded in this area of the chromatogram. Clearly, the accurate determination of background is very important for this measurement. For the purity measurement Fig.2 shows very clearly the necessity for an accurate background determination. In this figure the amount of elevation of background caused by 1 0 % impurity or 1 0 0 % impurity, spread over the whole chromatogram, is shown diagrammatically in relation to the main peak. The elevation of back­ ground caused by 1% of impurity spread over the whole range is not shown because it is virtually impossible to draw it in.

FIG.2. Elevation of base-line by evenly distributed impurities.

Coming back to Fig.l, we have two problems to solve in relation to the background, one philosophical and one technical. The philosophical problem is what we do with areas of raised background which do not appear to represent peaks of impurity. The technical one is the ac­ curacy with which we can measure and subtract the background from the influence which this has on our final figures. I think there is only one honest answer to the first problem. Where the background is raised for any reason, any numerical calculation must include this as part of the impurities present on the chromatogram, unless there is special knowledge about the properties of the chromatographic system and the m aterial being analysed which enables some kind of allowance to be made for this phenomenon. If, for example, one is able to remove some of the activity between the start line and the main peak, and show that on 118 MELLISH applying this to a fresh chromatogram the m aterial runs with a mobility corresponding to that of the pure compound, then it would be reasonable to say that this raised background corresponded to streaking of the pure m aterial and not to any impurity present.

STATISTICAL ACCURACY OF PURITY MEASUREMENTS

The parameters which are directly measured on the chromatogram are the counts in the main peak P, the other counts in the scan, which include a background contribution, and the background counting rate. Manipulation of these three quantities gives the purity of the m aterial:

Let counts in peak = P counts Background counting rate = G counts/sec Time taken scanning impurities = i sec (this is usually the whole scanning time less the time spent scanning the main peak) Time taken determining G = t sec Mean impurity count-rate during the scan = .1 counts/sec

The purity is measured by peak counts/total counts, and the error in purity will be the error in the calculation of the fraction

. ______Peak counts______Peak counts + impurity counts - background counts in impurity scan

(This assumes that the peak counts can be calculated directly, or that the subtraction of background from the total counts recorded in the peak can be done with negligible error. - This is generally true, as the background correction to the peak counts is small.)

P u rity = P + (I + G)i - Gt X i/t

I - Ii+ Gi - Gt X i/t P + (I + G )i - Gt X i/t

If the purity is greater than 90%, this approximates to

„ , И + Gi - Gt X i/t Purity = 1 ------p------<—

= 1 - a, where a is the fraction of impurity present in the sample.

In this expression we have three total counts which are directly determined: P, the total counts in the peak, (I + G)i, the rest of the counts in the scan, and Gt, the counts from which G is determined. The counting standard deviation of each of these quantities is its square root, so we can calculate IAEA-PL-336/10 119 the standard deviation of the impurity fraction by combining the standard deviation of these quantities by the usual rules (e.g. Cook and Duncan [1]).

...... И + Gi + Gt (i/ t)2 . P (fractional standard deviation of a) = ------^ or, substituting the approximation a

(Standard deviation of a)2- = ^

As the impurity fraction a is not normally more than 0.05 for satisfactory m aterials we can neglect the term <*2/P in comparison with a j P and obtain the approximate expression

2 , , a , G (it + i2 ) a (a) = - + V*t

w here a = fraction of impurity in the material

о (a) = standard deviation of a

Using this expression we can work out the standard deviation of the impurity fraction for any chromatogram from the directly determined measurements which occur in the equation. The two items in the ex­ pression for the standard deviation of a correspond roughly to having enough counts in the peak (first term) and counting an accurate back­ ground (second term). However, the two effects are a little confounded by the P 2 factor in the second term, which makes it difficult to formulate simple rules to maintain accuracy. Figure 3 shows a diagram of an imitation chromatogram for which a 2 a confidence limit has been calculated as follows:

Example: Imitation TLC plate.

Peak counting rate = 500 cps P = j X 500 Time scanning peak 80 sec = (base width just> 1 cm) X 80 = 20 OOOcounts Background rate G = 1 0 cps Time spent determining G 200 sec(about = 3 cm ofbackground trace) Time scanning impurities 800 sec = (roughly all the chromatogram excluding the peak) 0.03 10 . 640 0001 Purity = 97%, (i.e.a = 0.03) a 2 (a) = 20 000 4 X 10a 200 J

= 1.01 X 10" 4 ■

a (a) = 0.01 i.e . the 2ct confidence limits on the purity are 95 and 99%. 120 MELLISH

-500 c.p.s.

.FINISH START I

FIG .3. Diagrammatic chromatogram scan showing a statistical error of up to 2°jo in the derived purity figure. p = 20 000 counts; purity = O T ’/o ± 2°¡o.

FIG.4 . Graphs showing the dependence of the standard deviation of the impurity fraction on the total peak counts.

In this case the major contribution to the statistical error comes from the second term in the expression for the standard deviation. The background is not determined sufficiently well to give a really accurate purity figure. IAEA-PL-336/10 121

Although the calculation of background in this expression assumes a computation based on a total background count instead of drawing a line on the chart manually or using some kind of integrating device, it is a fair approach, for these other procedures cannot be more accurate than the total count method. The calculation here gives the minimum assessment of statistical error. The general conclusions from the form of the expression are not surprising. For good accuracy we need a high peak, low background, long counting times. We also need pure material, as accuracy depends upon a. The purer the material, the more accurate the analytical figure. In Figure 4 we have curves drawn showing the separate behaviour of the two terms in the expression for different parameters of the chromatogram. The Curves on the left-hand side plot the a obtained fro m the а /P term; the curves on the right-hand side plot the a obtained from the second term only. For any given chromatogram, of course, the standard deviation has to be obtained by squaring the two values of a obtained in this way, adding them and taking the square root as is indicated in the expression itself. One can see that the requirements of the second term of the expression are in general much more stringent than those of the first term. For these graphs a time was taken for scanning impurities of 1 0 0 0 sec, which is about a quarter of an hour, and a time taken to determine the background accuracy of 5 min. Under these conditions it is seen that whereas the first term begins to give satisfactory standard deviations with about 1 0 4 counts in the main peak, the reqùirement of the second term is that at least 3 X 104 counts are required in the main peak to bring the error satisfactorily low, even with a background as low as 1 count/sec. The limit lines on Fig.4 are drawn to give an idea of the conditions required for quoting purity figures where the 2 ct variation on impurity fraction is not more than 0.5%. That is to say, a quoted figure of 97% is unlikely to correspond in reality to a purity of > 97.5% or < 96.5%. The top limit line shows the limit when only one term in the expression is important, that is when the other term is very small in comparison. .The lower limit line shows the level that has to be met when the terms are of equal importance. When one wishes to quote the lim its of accuracy of an impurity present, rather than quote a purity figure for the chromatograms as a whole, the same expression can be used for determining the errors in­ volved, as the a (a) in Eq. (1) is the standard deviation of any one im­ purity if the total counts determined in the impurity peak are substituted for the total impurity counts obtained over the whole chromatogram. The equation for statistical error can equally well be applied to chromato­ grams evaluated by dissecting techniques; the only difference is that total counts are determined directly instead of being rates multiplied by tim e s.

DISCUSSION

The difficulty of obtaining satisfactory statistics on chromatograms revealed by these figures focuses attention on the methods of integrating scans of the type described. 122 MELLISH

FIG.6. Chromatogram from Fig.5 scanned on a more sensitive range. (X 6).

The calculation we have just been through shows the importance of the accurate determination of background more than anything else, but although that discussion was confined to the statistical difficulties in the subtraction of background, it is clear that there are other problems in IAEA-PL-336/10 123 performing this operation satisfactorily, and that the statistical error will not be the only error. If we consider Fig.l again, we can see that the simple procedure of drawing a line on the chart at the background rate position is not only open to criticism because of the statistical variation in the base line, but also because of the difficulty of measuring the very small areas of interest accurately. It is therefore valuable to consider how more sophisticated methods of integrating records of this kind will affect the accuracy of the kind of determination we are interested in. One immediate improvement in the analytical situation may be af­ fected by running the chromatogram on a more sensitive recorder range in addition to the one which gives the main peak as near as possible a full-scale deflection of the chart. This may be done either by à repeat scan of the chromatogram or by using a two^pen recorder recording both the original range and the more sensitive range simultaneously. These procedures give an immediate improvement in legibility and hence in accuracy in doing a manual area determination. On the more sensitive range the main peak is off scale, and the areas of impurities determined on this ránge are related to the main peak area through the calibration of the ranges on the ratemeters. Clearly, the cross calibration of the ranges on the ratemeter introduces a possibility of error and this cross calibration must be regularly checked. An alternative to running the chromatogram on two ratemeter ranges in this way is to run an aliquot sample as a comparison with the chromatogram run on the sensitive range. In this case the impurity area determined is compared not with the main peak area on the less sensitive range but with the size of the aliquot sample, one-tenth or one-hundredth of the size of the main sample, which is scanned on the same sensitivity. In this case the accuracy depends on the dilution procedure instead of the cross calibra­ tion of the ranges on the ratemeter. One manufacturer of radiochemicals routinely sends out scans of this type with his product showing the de­ flection corresponding to a 1% impurity by running a 1% aliquot of the sample used for the main chromatogram. The next step in sophistication is to use some kind of automatic recorder-tied integrator as part of the scan. This may be a simple integrating pen which moves across the scale from one side to the other, or it may be the more sophisticated kind used in the trace which gave the diagram in Fig.5. Here, the integrating pen moves with a slope which is proportional to the rate at which counts are being put in, but when it reaches the 10 division mark on the scale it reverses direction. Large areas may be integrated by counting the number of reversals of the integrating pen corresponding to the area being measured; for greater accuracy or to measure a small area, the number of divisions passed on the chart must be added in as well. Figure 5 shows a scan which has impurities which are very dif­ ficult to see, and do not inspire confidence in their significance. Al­ though the integrator has measured the 1.5% impurity near the start line, one is far more confident about its presence after seeing Fig . 6 where the same scan is repeated on a more sensitive ratemeter range. . Thus this measurement combines the two improvements mentioned, integration by an automatic analogue device and recording on more than one range for greater legibility and sensitivity. 124 MELLISH

Another method of obtaining integrated figures from chromato­ graphic results is to take samples of eluate at fixed times, or at fixed distances on a paper chromatogram. There is no doubt that this pro­ cedure can give the most accurate results of all, provided the sections cut or the samples taken are sufficiently numerous. P.B. Klein [2], at the Argonne Laboratory, has published some data on the relative per­ formance characteristics of different fractionation procedures, and this comes out very clearly from his figures. In the case of paper chromatograms, the cutting of reproducible small sections is not dif­ ficult; for thin-layer plates little work has been done on the reproducible and repetitive taking of small samples for counting, but Snyder [3] has developed a machine which will do this automatically. For most kinds of isotopic labelling, it is possible to take the samples from thin-layer or paper chromatograms and drop them into liquid scintillant for counting in a beta liquid scintillation counter. The only major excep­ tion to this is tritium compounds, where on a piece of paper the orienta­ tion of the paper sample in the liquid scintillator alters the efficiency of counting too much for this to be a practical proposition. These techniques, although they are very accurate, are probably too time-consuming for routine quality control. It is possible to simplify the procedures by autoradiographing the chromatograms, and then cutting up the sections of main peak and impurity separately. In this way the number of cuts and the number of counts is drastically reduced. There are serious objections to the adoption of this technique for chromato­ grams which have not been scanned, because the outlines of chromato­ graphic spots on autoradiographs are not very sharp and do depend on exposure times quite critically. A problem which has not been dealt with so far in this paper at all is that of peaks on the chromatogram, which are not completely resolved from each other, and impurities which are seen as shoulders on the side of main peaks. Although it may not be possible always to measure accurately such impurities, it is important for the analyst to see that they exist and to make some estimate of their size. Partly resolved peaks of this kind are not easily visible in the intensity shading at the edge of an autoradiographic spot. The possibility of completely automating the integration and analysis of the chromatogram is a very tempting one. The automation of analytical procedures is a live issue at the present time, and every exhibition of laboratory equipment shows a number of different sorts of equipment designed to carry out a large number of analyses automatically. Un­ fortunately, it is characteristic of the radiochemical and radiopharma­ ceutical business that we are concerned more with large numbers of small batches of products than with repetitive analyses on the same product. It is easy to get an electronic integrator to attach to a gas chromatograph which is monitoring the process stream of, say, an oil refinery. Essentially, this machine is carrying out the same analysis all the time, and the integrator can be set to recognize and deal with the peaks which always occur, in the same places, in different propor­ tions and with different shapes as the reaction proceeds. It is not any­ thing like so easy to obtain an integrator which will cope with measuring fifty chromatograms a day which are all of different materials and in different systems, so that the Rfs and peak shapes and likely impurities are all different. Again, it is not sufficient for a routine quality control IAEA-PL-336/10 125 organization to devise a scheme for a full computer analysis of chromato­ grams which allows for the diversity in the results by using very complex curve fitting routines, unless a computer of the size and complexity to carry out this analysis is available to give quick service on the results. With short radioactive decay times the gap between pre­ paration and delivery must be kept as short as possible, and it is no good having a computer which will calculate an analysis in 30 sec, if it takes a day to transport the data and the answers to and from the com­ puter. I feel confident, however, that automatic handling of the data and automatic calculation of purity figures must be our aim in the future. There is little difficulty in obtaining digital printout on punched paper tape of the data of the basic chromatogram; at the Radiochemical Centre we are putting in some effort to see whether simple computer programs can be devised which will give us worthwhile results with the type of simple computer which it is economic to have available for this kind of application.

REFE RENCE S

[1] COOK, G .B ., DUNCAN, J .F ., Modern Radiochemical Practice, O .U .P. (1952) 60. [2] KLEIN, P.B., Separation Science 1 (5) (1966) 511-29. [3] SNYDER, F ., Advances in Tracer Methodology 4 (1968) 81-104.

IAEA-PL-336/11

DETERMINATION OF INORGANIC RADIOIODIDES IN 131I-LABELLED COMPOUNDS BY MEANS OF THIN-LAYER CHROMATOGRAPHY

J. ALVAREZ Mexican National Nuclear Energy Commission, M exico N. G. B. de SALAS Argentine National Atomic Energy Commission Buenos Aires, Argentina P. RABAN Charles University, Prague, CSSR and A.E.A. MITTA Argentine National Atomic Energy Commission, Buenos Aires, Argentina

Short contribution

INTRODUCTION

Organic compounds labelled with a radioiodide for use in medical diagnosis or therapy generally contain inorganic radioiodides as their main impurities. According to current regulations, the radioiodide content should never exceed b%. Using thin-layer chromatography [1] , we have devised methods for separating radioiodides from organic molecules [2-5] . We have tried to reduce the chromatogram development time and looked for readily avail­ able solvents for use with most of these compounds; IN hydrochloric acid was found to be the most suitable. Chromatograms were prepared by us on glass plates using Silicagel G (Merck); the chromatogram was developed at ambient temperature and the substances localized by chemical and auto­ radiographic means. Quantitative determinations were made with a model 7200 Packard Radioscanner, which is capable of detecting 0. 5% radioiodide concentrations.

E xp erim en t

Adsorbent: Silicagel G (Merck) — grain size 250 цт Solven t: IN HC1 Ascending chromatography Development time: 15 min Rf value of the radioiodide : 0.98-1.00

127 128 ALVAREZ et al.

We give below a list of the labelled organic compounds chromatographed with this solvent and the corresponding Rf values

Labelled substance

1 Rf

1 . Para-aminohippuric acid -131I 0 . 0 131 2. Diphenylhydantoin- I 0 . 0

3. "Hypaque" Sodium-131I 0.0

4. Hippuran- 131I 0. 25

5. " Urografin" - 131I 0.0

6 . Album in-131I 0 . 0

7. Rose Bengal-131I 0.0

8 . "Biligrafin" - 131I 0 . 0

9. ' ' A lilin u lin a" - 1311 0.0

10. In su lin - 131I 0.0

11. T SH -131I 0 . 0

12. Sodium iodothalamate - 131I 0 . 0 ‘

h - CO "Diprocon" - 131I 0. 36 1 Я1 14. Bromosulphalein- I 0 . 0

15. Iododeoxyuridine- 131I 0 . 0

16. Chloram bucil-131I 0 . 0

17. Iodoform -131I 0 . 0

18. Gamma globulin-131I 0 . 0

19. Congo red - 131I 0 . 0

The typical graph in Fig. 1 shows a clear separation of the labelled substance from the inorganic radioiodides.

CONCLUSIONS

This method is considered suitable for rapid control procedures at the plant and in situations where it is not easy to obtain various chromato- graphically pure solvents.

NOTE

When the experiments are repeated under the same conditions but using glass fibre (ITLC), the displacement times are reduced from 15 to 3 min for the same distance. IAEA-PL-336/11 129

О 10 20 30 ДО 50 60 70 80 90 100

FIG. 1. Separation of the labelled substance from the inorganic radioiodides.

REFERENCES

[1] STAHL, E ., Pharmazie И (1956) 633. [2 ] MITTA, A .E .A ., CAMIN, L. L. , FRAGA, A .H ., CNEA Rep. No, 110, (1964). [3 ] STRUFALDI, B. , ENGELSTEIN, E ., BARBERIO, J.C . , MITTA, A.E. A ., Publicaçâo IEA No.120 (1965). [4] MITTA, A. E. A. , CAMIN, L. L. , TROPAREVSKY, M. L. P. , Radiochimica Acta 6 (1966) 111. [5 ] SALAS, G .N .B ., TROPAREVSKY, M. L. P ;, MITTA, A. E. A. , Radiochimica Acta 8 (1967) 224.

IAEA-PL-336/12

SELF-DECOMPOSITION OF SOME 131I-LABELLED RADIOPHARMACEUTICALS *

I. GALATZEANU ; Institute of Atomic Physics, Laboratory of Radiochemistry, Bucharest, Romania and G .B . COOK International Atomic Energy Agency, Vienna

Abstract

SELF-DECOMPOSITION OF SOME 131I-LABELLED RADIOPHARMACEUTICALS. The stability of radio-iodinated (13II) Rose Bengal, thyroxine, hippuran, and human serum albumin towards self-radiolysis was investigated by using external gamma irradiations and by checking the radio­ chemical purity after various periods of storage.

INTRODUCTION

Most labelled compounds used in medicine are labelled with gamma- emitting radioisotopes such as iodine-125 and iodine-131, cobalt-57 or cobalt-58, chromium-51, selenium-75 and mercury-203 or mercury-197 etc. Compounds labelled with pure beta-emitting radioisotopes are most commonly used in tracer investigations. It is known that a radioactively labelled compound decomposes during storage. Decomposition depends partly on the amount of energy absorbed by the compound during storage and also on the type of emitter and its specific activity. : The underlying causes of self-decomposition of radioactively labelled compounds were classified by Bayly and Weigel [1] as follows: (1) Prim ary (internal) radiation effects, ( 2 ) primary (external) radiation effects, (3) secondary radiation effects, and (4) chemical effects. Secondary effects are commonly the most damaging and fte decompo­ sition thus produced is very susceptible to minor variations of the environ­ mental conditions and the physical state of the labelled compounds, e. g. time and temperature of storage, storage in dry state or in aqueous solution, addition of various stabilizers and radioprotectors etc. The secondary decomposition or transformation of a molecule is due to its interaction with reactive species, for example free radicals, radiolysis products of water (solvent), produced as a result of primary radiation e ffe c ts. The purity requirements for radiopharmaceuticals depend upon the intended application and the scope of the particular experiments in which they are employed. The purity criteria of radiopharmaceuticals will be

* This work was performed under the auspices of the International Atomic Energy Agency in the IAEA Laboratory at Seibersdorf near Vienna, Austria.'

131 132 GALATZEANU and COOK considered under three aspects: radionuclidic purity, radiochemical purity and chemical purity. The absolute radionuclidic purity involves the absence of any radionuclide other than the one stated. Conventionally, presence of the activity of daughter nuclides is disregarded, e. g. strontium-90 always contains its daughter yttrium-90, ruthenium-106 its daughter rhodium-106, zirconium-95 its daughter niobium-95 and many other examples. A radiopharmaceutical which is considered radiochemically pure does not contain the radioisotope in any chemical form other than the one in question. ■ Thus the radiochemical purity is the proportion of the total activity which is attributed to the stated chemical form. The radiochemical purity can also be affected by the method of preparation of the compound and by the chemical purity of the starting compound before labelling. Chemical purity is the proportion of the material in a specified chemical form .and this term involves the absence of any chemical substance other than the carrier, or the buffer salts. It is very important to note that, in recent years at many international conferences on labelled compounds and radiopharmaceuticals, attention has been drawn to the needs and demands of the users for a better and more complete specification concerning radionuclidic and radiochemical purity. In terms of its tasks the IAEA Laboratory at Seibersdorf has initiated comparison of radiochemical purity of the different 131I-labelled pharma­ ceuticals and much attention has been paid to the determination of the self­ decomposition during storage. Iodine-131 labelled Rose Bengal, thyroxine, hippuran and radio- iodinated human serum albumin were ordered from different producer companies or national centres from the United States of America, USSR, France, United Kingdom, the Federal Republic of Germany, Italy, Spain, the Netherlands and Hungary. Before radiochemical analysis, the samples under investigation were analysed with regard to radionuclidic purity by gamma-spectrometry. Then chromatography and electrophoresis techniques were used for the determination of radiochemical purity. The quantitative distribution of the activity along the chromatograms and electrophoregrams was determined by scanning.

I. ROSE BENGAL-131I [2, 3]

Radioactively labelled Rose Bengal was obtained from four of the principal commercial suppliers, here designated А, В, С and D and analysed periodically during one month. Commercial inactive Rose Bengal is a mixture of high and low- halogenated compounds, used for the study of the hepatic function, but physiologically it is eliminated by the liver only in the form of the higher halogenated compound as tetraiodo-tetrachlorofluorescein. Therefore, a very high chemical and radiochemical purity is needed.

Experimental

Paper and thin-layer chromatography were the techniques used to analyse Rose Bengal preparations. The radioactivity of spots was deter­ mined by a scintillation counter and each spot was examined by gamma IAEA-PL-336/12 133

FIG .l. Thin-layer chromatograms of Rose Bengal-131I from suppliers A-D 1 d after receipt (measured by densitometric and scanning methods). RB4 = tetraiodo Rose Bengal RB3 = tri-iodo Rose Bengal spectrometric method (using a 3 in. X 3 in. sodium iodide crystal detector and a 400-channel RIDL analyser to detect any gamma-radioactive impurities). Analysis of each sample was carried out 1, 8, 19 and 28 d after the sample reached the laboratory. 134 GALATZEANU and COOK

TABLE I. ANALYSIS OF ROSE BENGAL 1 d AFTER RECEIPT (Thin-layer chromatography on silica gel plates, cf. Fig. 1)

Contents of 13lI (%)

Rf 0. 69 - 0. 75 Sample Rf 0. 8 - 0. 9 Rf 0. 9 - 0. 97 (I)

A n. d. 1 99

В 4 1 95

С 2 5 93

D 3 10 87

n, d. = not detected

FIG. 2. Thin-layer chromatograms of Rose Bengal-131I from suppliers A-D 8 d after receipt (measured by densitometric and scanning methods). IAEA-PL-336/12 135

FIG. 3. Thin-layer chromatograms of Rose Bengal-131I from suppliers A-D 19 d after receipt (measured by densitometric and scanning methods).

Using thin-layer plates of silica gel and n-butanol-acetic acid-wàter solvent system (4:1:1), periodic analyses of the four different preparations of iodinated Rose Bengal were made.

Results and discussion

The firsf control showed that sample A was radiochemically pure, by the presence of a single spot of R f 0. 96 on the chromatogram. The quantity of inorganic iodide was negligible (less than 0. 5%). All other samples, В, С and D, showed the presence of inorganic iodide of R f 0. 7 (Fig. 1). The distribution of iodine-131 among the various compounds was obtained by determining the radioactivity of the individual spots and is shown in Table I. The second control of Rose Bengal, 8 d after arrival, shows that an appreciable amount of inorganic radioiodide together with noticeable amounts of the lower iodinated homologues had been formed in sample A (Fig. 2). The situation is very sim ilar for samples В, С and D, but with increased amounts of inorganic iodide and the lower iodinated homologues being formed (Fig. 2). The results of the third control, 19 d after arrival (Fig. 3, Table II), give a quite different picture for all samples. With A, the amount of inorganic iodide has decreased, while at the same time two well-defined spots of Rose Bengal homologues (Rf 0. 84 and 0. 92) are visible (Fig. 3, Table II). The first is predominantly the di-iodo-, and the second a 136 GALATZEANU and COOK

TABLE II. ANALYSIS OF ROSE BENGAL AFTER 19-d STORAGE (Thin-layer chromatography on silica gel plates, cf. Fig. 3)

Contents of 13 !I (%) оГ о c~ 0 CO

Rf 0.6 - 0.7 1 1 Sample Rf 0. 8 - 0. 9 Rf 0.9 - 0.97 (I) i

A n. d. Traces 54 40 ( 0. 92)

В 2 (0 .6 2 ) n. d. 80 (0. 83) 1 9 ( 0 . 91)

С 1 0 (0 . 65) 25 (0. 73) 65 (0 .8 2 ) n. d.

D Traces 12 (0 .7 3 ) 88 (0. 84) n. d.

n. d. = not detected mixture of tri-iodo- and tetra-iodo-tetrachlorofluorescein. In the other cases, В, С and D, the same phenomena seems to take place but to a lesser extent. It was noticed that in all samples compounds not contairiing iodine were separated chromatographically from the radioáctive components and were visible under ultraviolet light. The fourth control after 28 d shows a continuation of the decomposition process, mainly with the formation of lower-iodinated Rose Bengal homo­ logues. Unlabelled decomposition products were also detected and increased in quantity as determined by the intensity by their fluorescence in ultra­ violet light.

II. THYROXINE-131I [2, 3]

Iodine-131 labelled thyroxine is used in the study of the metabolism and transport of endogenous thyroxine in humans and to determine the nature and amount of thyroxine-binding protein in plasma.

Experim ental

Samples were obtained from four of the principal producers (here noted А, В, E and F) and analysed periodically 1,. 7, 17, 26 and 33 d after receipt of the samples to follow self-decomposition and other change with time. The analyses were carried out by means of thin-layer chromato­ graphy, paper electrophoresis and gamma spectrometry. Thin-layer chromatography on silica gel plates was considered as the best method for separation, not only of free iodine released from radio­ active thyroxine but also of thyroxine derivatives in the solvent system: isopropanol - ethyl acetate - acetone - 6N ammonia = 35:30:25:20.

Results and discussion

The first control, 1 d after receipt, showed that the decomposition of samples A and E was less than that of the other two. F o r thyroxine A most of the organic iodine-131 (over 70%) was concentrated on the spot of IAEA-PL-336/12 137

A~ м Я ^ М И Ы ' Hiilli il li'il iHIHiimrir.I ...... ■«-......

FIG. 4. Thin-layer chromatograms of thyroxine-131I from suppliers A and E ld after receipt (measured by scanning).

Rf 0. 43 and for sample E on the spot of R f 0. 56 (75%), (see Fig. 4), both the spots are attributed to the thyroxine and triiodothyronine. There was a negligible concentration of diiodothyronine. For thyroxine В and F, however, clear indications of diiodothyronine in addition to thyroxine were obtained (Fig. 5). The second control showed that the amount of organic iodine on the spots of Rf 0. 85 - 0. 96 attributed to the derivatives of tyrolactic or tyroacetic acids was increased (Table III). With further storage the self-decomposition of thyroxine increases and leads to the formation of lower iodinated homologues of thyroxine like diiodothyronin (Rf 0. 56 - 0. 70) and triiodothyronine (Rf 0. 45 - 0. 55). Likewise small amounts of diiodotyrosine were also detected (Rf 0. 20 - 0. 38) (Fig. 6). Formation of inorganic iodine reached a maximum after about 9 d for samples В and about 17 d for samples A and F . Quantitative determination of the amounts of free iodine and other decomposition products for samples A and E is given in Table III. It is important to note that only laboratory F specified the expiration date for thyroxine-131I which was two weeks after its preparation. A possible reaction mechanism is as follows. During the first days of storage, the thyroxine undergoes de-iodination, probably from position 3' resulting in the formation of triiodothyronine and diiodothyronine. Parallel with the de-iodination, an oxidation of the alanine chain takes place leading to the de-amination and to the formation tri- and diiodo­ thyronine derivatives of lactic acid. At the same time an oxidation of the iodide ion to iodine takes place and the latter re-iodinates the lower iodo- thyronines. 138 GALAT ZEANU and COOK

FIG. 5. Thin-layer chromatograms of thyroxine-131I from suppliers В and F id after receipt (measured by scanning).

Effects of gamma radiation on the 131I-labelled thyroxine and derivatives

During the storage of the 131I-labelled thyroxine of high specific activity, it is important to determine the release of free iodine-131 as a result of self-decomposition. For this purpose a sample of low.specific activity was irradiated with a 60Co source at the doses of 0. 5 and 1 Mrad. The radiolysis products were separated by means of thin-layer and paper chromatography. Some decomposition products of thyroxine and its derivatives irradiated in 50% propylene glycol were determined by measurement of absorption spectra in visible and ultra-violet light. IAEA-PL-336/12 139

FIG. 6. Thin-layer chromatograms of thyroxine-131I from suppliers A and E 14 and 9 d respectively after receipt (measured by scanning).

Gamma irradiation causes more decomposition of tyrosine, diiodo­ tyrosine, thyronine, diiodothyronine, triiodothyronine and thyroxine in aqueous solution than in 50% propylene glycol solution. Irradiation of thyroxine and derivatives leads to the formation mainly of hydroxylated compounds, e.g. 3, 4-dihydroxyphenylalanine (DOPA), hydroxythyronines and hydroxythyroxine. At high doses of irradiation the thyroxine splits into two parts: a phenolic component and a derivative of phenylpyruvic acid. Table IV lists the quantitative data (in DOPA equivalents) of hydroxylation products determined by a specific reaction with a mixture of NaNÜ2 -NaMo04 (1:1) followed by spectrophotometrical determination of colour intensity at 510 nm. Another aspect of simulated radiodecomposition of high specific activity thyroxine which was studied, was the formation of carbonylated compounds. Determination of concentration of carbonyl groups in irradiated thyroxine and derivatives showed that the process of carbonylation increases with the applied dose of irradiation from 0. 1 to 1. 5 Mrad (Table V). The chromatographic behaviour of the carbonylated compound indicated that it was probably a derivative of phenylpyruvic acid. Concerning the stability of iodine-131 labelled thyroxine, it was determined that gamma irradiation at a dose of 0. 5 Mrad reduces the contents of organic iodine to approximately 50% (Fig. 7). Further irradi­ ation of thyroxine indicated a complete de-iodination at the dose 1-1. 2 Mrad. It was observed that the main process of radio-decomposition of iodine-131 labelled thyroxine during the gamma irradiation is the hydroxy­ lation of the benzene ring by substitution of iodine atoms with OH groups. A parallel process with the hydroxylation and de-iodination of thyroxine is an oxidation of the alanine chain leading to the formation of carbonylated compounds, analogues of phenylpyruvic acid. 140

TABLE III. RADIOCHEMICAL PURITY DATA DETERMINED BY THIN-LAYER CHROMATOGRAPHY ON SILICA GEL PLA TES [ solvent - isopropanol: ethyl acetate: acetone: 6N ammonia] (35:30:25:20)

Other Thyroxine Analogues T a il of T otal of Days organic Iodide and triiodo­ o f thyror organic organic

Sam ple • after radioactive GALATZEANU and COOK (1o) thyronine acetic acid iodine iodine receipt compounds (°!o) № do) (%) (°lo)

1 4 70 11 14 i 96

14 13 55 12 17 3 87 A 21 18 57 14 10 1 82

27 19 46 18 6 11 81

1 3 75 8 12 ' 2 97

9 11 60 15 13 1 89 В 16 17 58 11 13 1 83

23 17 57 12 13 1 83 TABLE IV. DETERMINATION OF HYDROXYLATION PRODUCTS OF IRRADIATED THYROXINE AND DERIVATIVES IN AQUEOUS SOLUTION

Tyrosine Thyronine Diiodothyronine Triiodothyronine Thyroxine

Optical Optical O ptical O ptical O ptical density (ig/m l+ density /ig/mla density /ig/mla density fig/ml a density |ig/mla at 510 nm at 510 nm at 510 nm at 510 nm at 510 nm AAP-3/2 1 4 1 IAEA-PL-336/12 0. 00 0. 010 - 0. 015 - 0. 050 0. 040 - 0. 050 -

0 ,2 5 0. 060 7 0. 055 13 0. 065 15 0 .1 0 5 26 0. 055 13

0. 50 ■ 0. 060 14 0. 075 19 0. 085 ’ 21 0 .1 1 5 29 0.1 3 5 38

1. 00 0. 080 20 0.085 21 0 .1 0 0 25 0 .1 6 5 41 0 .2 3 0 57 ■

2. 00 0.1 5 5 39 ' 0.1 6 5 41 0 .1 7 5 44 0 .2 5 5 64 0. 350 87

pH 10. 4 10. 6 1 0 .8 1 0 .5 10. 8

C oncen- tration 0. 01 0. 01 0. 01 0. 01 0. 01 M

a Data in equivalents of DOPA 142

TABLE V. SPECTROPHOTOMETRIC DETERMINATION OF CARBONYL GROUP CONCENTRATION4 IN IRRADIATED THYROXINE AND DERIVATIVES (In solution of 50% propylene glycol, at 480 nm. Spectrophotometry of 2, 4-dinitrophenylhydrazone)

D o s e (Mrad) - pH

substance (mg/ml) 0. 00 0 .2 5 0 .5 0 1. 00 1. 50 2. 00 COOK andGALATZEANU

Tyrosine 1 ' 2 .2 1 6 .2 6. 9 7. 6 8 .2 8 .1

Diiodotyrosine 1 4 .9 . 7. 0 7 .5 8 .2 9 .2 10. 0

Thyronine 1 1 .9 1 1 .7 1. 9 2 .1 2 .3 2 .2

Diiodothyronine 1 1 .9 1 10. 0 3 .5 6. 5 5. 5 -

Triiodothyronine 1 1. 8 1 4. 7 5. 0 - 5 .9 -

Thyroxine 1 1. 9 1 4. 7 5 .8 - 6. 0 -

Propylene ^ 50% - 0. 00 0 .2 7 0. 36 0 .4 0 0 .4 1 0 .4 3 glycol

a Calculated in rapport to the concentration of carbonyl group in the поп-irradiated substance, considered as 1 k Optical density IAEA-PL-336/12 143

A 0,5Mrad A lOMrad

¿,6 6 7

FIG. 7. Thin-layer chromatograms of irradiated 0. 5 and 1. 0 Mrad thyroxine-131I (aluminium oxide plates developed with solvent: n-butanol saturated with 6N-ammonia).

III. HIPPURAN-131!

- CO - NH - CH2 - COO" Na+

131J

Solutions of sodium o-iodohippurate-131I (hippuran) and o-iodohippuric acid-131! are widely used in medicine as a radioisotopic diagnostic agent in kidney function studies. It is well known that if' a fraction of the free radioactive iodine is present in inorganic forms such as elemental iodine or iodide, the useful­ ness of hippuran in the study of the renal function is severely limited because of significant changes in its clearance values when free iodine is present. The effect of free iodine on the variation of clearance value of the o-iodohippurate-131I was demonstrated by Bianchi and co-workers [3] and shows that inorganic iodine in an amount of 5% of the total activity causes a decrease of 49% in the clearance values. In this case it is very difficult or impossible to make a distinction between normal and pathological cases.

Experimental

Four commercial samples of hippuran or o-iodohippuric acid labelled with iodine-131 were obtained from four main producers in Europe, here noted in chronological order G, H, В and C. The samples of o-iodohippuran-131I, were stored in the dark at room temperature which varied from 20 to 22°C and were periodically analysed 1, 6, 12, 19 and 28 d after receipt. Activity ______L ______Act!vity FIG. 9. Paper electrophoresis o f hippuran-131! from the suppliers suppliers the from hippuran-131! f o electrophoresis Paper 9. FIG. В n С d fe receipt. after d 1 С and IAEA-PL-336/12 145

Thin-layer chromatography, electrophoresis and- autoradiography were chosen as the methods for control of radiochemical purity, since preliminary tests had demonstrated that paper chromatography may induce de-iodination of the hippuran-131I. For thin-layer chromatography on silica gel plates the best results were obtained with the solvent system: n-butanol saturated with IN acetic acid, and for paper electrophoresis with the buffer consisting from a 0. 025M Na2HP04 solution at pH 7. 8 and during 1 h at 250 V. The radionuclidic purity was checked by gamma spectrometry. The position of the spots was detected for radioactive hippuran by autoradi­ ography and with a solution of 1% of p-dimethylaminobenzaldehyde in acetic anhydride for the non-radioactive compound by preliminary tests. Determination of the activity of the radioactive spots was made using a thin-layer and paper chromatographic scanner.

Results and discussion

The stability of hippuran-131I solutions is much higher than that of thyroxine-131I, although both compounds have the iodine-131 bonded to the benzene ring. Particularly the higher radiostability of hippuran-131I may be due to its chemical structure which has a higher capacity to accept free radicals resulted from autoradiolysis of aqueous solution. The main factors influencing the release of free iodine during the storage of hippuran-131I solutions'are the specific activity and the radio­ active concentration. The results of the first electrophoretic'control listed in Table VI and illustrated in Figs 8-9, indicate that the hippuran-131I from producer С which initially had had in comparison with the other three samples (G, H and B) the highest radioactive concentration (1 mCi/0. 31 ml) is more decomposed. Thin-layer chromatography data indicate similar values of released free iodine and are not tabulated here. The second control 6 d after receipt showed a small increase of free iodine in samples G, H and В and a greater increase in sample С (Table VI and Figs 10-11). While the third control for the samples G and H indicated a constant release of inorganic io’dine-131, the samples В and С showed an increase of free iodine-131. This fact can be explained taking into account the great influence of the radioactive concentration on the stability of the iodine-131 labelled hippuran which in the case of samples В and С is higher than for other two (Table VI and Figs 12-13). The fourth and fifth controls (19 and 28 days, respectively after receipt) indicated for sample G and H a constant or diminished amount of free iodine while the samples В and especially С contained again higher amounts of free iodine. It is possible that the stabilizer (0. 2% sodium citrate) is responsible for the small contamination of hippuran with free iodine-131. It was found that the presence of appreciable concentrations of sodium chloride in sodium o-iodohippurate solutions labelled with iodine-131 appears to protect them partially from decomposition. As mentioned above and also in previous papers [4, 5] a specific activity of 0. 1 mCi/mg and lower did not show significant changes in the stability of the hippuran-131I; thus, it is very important to estimate the self-decomposition of this radiopharmaceutical by high specific activity. 146 GALATZEANU and COOK

TABLE VI. DISTRIBUTION OF THE ACTIVITY OF HIPPURAN-131I BY ELECTROPHORESIS MEASUREMENTS

Storage Unknown Hippuran-131I Producer Ï -°Jo (d) compound (%) (°ío)

1 1 - 99

6 2 - 98

G 12 Í - ‘ 99

19 2 - 98

28 n. d. - 100

1 1 99

6 2 - 98

H 12 2 2 96

19 2 3 95

28 2 4 94

1 1 - 99

6 2 2 96

В 12 5 2 93

19 3 1 96

‘ 28 2 ' 98

1 2 2 96

6 4 3 93

с 12 6 4 90

19 6 - 94

28 5 - 95

Irradiated Hippuran-131! Dose (Mrad)

0. 0 1 - 99

0 .2 5 2 93

G 0 .5 10 2 88

1. 0 13 2 85

1. 5 14 - 86

For this purpose different samples of low specific activity of the hippuran were irradiated at a dose of 0. 2, .0. 5, 1 and 1. 5 Mrad in a gamma source of cobalt-60, and then analysed by thin-layer chromatography and electro­ phoresis. The data on the free iodine-131 release from the irradiated hippuran-131! (Table VI and Figs 14-15) indicate that gamma radiation IAEA-PL-336/12 147

FIG. 10. Paper electrophoresis of hippuran-131! from the suppliers G and H 6 d after receipt.

Hipp BE

FIG. 11. Paper electrophoresis of hippuran-131! from the suppliers В and С 6 d after receipt. 148 GALATZEANU and COOK

FIG. 12. Paper electrophoresis of hippuran-131! from the suppliers G and H 12 d after receipt.

Hipp ВШ

J

F1G.13. Paper electrophoresis of hippuran-m I from the suppliers В and С 12 d after receipt.

i Activity Activity I. 4 Ppr lcrpoei ofhpua-3! raitd 2 ad 4 Ma, respectively. Mrad, .4 0 and .2 0 irradiated hippuran-131! f o electrophoresis Paper 14. FIG. I.1. ae eetohrss iprn11 irdae 1. n 1 Ma, respectively. Mrad, 5 1. and .0 1 irradiated hippuran-131! f o electrophoresis Paper 15. FIG. IAEA -PL-336/12 D В В 7 is /i 149 150 GALATZEANU and COOK causes a radiolysis of the carbon-iodine bond in aqueous media. The effect of the ionizing radiation on splitting the carbon-iodine link in radioiodinated compounds can be diminished by using various common scavengers and stabilizers.

IV. RADIO-IODINATED HUMAN SERUM ALBUMIN

Radio-iodinated-131I and -125I human serum albumin are widely accepted as a useful tracer in the estimation of blood and plasma volumes in humans. It has also been employed in localization studies of hepatic, pulmonary and cerebral tumours. Commercially the radio-iodinated human serum can be'obtained in the form of a crystalline ppwder or a sterile, buffered (pH = 7-8.5) isotonic solution which is clear and colourless. It can also be delivered in the form of sterile, isotonic suspension of macroaggregates with a pH = 5, used for scintigraphy of pulmonary infarcts.

Experimental

Samples of radio-iodinated-131I human serum albumin were obtained from the suppliers noted here as G, H, В and K. Analyses were carried out 1, 7, 14, 21 and 28 d after receipt using ascending thin-layer chroma­ tography on silica gel plates (solvent: isopropanol - ethyl acetate - acetone - 6N ammonia = 35:30:25:20) and paper electrophoresis (veronal buffer pH = 8. 6, 200 V during 1 h). Autoradiography, using Kodirex X-ray films, was used to localize the radioactive spots. Quantitative estimates were made by scanning of the plates' and paper strips, followed by inte­ gration of the recorded radioactive peaks. To determine the presence of any gamma radioactive impurities, the samples of radioiodinated human serum albumin were controlled by gamma spectrometry.

Results and discussion

Serum albumin is the major component (55-60%) of the proteinic complex in the human serum. ( Generally, human serum albumin contains about 560 amino-acid residues disposed in a single chain having a helicoidal structure. From these amino acids, 18 are tryosyl groups which may be substituted by radioiodine (131I or 125I) on iodination, leading to the formation of diiodotyrosine mainly. Therefore, the maximum degree of iodination can be 36 iodine atoms per molecule of albumin. Though in some cases this theoretical degree of iodination is approached, the usual commercial products contain less than one atom of radioiodine per molecule of albumin. The work of a number of investigators had demonstrated that the biological behaviour of a plasma protein may be altered during the purifi­ cation,- iodination, thermal sterilization and self-irradiation. Therefore, it is very important to check the preparations of radio-iodinated human serum albumin for radiochemical and radionuclidic purity. In this case although it has a complex structure, it is usually considered that the main impurity in radio-iodinated human serum albumin is the inorganic radio­ iodide. It is very important to have a quick, accurate and simple method IAEA-PL-336/12 151 for its detection and quantitative determination. It was found that thin- layer chromatography and paper electrophoresis are satisfactory for this purpose.

1. Thin-layer chromatography

The first control was made one day after receipt of the samples in the laboratory. The results listed in Table VII have shown that the sample В contains a somewhat larger amount of freé inorganic iodine than the other three. As it was mentioned in a previou chapter, high specific activity and high radioactive concentration of iodine-131 labelled radio­ pharmaceuticals lead to an increase of self-decomposition of such com­ pounds. The high content of inorganic radio-iodide in sample B, which had a higher radioactive concentration than the other three samples, can be explained from this point of view. The second and third controls, 7 and 14 d respectively after receipt indicated an increase of the free iodine-131 content (Table VII). The fourth and fifth controls marked also an increase of de-iodination compara­ tively to the other controls of serum albumin (Table VII). After four weeks of storage at room temperature no splitting of human serum albumin was observed during the chromatographical development. The solvent system used gave a good separation of inorganic iodide (Rf = 0. 7 - 0. 8) • from the radio-iodinated human serum albumin which remains on the start line.

2. Paper electrophoresis

The usefulness of electrophoretic techniques for the separation of proteins is well established. A veronal buffer allowed a good separation of free inorganic iodine-131 which migrated. 7. 5 cm whereas serum albumin migrated only 1.5-2 cm. It is interesting to note that the percentages of free iodine listed in Table VII by scanning of electrophoretic paper strips for all samples are higher than those obtained by thin-layer chromatography. In order to determine the possible influence of the buffer type, experiments were carried out using phosphate (pH = 7. 8) and borate (pH = 8. 7) media, but identical results were obtained as with veronal.

TABLE VII. RADIOCHEMICAL PURITY OF RADIO-IODINATED HUMAN SERUM ALBUMIN

G H В К Days of storage Free iodine-lSl^o Free iodine-lSl^o Free iodine-lSl^o Free iodine-lSl^o TLC EF TLC EF TLC EF TLC EF

1 0 .5 traces 2. 0 2 .5 2 .5 3. 0 1 .0 1 .5

7 2 .0 3 .0 2 .5 3. 5 3 .0 5 .0 2 .0 3. o-

14 2 .0 3 .0 2 .5 3. 0 3 .5 5 .0 3 .0 3 .0

21 1 .5 2 .5 3. 0 5 .0 3 .5 5 .5 3 .0 4 .0

28 2 .5 3 .5 3. 0 4 .5 3. 0 5 .5 3. 0 4 .5 152 GALATZEANU and COOK

It is possible that the higher percentages for inorganic iodine in this case are due to the partial de-iodination of the radio-iodinated human serum albumin during the paper electrophoresis, although the serum albumin migrated in a single spot and no splitting was observed.

REFERENCES

[1] BAYLY, R .J., WEIGEL, H., Nature (London) 188 (1960) 384. [2 ] COOK, G. B ., GALATZEANU, I . , " Study of the self-decom position of 131I-lab elled Rose Bengal and thyroxine", Paper presented at Radiochemical Conf. , Bratislava (CSSR), 6-10 Sept. 1966. [3] COOK, G. B. , GALATZEANU, I., "Contrôle de la pureté radiochimique du Rose Bengal et de la Thyroxine marqués a riode-131”, Paper presented at 2nd Int, Conf. Methods of Preparing and Storing Labelled Compounds", EURATOM, Brussels, 28 Nov. - 3 Dec. 1966. [4 ] BIANCHI, G., HEGESIPPE, E ., MEOZZI, A ., ROSA, U ., SOSI, S ., Minerva Nucl. 9 (1965) 152. [5 ] ANGHILERI, L .J ., J. nucí.M ed. 4 (1963) 155. IAEA-PL-336/13

RADIOCHEMICAL PURITY AND STABILITY OF SOME RADIOPHARMACEUTICALS

J. CÍFKA Nuclear Research Institute of the Czechoslovak Academy of Sciences, Rez, ÍS S R

Abstract

RADIOCHEMICAL PURITY AND STABILITY OF SOME RADIOPHARMACEUTICALS. The stabilities of the sodium salts of o-iodo-hippuric acid, diatrizoate, diiodofluorescein, Rose Bengal and oleic acid labelled with 131I, as well as of radioactive chlormerodrm, colloidal radiogold, sodium phos­ phate and chromate, were investigated by various methods for determining the radiochemical purity.

1. INTRODUCTION

This paper deals with some results obtained during recent years in the Czechoslovak State Institute for Control of Drugs with the partial collabo­ ration of the Nuclear Research Institute of Czechoslovak Academy of Sciences. The work on control of radiopharmaceuticals commenced in 1963 and, in addition to the routine control of com m ercial products, deals with the problems of purity and stability of some radiopharmaceuticals. The major effort has been concentrated on the radiochemical purity and factors influencing it. Some work was done in connection with preparation of monographs [1 -6] for the Third Edition of Czechoslovak Pharmacopoeia, which is now in press. Other papers here have discussed the control of radiopharmaceuticals in production centres. I should like to point out that the State Institute for Control of Drugs is subordinate only to the Ministry of Health and is inde­ pendent of any producer of drugs including radioactive pharmaceuticals. This means, first, that the radiopharmaceuticals have been analysed chiefly from the u ser's point of view and, second, that it is not only com­ m ercial products that have been used to solve the problems.

2. ACCELERATED DECOMPOSITION EXPERIMENTS

The stability of a given radiopharmaceutical can be studied either by the estimate of an over-all decomposition under stated conditions of storage, or by estimating the respective contributions of temperature, light, radiation, etc., simultaneously. The latter method is based on the simplifying assumption that the effects of all processes are additive; however, by this method it is possible to study the thermal and radiation stability by means of so-called accelerated experiments. The method of accelerated experiments is well known and often used to study the stability of non-radioactive pharmaceuticals [7-9]. In the evaluation of thermal

153 5 CÍFKA 154

TABLE I. ABSORBED ENERGY (in 1017 eV/ml) IN SOLUTIONS HAVING THE INITIAL RADIOACTIVE CONCENTRATION OF 1 mCi/ml; . (The values refer to the geometrical unit)

Days of storage Radioisotope 1 2 4 8 12 16 20 24 28 40 60

32 p 1 9 .5 3 7 .8 7 2 .4 132 181 221 - - -

125 i 0 .6 4 - 2 .5 4 .9 7 .2 9 .4 1 1 .4 1 3 .4 1 5 .3 2 0 .5 2 7 .8

131 J 5 .7 1 1 .0 2 0 .2 3 4.6 4 4 .7 5 1 .9 5 6 .9 6 0 .5 6 3 .0 -

1£"H g 2 .2 5 3 .9 9 6 .3 7 8 .6 5 9 .4 6 9 .7 8 - - - ‘ - -

203Hg 3 .2 5 6 .5 1 1 2 .4 2 4.1 3 5 .2 4 5 .6 5 5 .6 6 4 .7 7 3 .4 9 6 .6 127 IAEA-PL-336/13 155 stability this method consists of estimating the decomposition rate at elevated temperatures followed by extrapolation to the usual storage temperature. j In the study of the self-radiation decomposition, the effect of internal ionizing radiation is simulated by an irradiation of the sample from the external ionizing radiation source. Most radiopharmaceuticals are supplied in aqueous solution. There­ fore it seems reasonable to, distinguish two stages of radiation decomposition [10, 11]. A. The radioactive isotope disintegrates independently of its chemical state. The emitted ionizing radiation is partially absorbed in the solution. The total energy absorbed in the solution in a certain time interval depends on the radioactive concentration (mCi/ml), energy and type of the radiation emitted, total volume and geometrical shape of the solution. These pro­ cesses are common for all radiopharmaceuticals containing the same ■ radioactive isotope. ; B. The solution of the ^radiopharmaceutical is decomposed owing to the action of ionizing radiation. The extent of decomposition of the given radiopharmaceutical is a function of total absorbed energy (radiation dose) and the radiation yield of decomposition. (G(_M) ' value). The radiation yield of decomposition depends in general, on the concentration of the radio­ pharmaceutical, on the presence of other-substances (including dissolved oxygen), on the intensity of radiation (dose-rate), and in some cases on the linear energy transfer (LET). Only the last two dependences mentioned in paragraph В can limit the applicability of the method of accelerated experiments. Fortunately, in the case of dilute aqueous solutions of radiopharmaceuticals, the action of radiation on the dissolved compounds can be regarded as indirect, caused by radicals and other species created by radiation from water. In the interval of L E T from 0.01 to 0.2 eV/A, which is characteristic for the most beta or beta-gamma em itters (with the exception of tritium), the changes in radiation yields of these radicals do not exceed 25% of their value [12]. The remaining limitation, i.e. the dependence or independence of the radiation decomposition yield on the dose-rate is to be experimentally proved, because the dose-rate during the self-decomposition is often lower by one or two. orders of magnitude than those used in external irradiation. The accelerated radiation experiments were carried out as follows. The solution of a given radiopharmaceutical was diluted to a radioactive concentration of 5,- 30 /uCi/ml; then the decomposition effect of its own radiation could be neglected and the radioactivity served only for the ana­ lytical purposes. Water, solutions of non-radioactive carriers, or solutions of various additives were used for dilution. The solutions were then irradiated with cobalt :60Co gamma-rays under well-defined conditions, and analysed by means of-paper chromatography. Thus, the decomposition yields and the formation yièlds of the newly created compounds (if labelled by radioactive isotopes) were obtained as functions of concentration, additives,' dose-rate etc. In the first part of these studies, the radiation energy absorbed in solutions of phosphorus-32, iodine-125 and -131, mercury-197 and -203 was calculated and tabulated (Table I) for various storage times [11]. The influence of the geometrical shape of solutions was also evaluated. A cylinder 2 cm in diameter and 1 cm high was chosen as the geometrical 156 CÍFKA

unit because most radiopharmaceuticals are packed in standardized multi­ dose glass vials, the diameter of which is approximately 2 cm. The cal­ culations were verified by means of chemical dosimeter containing iodine-131 and found reliable.

3. REVIEW OF RESULTS OBTAINED FOR INDIVIDUAL RADIOPHARMACEUTICALS

Sodium iodohippurate [13]

Several chromatographic solvents were compared [14-16]. The best results were obtained with the USP solvent (benzene-acetic acid-water 2 :2 :1 ) , which made the separation of inorganic iodine (Rf 0), iodohippurate (Rf 0. 3), iodobenzoate (Rf 0. 90) and one still unidentified compound (Rf value 0.8, content comparable with iodobenzoate, i.e. 0.3-1.0% of total activity) possible. It was found that the acetone-n-butanol-water ( 5 :5 :1 ) mixture [17] gave erroneous results; the separation was caused by over­ loading of the paper.

FIG .l. The radiation decomposition of iodohippurate as a function of absorbed energy dose. The concen­ trations of iodohippurate were as follows: (a) 4 .2 x 10"4M, (b) 1.4X 10-4 M, ( c ) 3 . 4 x 10-3 M, (d) 1 .0 4 X 1 0 " ZM, (e) 3 . 0 x 10"2 M. — _ _

Before the application of the analysed solution, 0.01 ml of 0.05N solu­ tion of sodium thiosulphate was applied on the starting line to prevent the loss of inorganic iodine, which could be present partially in elemental form. This procedure can be recommended for all iodine compounds'. The thermal stability of iodohippurate-1311 was studied for 1. 5 X 10 "2 M to 3. 2 X 10'4 M solutions. The light increase in inorganic iodine was observed only for the lowest concentration stored at 37°С for time longer than one week. Autoclaving at 120°С repeated five times was without any effect for 1. 5 X 10-2 WI solutions; an increase of inorganic iodine up to 3.7% was observed for 3. 2 X 10"4 M solutions. Inorganic iodine was' found to be the single product of radiolysis. The radiation decomposition of iodohippurate was measured for various chemical concentrations and various radiation doses of external gamma- rays. By plotting the logarithm of remaining iodohippurate versus radia­ tion dose a set of straight lines was Obtained (Fig. 1). From these values the initial radiation yield of decomposition of iodohippurate (G(_M)) was calculated (Fig. 2). IAEA-PL-336/13 157

MOLARITY

FIG.2. The initial radiation decomposition yield of iodohippurate as a function of concentration.

FIG.3. The percentage of inorganic iodine formed as a function of the concentration of added benzylalcohol. The concentration of iodohippurate was 4 .0 x l0 "4M, the absorbed energy dose was 1 .72x l018 eV/ml.

Benzyl alcohol had a protective effect on the iodohippurate during radiolysis. Figure 3 shows the decreasing percentage of inorganic iodine with increasing concentration of benzyl, alcohol; all samples were exposed to the same radiation dose 1. 72 X 1018 eV. The interpolated values of G(_m) were used for the calculation of the decomposition of iodohippurate at various concentrations and doses. The results are presented in Fig. 4. The arrow shows the value of absorbed energy in solution of iodine-131 with the initial radioactive concentration 1 mCi/ml during 24-d storage in a vial (6.05X 1018 eV/ml). The values presented in Fig. 4 agree with values obtained for stored labelled iodo­ hippurate- !3il by Kato et al. [18]. The values in Fig. 4 aré the maximum values; in the presence of preservatives like benzyl alcohol a lowered decomposition is observed.

Sodium diatrizoate [19]

The paper chromatography of sodium 3, 5-diacetylamino-2, 4, 6-tri- iodobenzoate was carried out in the mixture n-butanol-pyridine-water- (10:3:3). The R f values of diatrizoate and iodide are 0.13 and 0.39 respec­ tively, the radioactive spots (Rf 0.03 and 0.8) of decomposition products have not yet been identified. Indirect evidence shows that the first product of decomposition, the derivative of diiodobenzoic acid, is not separated in this chromatographic solvent from the spot of diatrizoate. 158 CÍFKA

FIG.4, The calculated radiation decomposition for various concentrations (mg/ml) of iodohippurate as a function of absorbed energy.

FIG.5. The radiation decomposition of diatrizoate and formation of decomposition products as a function of absorbed energy dose, (a) Diatrizoate, (b) inorganic iodine, (c) unidentified compound, Rf0.8, (d) unidentified compound, Rf0.03. The initial concentration of diatrizoate was 3.25 x 10"3M.

The thermal stability of neutral solutions of diatrizoate is very 'good, and the percentage of inorganic iodine is lower compared with iodohippurate. The formation of radiation decomposition products and decrease in the diatrizoate content during gamma-ray irradiation is demonstrated in Fig. 5. The decrease in diatrizoate concentration was not linear even in the semi- logarithmic plot. The formation of inorganic iodine is not completely balanced by the activity in the spots with Rf 0. 03 and 0. 8; this can be con­ sidered as further evidence that, in the spot of the diatrizoate, other less iodinated compounds may be present. The initial radiation yield of the decomposition of diatrizoate as a function of concentration is presented in F ig .6. Figure 7 shows the calculated decomposition curves of diatrizoate as functions of absorbed energy for various concentrations of diatrizoate. The formation of other iodinated compounds during storage of diatrizoate supported the opinion that the percentage of activity in the inorganic form gives only a very rough indication of the decomposition of iodinated substances. IAEA-PL-336/13 159

MOLARITY FIG. 6. The initial radiation decomposition yield of diatrizoate as a function of concentration.

FIG.7. The calculated radiation decomposition for various concentrations (mg/ml) of diatrizoate as a function of the absorbed energy. '

Sodium diiodofluorescein [20]

The phosphate buffer of pH 7. 2 was used as the solvent for the paper chromatographic separation of the decomposition products. Table II shows the Rf values and peaks of optical absorption of individual spots including the two unidentified compounds. The triiodofluorescein remained at the start point; better resolution of di- and triiodofluorescein was obtained by repeated development of thé chromatogram (after counting the activity) with the same solvent.

TABLE II. Rf VALUES AND PEAKS OF OPTICAL ABSORPTION (IN BORATE BUFFER OF pH 9. 0) OF COMPOUNDS FOUND IN.GAMMA- IRRADIATED SOLUTION OF DIIODOFLUORESCEIN

'Чпах Rf Compound Observed Theor.

0 + 508 -5 1 5 517 (Triiodofluorescein)

0 .1 2 + 508 508 Diiodofluorescein

0 .3 3 ■ + 497 498 Monoiodo fluorescein

0 .5 0 0 490 490 Fluorescein

0 .5 5 - 0 .7 0 + 498 -5 0 3 - Unidentified, I

0 .7 5 - 0 .9 2 + 488 - Unidentified, II

0 .8 5 + - - Iodide 160 CÍFKA

FIG. 8. The radiation decomposition of diiodofluorescein and formation of decomposition products as a function of absorbed energy dose, (a) Inorganic iodine, (b) mono-, (c) di-, (d) tri-and tetraiodo- fluorescein, Ce) unidentified decomposition products. The initial concentration of diiodofluorescein was 3X10"3M.

FIG. 9. The initial radiation decomposition yields of (a) diiodofluorescein, (b) formation of inorganic iodine and (c) monoiodofluorescein as functions of the concentration of diiodofluorescein.

The decomposition of diiodofluorescein during autoclaving increased with the decreasing pH value of the solution. The formation of triiodo- fluorescein was observed together with the creation of monoiodofluorescein and inorganic iodine. The decrease of diiodofluorescein and increase of individual decomposi­ tion products during the gam ma-ray irradiation is shown in Fig. 8. The initial radiation yields of decomposition of diiodofluorescein and formation of monoiodofluorescein and inorganic iodine as functions of the concentration of diiodofluorescein are presented in Fig. 9. The independence of radiation decomposition of diiodofluorescein on the dose-rate was found as well as the independence on the pH of the solution in the pH 6 -8 range (Fig. 10). The protective effect of benzylalcohol is demonstrated in Fig. 10. IAEA-PL-336/13 161

FIG.10. The radiation decomposition of diiodofluorescein as a function of absorbed energy. □ pH 6; + pH 7; О pH 8; Д solution contained 0 .9*7o of benzylalcohol.'

Z 100 UJ a ш C£ o 98 3 -J u_

03 06 1 15 2 3 12 15 И A V \ , \ \\W\N 0 1 2 3 lo'9 eV/ml

ш / ; / z аз ; об 1 15 2 Q О

О 3 ^ z s 6 - 8 ^ 12 f

lili 1 1 1 I

Ю19 e V /m l FIG .11. The calculated radiation decomposition of diiodofluorescein and formation of inorganic iodine as functions of absorbed energy. Individual initial concentrations of diiodofluorescein are expressed in mg/ml.

The decrease of diiodofluorescein and increáse of inorganic iodine as functions of absorbed radiation energy were calculated from the measured values for individual concentrations of diiodofluorescein (Fig. 11).

Rose Bengal [21, 22]

Sodium 2', 4', 5', 7'-tetraiodo-3, 4, 5, 6-tetrachlorofluorescein contains usually the less halogenated components which, according to several 162 CÍFKA

TABLE III. RELATIVE Rf VALUES (REFERRED TO THE TETRAIODO- TETRACHLOROFLUORESCEIN) OF INDIVIDUAL FLUORESCEINS IN THE CHLOROFORM-FORMIC ACID MIXTURE (87 : 13) ON SILICA GEL G AND THE PEAKS OF OPTICAL ABSORPTION IN BORATE BUFFER OF pH 9. 0 [22]

Fluorescein derivatives

Number of incorporated atoms Rf rel. ^m ax' nm

Iodine Chlorine

4 4 1 .0 0 549

3 4 0 .8 5 539

2 4 0 .7 0 529

1 4 0 .3 9 519

0 4 0 .1 5 5 0 9 .5

4 2 0 .9 4 5 4 4 .5

3 2 0 .77 535

2 2 0 .5 9 525

1 2 0 .2 8 515

0 2 0 .0 8 506

4 0 0 .8 5 527

3 0 0 .5 5 517

2 0 0 .2 0 508

1 0 0 .0 2 498

0 0 0 .0 0 490

authors [23, 24], have different excretion rates. Thin-layer chromatography proved to be the best method for separating all twelve possible radioactive components. The mixture of chloroform and formic acid (87 : 13) was used for most of the analyses on silicagel G. Very close or identical Rf values (or better relative values, referred to the tetraiodo-tetrachlorofluorescein as the substance with greatest Rf) were found for compounds with different content of halogens. The Rf value alone cannot therefore be sufficient to characterize impurity. The elution of the spot and determination of the peak of optical absorption (in borate buffer of pH 9.0) supplied the additional evidence for identification (TableIII). By changing the composition of developing mixture (i.e. the ratio chloroform-formic acid) the Rf values can be varied. Less acid mixtures resolve di-, tri- and tetraiodofluoresceins better, solvents containing higher concentrations of formic acid separate the non-iodinated fluoresceins and mono- and diiodofluoresceins more effectively. Í IAEA-PL-336/13 163 í It was found that the mobility and separation of individual spots was influenced markedly by the'concentration of acid vapours in the gas phase, and hence by saturating the silica gel layer by this acid vapour before the development of the chromatogram. Drying the prepared plates at 130° С before applying the sample, and especially the good saturation of the thin layer by the vapours of the solvent (15 min in a chamber previously well saturated with vapour of a mixture) before the development, proved to be very important to obtain good reproducibility of results.

Rt FIG.12. The activity distribution along chromatograms of Rose Bengal -131I from various producers. (a) Producer 1 (from Eastern Europe) in 1965; (b) The same producer in 1967; (c) Producer 2 (from Western Europe) in 1968; (d) Producer 3 (from Eastern Europe) in 1969. (A) T etraiodo-, (B) triiod o-, (C) diiodotetra- chlorofluorescein, (D) unidentified red compound containing iodine.

As an example, the radioàctivity distribution along chromatogram of sodium Rose Bengal-131I from various producers is shown in Fig. 12. According to the experience of the State Institute for Control of Drugs, the quality of Rose Bengal has gradually improved during the last years. Today it is quite possible to obtain (at least in Europe) the limit of 7 5% of the total activity in the tetraiodotetrachlorofluorescein spot and 90% in . spots of tri- and tetraiodotetrachlorofluorescein. The above-mentioned TLC method has also been used for the analyses of commercial products of labelled sodium diiodofluorescein.

Iodinated oils [25] !

Compounds of this group, i.e . iodinated oleic acid, olive oil and triolein labelled with iodine-131 o r ; 125I, were analysed not only for presence of free inorganic iodine but alfeo for the distribution of iodine among individual groups of organic compounds. Paper chromatography, on paper impregnated with paraffin oil was used to analyse labelled oleic acid. The mixture of acetic acid-water (90 :10) saturated with paraffin oil [26] proved to be best for separation. Differences were found'in products from various producers. Some examples are given 164 CÍFKA

Rf

FIG. 13. The activity distribution along chromatograms of iodinated oleic acid, (a) Oleic acid iodinated in our laboratory with iodine monochloride-1311; (b) 125I-labelled commercial product from Eastern Europe, 1967; (c) 1311-labelled commercial product from Western Europe, 1968.

in-Fig. 13. The main activity was found in the spot of oleic acid, (Rf 0. 24 - 0. 26). Other spots have not yet been identified exactly. The spots with Rf value higher than 0. 6 are probably not caused by a further impurity, but by a different method of iodination. The iodination is usually carried out with iodine monochloride giving 9-chloro-l 0-iodostearate as the main product. In the presence of water, iodo-hydroxy-stearate can be formed, the Rf of which, under slightly different conditions, is approximate­ ly 0. 9 [27]. The main question, whether or not the chloro-iodostearate has a different absorption rate in the organ than hydroxyiodostearate, remains unresolved. The free inorganic iodine content was determined either by means of paper electrophoresis or by extraction of the solution of iodinated acid in chloroform with an aqueous solution of thiosulphate. The values given by both methods were nearly identical and in all cases below 1% of total activity. Labelled oils and triolein were analysed by thin-layer chromatography on silicagel G, using the mixture of petroleum ether-ether-acetic acid (70 : 30 : 2) as the solvent [28]. The activity record of various products is shown in Fig. 14. It is interesting to observe that iodo-chlorination of olive oil gave a higher content of activity in triglyceride fraction than the iodochlorination of commercial triglyceride. The activity content in indi­ vidual fractions did -not depend on the degree of iodination; the same activity distribution was obtained with m aterial (both olive oil and triolein) iodinated with excess of iodine monochloride as well as with material iodinated to IAEA-PL-336/13 165 only 2% of theoretical capacity (Table IV). The percentage of activity in individual spots depends considerably on the method used for iodination as demonstrated for four various methods (see Table V).

FIG. 14. The activity distribution along chromatograms of iodinated triolein and olive oil. (a) Commercial triolein, and (b) commercial olive oil iodinated in our laboratory with iodine monochloride-1311 according to Wijs; (c) 1251-labelled triolein, commercial product from Eastern Europe, 1967; Cd) 1311-labelled triolein, commercial product from Western Europe, 1968.

TABLE IV. PERCENTAGE OF ACTIVITY IN INDIVIDUAL CHROMATO­ GRAPHIC SPOTS AS A FUNCTION OF THE DEGREE OF IODINATION WITH IODINE MONOCHLORIDE (WIJS METHOD)

Per cent of total iodine-131 activity Degree of . Iodinated in spots with: iodination compound of theory) Rf 0 .0 R f 0 .3 5 Rf 0 .8 5 Residuea

100 '■ 0 .5 9 .5 8 0 .5 9 .5 Triolein 3 2 .5 (1 0 .0 ) 8 0 .5 (7 .0 У

100 5 .5 3 1 .0 5 2 .5 1 1 .0

2 4 .5 -28.0 5 3 .0 14.5

a The activity between the spots ! 166 CÍFKA

TABLE V. PERCENTAGE OF ACTIVITY IN INDIVIDUAL CHROMATO­ GRAPHIC SPOTS AS A FUNCTION OF THE METHOD USED FOR IODINATION OF TRIOLEIN

Per cent of total iodine-131 activity System used for in spots with: Reference iodination Rf 0 .0 3 Rf 0 .2 2 Rf 0 .3 7 Rf 0 .8 0 Residue3

Iodine monochloride [29] 7 26 14 40 13 in ether

Iodine monochlorideO) in water-ethanol- [30] 7 30 12 37 14 chloroform mixture

Elemental iodine in water-ethanol [2 7 ,3 1 ] . 16 20 26 22 16b mixture

Iodine monochloride in chloroform-acetic [32] 6 25 7 46 16 acid mixture

a The activity between the spots. b Including the well-separated spot with Rf 0.12 and containing 11% of total activity.

The identification of the various spots is not simple. Paper chromato­ graphy with the solvent acetone-water 7 : 3 [33] was used for the separation of mono-, di- and triglycerides. The Rf values of these groups had to be 0.95, 0.4 and 0.0, resp ectively . No spots of diglyceride w ere found in this system for the above-mentioned labelled oils, so the thin-layer spot with Rf 0. 3 can hardly be regarded as diglyceride. The question of the comparison of in-vivo resorption of chloro-iodo and hydroxo-iodo triglyceride has also not yet been resolved.

Chlormerodrin [34, 35]

The chromatographic behaviour of chlormerodrin (3-chloromercuri- 2-methoxypropylurea, I) and of the expected radiochemical impurities was checked with several chromatographic solvents. In some solvents more than three spots were observed. I think that chlormerodrin can serve as a good example to show the complex nature of the problem of radiochemical purity.

Cl-Hg-CH2-CH-CH2-NH-CO-NH2

OCH3 chlormerodrin, I IAEA-PL-336/13 167

The main organic impurity was identified as 3-chloromercuri-2-hydroxy- propylurea, II.

Cl-Hg-CH2-CH-CH2-NH-CO-NH2

OH II

The Rf values of chlormerodrin and its impurities are presented in Table VI. All these test substances were applied either as solutions in pure water or in 0. 9% solution of sodium chloride, or as a solution containing equivalent amounts of theophylline, to evaluate the influence of these additives, which are often present in commercial products. The presence of these additives was the reason for the change in R f values and occurrence of double spots; a number of spots found in some chromatographic solvents and reported in the literature [41] could be explained in this way. The excess of sodium

FIG.15. The thermal decomposition of solutions of chlormerodrin. Chlormerodrin alone, heated at (a) 100°C, (b) 120°C. Chlormerodrin with theophylline in molar ratio 1: 1, heated at 120°C (c). The initial concentration of chlormerodrin was 10~2M. Í chloride or theophylline changed the Rf values completely, as is seen from the results of Table VII. The substitution of chlorine in chlormerodrin by an OH group or another anion can easily take place in solution, as fol­ lows from the simple preparation of various substances differing only in the nature of the anion. The migration of chlormerodrin as 3-hydroxy- mercuri-2-methoxypropylurea in an alkaline chromatographic solvent can therefore be expected. The addition of chloride ions to the solvent' contain­ ing a comparatively low concentration of hydroxide ions suppressed the above-mentioned substitution and chlormerodrin migrates unchanged as the 3-chlorom ercuri compound. Therefore it is recommended that only solvents without the above-mentioned effect (see the pyridine containing solvents in Table VI), are used, or an alkaline solvent with added sodium chloride. Descending paper chromatography and a mixture of 80 ml ethanol, 18. 5 ml water, 1. 5 ml çonc. ammonia and 1 g sodium-chloride is now used for the routine analyses. The 3-chloromercuri-2-hydroxy compound is formed in chlormerodrin not only during the synthesis but also during storage and autoclaving. The rate of transformation from1 2-methoxy to 2-hydroxy compound (Fig. 15) increased in the presence of theophylline and decreased in the presence of 6 CÍFKA 168

TABLE VI. Rf VALUES OF CHLOKMERODRIN (A), 3-CHLOROMERCURI-2-HYDROXYPROPYLUREA (B), AND MERCURIC IONS (C) IN SOME DESCENDING CHROMATOGRAPHIC SOLVENTS

Rf Front after Solvent Reference A В С (cm)

Ethanol-water-ammonia [36] 0 .4 0 a 0 .2 9 a 0 .1 a 2 9 - 3 0 6 : 2 : 1

Ethanol-1.5N ammonia [37] 0 .4 2 a 0 .2 8 a 0 - 0 . I a 3 0 - 3 3 8 :2

Ethanol-phosphate buffer 0 .7 7 0 .6 5 0 - 0 . 15a 2 6 - 2 7 [38] (pH 7.4 ) 0 . 74b 0 . 62b 0 - 0 . 15b 1 : 1

butanol-pyridine-water-ammonia [39] 0 .3 2 0 .2 0 0 .0 9 a 3 2 - 3 3 5 : 7 ; 1 : 3

butanol-pyridine-water [40] 0 .5 5 0 .3 7 0 .9 0 3 1 - 3 2 1 0 : 3 : 3

a Rf values were influenced by the composition of the analysed solution k Ascending chromatography IAEA-PL-336/13 169

TA BLE VII. CHANGE IN R f VALUES OF CHLORMERODRIN AND ITS RADIOCHEMICAL IMPURITIES DUE TO THE ADDITION OF COMPLEXING SUBSTANCE TO CHROMATOGRAPHIC SOLVENT ETHANOL-1. 5 N AMMONIA (8:2)

Rf Addition A В С

None 0 .4 2 0 .2 8 0 - 0 . 1

2 g of sodium chloride per 100 nil of solvent 0 .4 9 0 .3 7 0 .0

0.2 g of theophylline per 100 ml of solvent 0 .6 4 0 .5 0 0 .4 0

А, В, C - see Table VI.

sodium chloride. Theophylline influenced the transformation rate markedly even at room temperature (Table VIII). The formation of the 2-hydroxy compound can be explained by the following mechanism. Chlormerodrin is at first decomposed into the original compounds, i.e. allylurea, inorganic mercury and methanol:

Cl-Hg-CH2-CH-CH2-NH-CO-NH2 + H20->

OCH3

Cl-Hg-OH + CH2 = CH-CH2 - n h - c o - nh2 + CH3OH

This decomposition is accelerated by acids. The inorganic mercury can be again added to allylurea. Owing to the fact that this addition occurs in aqueous solution of components, the 2-hydroxy compound is formed:

Cl-Hg-OH ;+ CH2 = CH-CI^-NH-CO-NH^—»

Cl-HgT:CH2 -CH-CH2 -NH-CO-NH2

OH

The above-mentioned mechanism fully accords with the earlier isotopic exchange results [37]. The products of radiation decomposition of chlormerodrin were mostly mercuric ions and, to a lesser extent, 3-chloromercuri-2-hydroxypropylurea. A white opalescence or precipitate was observed in gamma-irradiated solutions of chlormerodriri. The curves representing the decomposition of chlormerodrin as func­ tions of absorbed dose consist of two parts; the results are presented in Fig. 16. The yields of radiation decomposition of chlormerodrin were cal­ culated for both stages of decomposition; the values are plotted in Fig. 17. The break on the curves has not been explained. It was found that this type of curve was unchanged by the presence or absence of oxygen and sodium chloride. On the other hand, in the presence of benzyl alcohol the initial 170 CÍFKA

TABLE VIII. AMOUNT OF 3-CHLOROMERCURI-2-HYDROXYPROPYL- UREA FOUND IN SOLUTIONS OF CHLORMERODRIN ON STORAGE IN DARKNESS AT ROOM TEM PERATURE

Activity found in The composition of Period • 2 - hyd roxy -compound the solution (d) • m

10"2 M chlormerodrin 3 0 .8

39 3 .0

10"2 M chlormerodrin 4 9 .2

and 1 0 "2 M theophyl­ 13 3 0 .2

line 39 5 5 .6

10 e V /m l

FIG.16. The radiation decomposition of chlormerodrin as a function of absorbed energy dose. The concen­ trations of chlormerodrin were as follows: (a) 3X10"4 M; (b) 1X10"3M; (c) 3xlO "3M; (d) 6xlO"3M; (e) lxlO -2 M; (f) 1Л 7х10-2 М . Chlormerodrin dissolved in water (+) or in 0. 9% solution of sodium chloride (O). The initial curves are shifted along the у-axis by steps amounting to 10%. yield of radiation decomposition was increased. The influence of theo­ phylline could not be quantitatively estimated owing to the formation of voluminous non-homogenizable precipitate. The influence of additives on the formation of 3-chloromercuri-2-hydroxypropylurea was observed; its formation was supported by the presence of the benzyl alcohol and hindered by the presence of sodium chloride. The radiation decomposition as a function of absorbed dose was cal­ culated for the individual concentrations of chlormerodrin using the initial yield of radiation decomposition; the results are shown in Fig. 18. IAEA-PL-336/13 171

MOLARITY FIG. 17. The radiation yield o f decomposition of chlormerodrin as a function o f concentration. (+) The initial radiation yield; (O) the radiation yield in the second stage.

FIG. 18. The calculated radiation decomposition of chlormerodrin as a function of the absorbed energy for various concentrations (expressed in m g/m l).

It should be emphasized that the significance of radiochemical purity of chlormerodrin is not merely theoretical. Herzmann et al. [42] have reported the higher uptake and slower excretion of the 2-hydroxy compound in kidney as compared with chlormerodrin.

Colloidal radio-gold [43]

Soluble, ionic gold (mostly in the form of chloroauric complex) is re­ garded as the radiochemical impurity in colloidal solutions of radio-gold. The presence of ionic gold is taken as the evidence of incomplete reduction during the production process. Paper chromatography or TLC is usually used to separate colloidal and ionic gold. The solvent acetone-water-hydrochloric acid (70 : 20 : 10) [44, 45] is the most widespread mixture in which the colloidal .particles should remain at the starting line, whilst ionic gold moves with the front. In further studies, it has been recommended that less acid solvent be used [46]. For the third Edition of the Czechoslovak Pharmacopoeia the use of a mixture of acetone - 0. IN hydrochloric acid 70 : 30 [6] was therefore recommended. In our studies we compared the behaviour of various colloidal solutions and also the solutions of cñloroauric ions in several chromatographic 172 CÍFKA

TABLE IX. THE RETENTION OF GOLD ON THE START LINE AS A FUNCTION OF THE APPLIED QUANTITY OF IONIC GOLD

Au (Mg) Chromatographic mixture Retained Au Applied Retained №)

Acetone-water-hydrochloric acid 0 .0 1 0.0073 73 7 0 : 2 0 :1 0 0 .2 0 .0 9 8 49

1 .0 0 .2 0 20

1 0 .0 0 .2 0 2

Acetone-O.IN hydrochloric acid 0 .0 1 0 .0047 47 7 0 :3 0 _ ■ 0 .2 0 .0 6 0 30 .

1 .0 0.0 8 0 8

1 0 .0 0 .0 5 0 .5

TA BLE X. PERCENTAGE OF THE IONIC GOLD FOUND IN THE FRONT OF CHROMATOGRAMS IN THE PRESENCE OF VARIOUS AMOUNTS OF ASCORBIC ACID (a. a. )

Au Au in the front (%) Chromatographic applied mixture No a .a . 1 /ig a .a . 10 Mg a .a .

Acetone-water-hydrochloric acid 0 .0 1 11 12 5 70: 20: 10 0 .2 13 9 3

1 .0 43 42 6

1 0.0 88 88 83

Acetone-O.IN hydrochloric acid 0 .01 21 20 16 70: 30 0 .2 38 34 5 1 .0 75 73 15

1 0.0 96 98 87

solvents. We used not only commercial products but also colloidal solu­ tions with modified properties (particle size, content of, gelatin, content and kind of reducing agent). The following results were obtained. (1) The ionic cold (i.e. the chloroaurate) is partially adsorbed on the paper; the percentage of gold in the front decreases with decreasing amount applied at the start (Table IX). Ionic gold is reduced during the chromato­ graphy by reducing agents present, for example ascorbic acid. This pos- . sible effect was studied so that ascorbic acid was placed 2 cm below the start line with applied chloroauric acid; the ascorbic acid also moves with the front of the solvent. The results in Table X are expressed in per cent of gold found near the front (0. 8 - 1. 0) because the activity is tailed from the start to the front owing to the action of the ascorbic acid during the development of the chromatogram. IAEA-PL-336/13 173

100 0.04 p g 0-1M9 > 80

Isoо

fe «>

20

0,

7. OF GELATIN

FIG. 19. The changes in activity distribution of ionic gold as a function of the gelatin content in solution. Chromatograms developed with the mixture acetone-0.1 hydrochloric acid 70:30. The respective amounts of applied chloroaurate are expressed in jjg of gold. (O) Activity on the start line; (x) activity between Rf 0.1 and 0. 8; (+) activity near the front, RfO.8-1.0.

2000r

o------;------1------1------0 , 5 10 15 7. OF HCI

FIG.20. The activity found in chromatographic solvents containing acetone and various concentrations of hydrochloric acid after 5 min immersing of irradiated gold wire under mixing.

The behaviour of ionic gold is also changed in the presence of gelatin, especially the ratio of the amount adsorbed on the start line to the amount adsorbed in the part between Rf 0. 1 - 0. 8. The results obtained for various solvents were analogous - examples are presented in Fig. 19. All the phenomena mentioned above have also been observed during paper electrophoresis. (2) In an acid solvent the colloidal particles are partially dissolved pro­ portionally to the content of the acid. This can be demonstrated not only with colloidal but also with compact gold. Figure 20 shows the increasing amount of activity which wa's dissolved from a gold wire (contacted with chromatographic acetine solvents) with increasing percentage of hydro­ chloric acid. The addition of ionic gold as ca rrier is not suitable because of the rapid isotopic exchange between the colloidal and ionic gold. Gelatin markedly influenced the mobility of colloidal particles on the paper. Some results are shown in Fig. 21 for com m ercial products. The colloidal particles are carried by the gelatin from the start line; this effect can be easily noted, even visually. (3) In the isotopic-exchange experiments two stages in the exchange were observed. Together with knowledge concerning the composition of the 174 CÍFKA

colloidal particles, this can be taken as additional evidence of inhomogeneity of colloidal particles. During the drying of applied colloidal solution on the start (on paper as well as on thin layer) the inner part of electric double-layer (in solution formed from complexes of monovalent gold) is gradually destroyed. Monovalent gold is either reduced to metallic gold or transformed by disproportioning partially to complexes of trivalent gold during coagulation of the colloid. The amount of gold in electric double layer is a function of the surface of the particle, i.e . of the radius of the colloidal particle. In addition, this amount can be also influenced by the method of preparation (concentration of gelatin, reducing agent etc. ). This is probably the source of ionic gold, which is found as a result of chromatographic analysis.

£ 2 со ои >- I— >

О <

FIG.21. The changes of mobility of colloidal gold observed chromatographically for various samples. (a) Colloidal solution containing 0.3 °Jo of gelatin (reduction was carried out with ascorbic acid); (b) the same sample after autoclaving; (c) colloidal solution containing 3.0 °jo of gelatin, prepared by means of glucose; (d) the same sample developed on the paper impregnated by 4% solution of lead acetate; (e) the commercial product having the same composition as the sample C; (f) sample e developed immediately, without drying the applied aliquot.

(4) The chromatographic value for the content of ionic gold was compared with the percentage-of activity remaining in the blood of rats after applica­ tion of the colloidal solution. These experiments were based on the well- known fact that chloroaurate remains in the blood fixed on the protein molecules while the colloidal particles are removed from the blood by the cells of the reticuloendothelial system. However, we are aware of the fact that under certain circumstances (small particles, excess of gelatin) even colloidal particles can remain in blood. In these experiments, the increasing amounts of chloroaurate-198Au were given to rats and after exactly 60 min the animals were killed and the activity of 1 ml of heparinized blood counted. The results are presented in Fig. 22. The total volume of the blood was calculated from the weight of each animal multiplied by the factor 0.075. Each point is the average of 5 or 6 animals. In addition, the chloroaurate was applied to rats which, 5 min earlier, received the colloidal radiogold (prepared from the same irradiated gold, to avoid isotopic-exchange). In all these experiments an IAEA-PL-336/13 175

average of 58.8% of the applied'activity was found. Owing to the close linearity between the administered and found activity, the source of the difference was not investigated. The blood activity found after the application of various colloidal solutions of radio-gold is presented in Table XI, together with the results of chromátography. If it was necessary to apply diluted colloidal solution (diluted with 0. 2% solution of gelatin), both original and diluted solution were analysed. No systematic correlation between the blood activity and the activity found between Rf 0. 8-1 (or between R f 0. 1-0. 8) was observed.

pg Au applied

FIG.22. The relation between the administered and ionic gold found in blood of rats 60 min after intra­ venous application. (+) Ionic gold applied 5 min after application of colloidal gold.

It can be said that the value of chromatographic analysis is very doubt­ ful, even if only the activity of the spot between Rf 0. 8-1. 0 is taken into consideration for the calculation [47]. It was shown that the presence of activity need not be related to the presence of ionic gold in the colloidal solution. In fact, in only one sample did we find the content of ionic gold to be higher than 5%. In this sample measurement of the redox-potential showed the absence of an excess of ascorbic acid. The evaluation of the radiochemical purity of the' colloidal radiogold solutions by means of paper chromatography is restricted to only those cases when marginally defective batches are to be excluded by means of a limit deliberately agreed upon. In no case can this method be taken as a universal one; the result is strongly influenced, for example, by the gelatin content.

Sodium phosphate~32P [48]

The main effort was devoted to accelerating the chromatographic deter­ mination of the pyrophosphate and higher condensed phosphates. The commonly used method takes 14-1-6 h. A thin-layer chromatogram on cellulose can be developed in 40-60 min using a mixture of 92. 5 ml ethanol, 7. 5'ml water, 5 g trichloroacetic acid and 0. 3 ml conc. ammonia: The separation can also be achieved on Whatman No. 4 paper within 3 - 4 h using the same mixture. Figure 23 shows the change in R f values of individual compounds as a function of the content of water in the mixture. The addition of pyrophosphate carrier can be recommended. : 7 CÍFKA 176

TA BLE XI. COMPARISON OF THE CONTENT OF IONIC GOLD AS FOUND CHROMA TOGRAPHICALL Y WITH ACTIVITY REMAINING IN BLOOD OF RATS AFTER APPLICATION OF COLLOIDAL RADIOGOLD

Activity found chrom atographically (‘Уо) A ctivity Maximum content Sam ple Dilution Mixture I Mixture II found in blood of ionic.gold s m Rf 0 .8 -1 R f 0 .1 - 0 .8 Rf 0 .8 -1 Rf 0 .1 - 0 .8

A 0 1 .1 4 ± 0 .1 5 0 1 .2 6 ± 0 ,1 1 0.036±0.003 0 .0 6 0

В - 0 1 .3 7 * 0 .0 1 0 1 .6 ± 0 .8 0.048 ± 0.005 0 .0 8 0

С - 0 .0 6 ± 0 .0 6 3 .3 ± 0 .5 0.20 ± 0.01 1 .3 ± 0 .2 - -

1 : 36 0.53 ± 0.12 1 3 .0 ± 1 .7 0.56 ± 0.06 1.45 ± 0.11 0 .3 1 ± 0 .0 4 0 .5 1

D - 0 9 5 ,6 ± 0 .1 0 9 4 .8 ± 0 .1 -

1 :4 0 2 .1 ± 0 .9 6 4 .5 ± 4 .5 0 6 9 .5 ± 1 .2 0 .6 6 ± 0 .0 6 1 .1

E - 0.36 ± 0.02 2 3 .5 ± 2 .6 0 .3 2 ± 0 . 02 3 8 .0 ¿ 2 .4 - -

1 : 50 0 .8 7 ± 0 .1 2 1 8 ,5 -± 0 .1 1 .2 4 ± 0, 21 0 .9 ± 0 .5 ■ 0. 059 ± 0 . 005 0 .1 0

a Calculated from the blood values which, under given conditions, amounts to 60°!o of the actual value. SamplesA, В, С, E contained 0.3% gelatin and were prepared by means of ascorbic acid. Sample D contained 3<7<> gelatin. Samples С and D were commercial products. Sample В was aerated under heating to destroy the excess of ascorbic acid. Mixture I: acetone-water-hydrochloric acid 70: 20: 10 Mixture П: acetone-0.IN hydrochloric acid 70: 30 IAEA-PL-336/13 177

1.0 10 a b ^ _-Q— о R f 0.8 0.8 О / // © 0.6 0.6 у tS / / ' 7 / • OA 0 Í • / -® / / 0.2 _ / ' * / ОС / ■9 / Á * 1 1 1 0 0 1 ------1— О 10 : 20 30 О 10 20 30 ml Н20 ' FIG. 23. The change in Rf values of individual compounds as a function of the water content in the ethanolic solvent (for composition see the text). (a) Paper chromatography, (b) thin-layer chromatography, (o) ortho-, (®) pyro-, (в) tri-, (•) tetraphosphate.

Sodium ch rom ate-51Cr [49]

At present there exist, in principle, three methods for determinating the presence of C r3+ in the solution of radiochromate. The paper chromato­ graphic method [23, 50] is based on the fact that, in alkaline aqueous solvents, Cr3+ ions remain on the start line while CrO|" ions move with a Rf of 0. 9. The method of British Pharmacopoeia [51] separates both ions on the strongly basic anion exchange resin after the addition of chromate and chromic chloride ca rrie rs. The USP XVII [16] recommends the addition of chromate carrier and the precipitation of chromate as lead chromate, leaving C r3+ ions in the solution. We compared all three1 methods using commercial samples of radio­ chromate from various producers and the solution of chromic chloride-51Cr (slightly acid, pH 3-4) as a; "pure" impurity. In addition, we' analysed the mixtures prepared by mixing solutions of sodium chromate-51 Cr and chromic chloride-51Cr.

TABLE XII. THE Cr3+ CONTENT FOUND IN SODIUM RADIOCHROMATE BY DIFFERENT METHODS

Per cent of Cr3+ found by method of Main chemical Samples form Paper chrom. BP USP

A СЮ2- 4 6 .0 6 ± 0 .3 0 6 .7 ± 0 .9 0.090 ± 0.010

В CrO^- 3.05 ± 0.10 4 1.90 ± 0.05 0.13 ±0.001

С с ю ;4 - 2 .8 ± 0 .1 0 2 .0 ± 0 .1 5 -

D Cr3+ 9 7 .4 ± 1 .1 9 2 .4 ± 0 .4 0 .3 6 ± 0 .1 4

Ea Cr3+-CiC?4' 2 6 .6 ± 0 .8 1 6 .2 ± 0 .4 -

Fb Cr3+- CrO^“4 2 7 .1 ± 0 .8 2 4 .6 ± 0 .2 -

a Samples С and D mixed to give approx. 14% of activity in Cr:r+. k Samples С and D mixed to give approx. 28% of activity in Cr3+. 1 7 8 CÍFKA

The samples of sodium chromate A and В and the sample of chromic chloride denoted as D were analysed by all three methods on the same day to eliminate the possible decomposition of the sample. The results are given in Table XII. The remaining samples С, E and F were analysed by only paper chromatography and the BP method because it was with certainty found that the USP [16] method gives erratically low results. The analyses of the mixtures were carried out immediately after mixing to avoid the possible slow reaction of both chemical forms of chromium. The retention of activity at the start line, not initially connected with the Cr3+ form, was studied in more detail. A set of solutions containing the same quantity of sample A but different quantities of non-radioactive chromic chloride carrier were analysed by paper chromatography. The results are presented in Fig. 24. The "plateau" value probably depends not only on the C r 3+ content but also on the concentration ratio of Cr3+ and CrO|‘ . Further experiments are necessary, however, to confirm this assumption.

FIG.24. The percentage of total activity of chromate-51Cr retained on the start line as a function of added amount of the trivalent chromium carrier.

According to our experience, only the method of the British Pharmacopoeia gives good results in the whole range of C r3+ -CrO|" ratios tested. The paper chromatography afforded good results for the Cr3+ content in the 1 - 5% range of the total activity. The USP method seem s to be inconvenient owing to the nearly complete co-precipitation of C r3+ with lead chromate. No differences were found between the thin-layer chromatography on silica gel G and on cellulose; the reduction of chromate by the paper can therefore be neglected [52].

4. PRESENT WORK

At present the study is being completed on the behaviour of colloidal chromic phosphate and its possible radiochemical impurities when chromatographed on paper. Work on the control methods for generator-isotopes and related labelled compounds is being carried out.

REFERENCES

[1] BURIANEK, I., CIFKA, J., Radioactivity and its measurement, Cslká Farm. ±4 (1965) 135 (in C zech ). [2] BENES, I., CIFKA, J ., Natrium radiophosphoricum (32P) injectio, Cslká Farm. 14 (1965) 533 (in Czech). [3] BENES, I . , CÍFKA, J. , Natrium radiojodatum (131I) injectio, Cslká Farm. 14 (1956) 526 (in Czech). IAEA-PL-336/13 179

[4] BURIANEK, J., Natrium radiojodhippuricum (131I) injectio, Cslká. Farm. 14 (1965) 526 (in Czech). [5] BENES, I., CÍFKA, J ., Natrium radiochromicum (51Cr) injectio, Pharm. Bohemoslovenica, 3rd Edition, in press (in C zech ). [6] VESELŸ, P., Radioaurum (198Au) colloidale injectio, Cslká Farm. 16 (1967) 105 (in Czech). [7] SCHOU, S.A ., MORCH, J., Stability of pharmaceutical preparations, Arch. Pharm. Chem. 66 (1959) 231, 287, 371, 423, 503. [8] SCHOU, S.A ., Stability and stabilization of pharmaceutical preparations, Helv. chim. Acta 34 (1959) 309. ~ [9] SCHOU, S.A ., Decomposition of pharmaceutical preparations due to chemical changes, Helv. chim. Acta 34 (1959) 398, [10] BURIANEK, J ., The stability of radiopharmaceuticals, Thesis, Faculty of Pharmacy, Commenius' University, Bratislava (1968) 92 pp,(in Czech). [11] CIFKA, J . , BURIANEK, J . , A study o f the stability and radiochem ical purity of some radiopharmaceuticals. 1. Calculations of absorbed radiation energy, J. labelled Comp. 4 (1968) 107. [12] ALLEN, A .O ., The Radiation Chemistry o f Water and Aqueous Solutions, D. van Nostrand C o ., Princeton, New Jersey (1961) Ch.5. [13] BURIANEK, J., CÍFKA, J ., "Stability of sodium iodohippurate-131I", Coll. Wks State Inst. Control of Drugs, Prague (1967) 130 (in C zech). [14] MANGNUSSON, G ., Purity and stability of commercial sodium iodohippurate labelled with iodine-131, Nature (London) 195 (1962) 591. [ [15] PRITASIL, L., FILIP, J ., MRLÍK, A ., VYSATA, F., Preparation of hippuran-131I and Rose Bengal-^q, Rep. Institute for Research, Production and Uses of Radioisotopes, Prague (1964) 72 pp (in Czech). [16] United States Pharmacopoeia, XVIIth Revision (1965). [17] ANGHILERI, L .J ., A chromatographic study of the stability of iodine-131 labelled sodium o-iodohippurate, J. nucl. Med. 4 (1963) 155. [18] KATO, S ., KURATA, K ., SUGISAWA, Y ., Stability of sodium o-iodohippurate (o-131I), J. pharm. Soc. Japan 85 (1965) 935 (in Japanese). [19] BURIÁNEK,T , CÍFKA, J . , Unpublished results.* [20] RYBAKOW, Z ., CÍFKA, J., A. study of the stability and radiochemical purity of some radiopharma­ ceuticals. 3. Labelled diiodofluorescein, J. labelled Comp., to be published. [21] VESELY, P., SKORKOVSKA, Z ., |,The separation of some iodinated fluoresceins by means of thin-layer chromatography, presented at Meeting on use of Chromatography and High-Voltage Electrophoresis in Analysis and Control of Radioactive Preparations, held by Council of Mutual Economic Co-operation in Mariánské Lázne, Czechoslovakia,: Oct. 1966 (in Russian). [22] VESELY, P., The analytical study of some halogenated fluoresceins, Rep. of State Institute for Control of Drugs, Prague (1968) 68 pp (in Czech). [23] COHEN, Y ., "Chemical and radiochemical purity of radioactive pharmaceuticals related to their biological behaviour", Proc. Symp. Radioactive Pharmaceuticals, Oak Ridge, Nov.1965, USAEC Symp. Ser. No.6 (1966) 67. [24] RABAN, P ., GREGORA, V ., New Method of labelling of Rose Bengal with iod ine-131I and - 125I, Cslká Farm. 16 (1967) 385 (in Czech). [25] VANKA, M ., CÍFKA, J ., Unpublished results. [26] BALLANCE, P.E., CROMBIE, W .M ., Paper chromatography of saturated and unsaturated fatty acids, Biochem. J. 69 (1958) 632. [27] RANKOFF, G ., RANKOFF, D ., The paper chromatographic separation of "critical pairs" of higher fatty acids by using products obtained by Margosches method for rapid determination of iodine values of fats, Fette Seifen Anstrmittel 66 (1964)1 912 (in German). [28] TUNA, N., MANGOLD, H .K., MOSSER, D .G ., Re-evaluation of the I131-triolein absorption test. Analysis and purification of commercial radioiodinated triolein and clinical studies with pure prepara­ tions, J. Lab. clin. Med. 61 (1963) 620. [29] VEIBEL, S ., The Identification of Organic Compounds. A Manual of Qualitative and Quantitative Methods, G.E.C. Gad Publisher, Copenhagen (1961). [30] VEALL, N., VETTER, H ., Radioisotope Techniques in Clinical Research and Diagnosis, Butterworth, London (1958) 344. [31] RUSSIN, K ., SZUCHNIK, A ., KOLACZKOWSKI, A ., WITKOWSKA, K ., Preparation of olive oil and oleic acid labelled with 131I, Rep. Inst. Bad. Jadrowych, Polish Acad. S ei., PAN-390/XIII (1962) 17 pp (in G erm an), [32] MARGOSCHES, B .M ., HINNER, W ., FRIEDMANN, L ., Rapid method for determination o f iodine value of fatty oils by iodine and alcohol,. Z. angew. Chem. 37 (1924) 334 (in German). 180 cI fk a

[33] JAKY, M ., PEREDI, J . , PALLOS. L ., Investigation o f a fat originating in period of ancient Rome, Fette Seifen Anstrmittel 66 (1964) 1012 (in German). [34] BURIANEK, J., CIFKA, J., "The stability of injections of chlormerodrin-203Hg," Presented at Meeting on Use of Chromatography and High-Voltage Electrophoresis in Analysis and Control of Radioactive Preparations, held by Council of Mutual Economic Co-operation in Mariánské Lázné, Czechoslovakia, O ct.1966, (in Russian). [35] BURIÂNEK, J . , CIFKA, J . , A study of the stability and radiochem ical purity of some radiopharma­ ceuticals, 2. Labelled chlormerodrin, J. labelled Comp., in press. [36] Radiochemical Centre Amersham, Specification MB. 19P. (Nov. 1963). [37] CÍFKA, J . , KACENA, V ., KRONRÁD, L ., "Preparation of some organo-mercurials labelled with mercury isotopes 197Hg and 203 Hg", Proc. 2nd int. Conf. on Methods of Preparing and Storing Labelled M olecules, Brussels 28 N ov.-З D ec.1966, EURATOM 3746 P - e - f (1968) 461. [38] ANGHILERI, L .J ., A study o f the stability of neohydrin (3-chlorom ercuri-2-m ethoxypropylurea) labelled with mercury-203, J. nucl. Med. 4 (1963) 410, [39] Radiochemical Centre Amersham, Specification M .19P., (Dec. 1965). [40] PRITASIL, L., MATUCHA, M ., Preparation of neohydrin-203Hg, Rep. of Institute for Research, Produc­ tion and Uses of Radioisotopes, Prague (1965) 49 pp (in C zech). [41] HEINRICH, H .C ., GABBE, E .E ., MEINEKE, B ., KUHNAU, J.,Jr-, M etallorganic Hg-compounds, their purity and whole body metabolism, Proc. 2nd int. Conf. on Methods of Preparing and Storing Labelled M olecules, Brussels 28 N ov.-З D ec.1 9 6 6 , EURATOM 3746 d -e -f (1968) 1063 (in German). [42] HERZMANN, H ., LOECHEL, M ., SCHWARTZ, K .D ., Som e experience with the use o f new mercury- labelled compounds for diagnostic purposes, Radioaktive Isotope in Klinik u. Forschung, Strahlen­ therapie 65 (1967) 480 (in German). [43] SPÉVACEK, V., VESELY, P., CÍFKA, J., Chromatographic determination of the radiochemical purity of colloidal radiogold-198Au, Rep. of Nuclear Research Institute of Czechoslov. Acad. Sei. (1968) 96 pp (in C zech). [44] COHEN, Y ., The control of artificial radioelements for medical use in France, Annls pharm, fr. 27 (1959) 250 (in French). [45] Pharmacopoeia Gall. (1964). [46] KHARLAMOV, V .T ., SHUBNJAKOVA, L.P., "Study of the radiochemical state of the colloidal solution of gold labelled with isotope 198Au”, Atomizdat, Moscow (1965) 162 (in Russian). [47] IYA , V .K ., GOPAL, N .G .S ., CHERIAN, S ., PATEL, K .M ., POHUJANI, S .M ., PRAYAGKAR, V .H ., Quality control of medical radioisotopes, Atomic Energy Establishment Trombay, Rep. No.43 (Radio­ chemistry) (1962). [48] BURIÁNEK, J., CÍFKA, J., Rapid determination of radiochemical purity of orthophosphate-32P, Z. anal. Chem. 213 (1965) 1 (in German). [49] C ÍFK A .77T BURIANEK, J . , Unpublished results. [50] COHEN, Y . , INGRAND, J . , Study and checking o f the labelling of red blood cells with chrom ium -51. Rev. Hemat. 15_ (1960) 217 (in French). [51] British Pharmacopoeia (1968). [52] BURIÁNEK, J . , The determination of radiochem ical purity of sodium chrom ate-51Cr injections by thin- layer chromatography, Cslká Farm. 15 (1966) 130 (in Czech). IAEA-PL-336/14

DEGRADATION OF 1311-HIPPURAN, 1311-LIPIODOL, 131I-ROSE BENGAL AND 198Au-COLLOIDAL GOLD

. F. CASAS MEDINA, D. V. REBOLLO CARRIDO, M. del VAL COB, Junta de Energía Nuclear, Madrid, Spain

Abstract

DEGRADATION OF I311-HIPPURAN, l34-LIPIO D O L, 134-R O SE BENGAL AND «»Au-COLLOIDAL GOLD. The control procedures for hippuran, Rose Bengal and Lipiodol labelled with 1311 and for colloidal gold (198Au) are described. Stabilities are examined by determining radiochemical purities after various conservation periods at different temperatures and in the presence or absence of light.

1. INTRODUCTION

The purpose of the present study is to determine the possible changes which physical agents such as temperature and light may produce in the radiopharmaceuticals under study. Among the types of deterioration to which these radiopharmaceuticals are liable, in the case of 1311-Hippuran, iSiI-Lipiodol and 131I-R ose Bengal, the most likely one is probably the presence of 13 Ц in the form of iodide. When the concentration of this ion exceeds 5% of the total activity of the radiopharmaceutical, physicians should not use it on humans. Similarly, colloidal 1 9 8 A u may manifest its deterioration by variations in the size of its colloidal particle and also by increased concentration of 198Au in ionic form. The storage period is influenced by the half-life of the radionuclide concerned as most radiopharmaceuticals are rejected by the user when the radioactive concentration is below certain limits, thus rendering its in-vivo detection difficult unless large amounts are administered. In the case of 1311 Hippuran, the ádministration of large amounts would produce a marked diuretic effect on the patient. When large quantities of colloidal 98Au are administered, the concentration of gelatine in the organism is increased and the likelihood of secondary and undesirable effects is greater. . Therefore, the aims of the experiment were to establish the optimum storage and conservation conditions for these pharmaceuticals in order to obviate deterioration as far as possible and to determine the permissible storage life.

2. EQUIPMENT

2.1. Apparatus

The apparatus used in the experiment consisted of the following items:

Chromatogram analyser • pH meter RADIOMETER, Copenhagen, electrode type GK 2025 С Electrophoresis equipment:

181 182 CASAS MEDINA et al.

Chromatography cameras Culture oven (40° C) Culture oven (20°C) Refrigerator Maximum and minimum thermometers SIEMENS Elmiskop I electron microscope 2 0 -watt fluorescent tubes

2.2. Ancillary equipment and reagents

Additional ancillary equipment used is listed below.

1311-Hippuran solution JEN. 1311-Rose Bengal solution JEN. 131I-lipiodol solution JEN. 198Au-colloidal gold solution JEN. Whatman No. 3 MM and No. 1 paper. 5- and 10-jul micropipettes n-butanol-acetic acid-water solution (4:1:1), pH 2.4. Veronal - sodium veronal solution, pH 8 . 6 . Acetone-water-conc. HC1 solution (70:20:10). Industrial X-ray film, KODAK type AA. Rapid X-ray developer, KODAK. Rapid acid fixer, KODAK. Ammonium citrate - water solution (1:50), NH4 OH q .s . for pH 7.

3. EXPERIM ENT

The conditions of the experiment were similar for the three radio­ pharmaceuticals. They were subjected to three different temperatures (0, 20 and 40°C). Two 20-W fluorescent tubes were used as a light source and the samples were placed 10 cm away from them.

3.1. Hippuran

The initial 131I hippuran was prepared according to Ref.[l] and had the following characteristics:

Radioactive concentration: 5.5 mCi/ml Chemical purity: 99. 9% Radiochemical purity: over 98% pH: 7.. 6 Isotonic, sterile and pyrogen-free

The possible impurity of 131I in the form of iodide ion was determined by chromatography using n-butanol-acetic acid-water (4:1:1) with pH = 2. 4 as chromatographic solvent. The chromatography paper used was Whatman No. 3 MM and the development time between. 3 and 3.5 h [2]. Impurity was determined quantitatively by analysing the radioactivity of the chromatogram. Autoradiographs were made simultaneously to locate the different radioactive compounds. Table I shows the percentages of free iodine for the different experimental conditions.' IAEA-PL-336/14 183

TABLE I. DEGRADATION OF 131I HIPPURAN AS A FUNCTION OF TEMPERATURE AND EXPOSURE TO LIGHT

Percentage of free 131I Time elapsed since preparation 0°C 20 °C 40°C Light, 25°C (d) C a .l.f (a.i.) (a.i.) (40 W - 10 cm)

0 1 .0 - -

2 2 .3 2 .0 2 .7 2 .7

9 3 .4 3 .0 3 .2 5 .0

a a . 1, = in the absence o f light

TABLE II. DEGRADATION OF Ш1 ROSE BENGAL AS A FUNCTION OF TEMPERATURE AND EXPOSURE TO LIGHT

Percentage of free 131I Time elapsed since preparation 0°C 20 °C 40° С Light, 25°C (d) ' ( a .l .) a ( a .i .) ( a .i .) (40 W - 10 cm)

0 - 0 .4 - -

5 0 .5 0 .6 0 .8 1 .2 5 .

10 0 .7 5 0 .8 1 .3 2 .4

a a .i. = in the absence of light

3.2. Rose Bengal

The initial 131I Rose Bengal was prepared according to Ref.[3] and had the following characteristics:

Radioactive concentration: 2 mCi/ml Chemical purity: 99. 9% Radiochemical purity: over 98% pH: 7.5 Isotonic, sterile and pyrogen-free

Chromatography was employed to determine free 131I, using ammonium citrate-water (1:50) as chromatographic solvent to which was added suf­ ficient ammonium hydroxide to obtain a .pH of 7. The chromatographic paper was Whatman No.3 MM, and the development period approximately 2. 5 h [2]. The quantitative determination of the radioactivity was the same as for 1311-hippuran. Table II gives the percentages of free iodine for the different experi­ mental conditions. The autoradiographs obtained are shown in Fig. 1. 184 CASAS MEDINA et a l.

í h

»1 »2 .Э iI. ♦В Uft ? iОд i

FIG .l. Autoradiograms of 131I Rose Bengal.

1. Od 5. 5 d, light, 25°C 9. 10 d, light, 25°C 2 . 5 d, 0°C 6. 10 d, 0°C A. 131I Rose Bengal 3 . 5 d, 20°C 7. 10 d, 2 0 DC B. Free 131I 4. 5 d, 40°C 8. 10 d, 40°C C. 1% dilution

3.3. 1311 lipiodol

Samples of 1311-lipiodol (F) and UF) were prepared according to Ref. [5] and-had the following characteristics:

Radioactive concentration: 1-4 mG¿/ml Radiochemical purity: over 98% Sterile and pyrogen-free

TABLE III. DEGRADATION OF Ш 1 LIPIODOL AS A FUNCTION OF TEMPERATURE

Percentage of free 131I Time elapsed since preparation 3 eC 6°C 9°C 20°C 37°C 4 0 eC Cd)

5 0 .1 - - - 1 .2 1 .3

10 0 .1 5 0 .5 0 .8 1 .1 2 .1 3 .0

15 0 .2 0 .8 1 .1 1 .7 3 .5 4 .7

20 0 .3 0 .9 1 .4 1 .5 5 .5 8 .0

25 0 .3 5 1 .1 1 .8 3 .5 7 .8 1 0 .5

30 0 .4 1 .5 2 .1 4 .3 9 .5 - IAEA -PLN336/14 185

Iodine-131 in the form of iodide ion was determined by electro­ phoresis with veronal - sodium veronal as electrolyte, pH 8.6, Whatman No.l paper, current density 0.3 mA/cm and development time 1.5 h. The impurity was determined quantitatively by analysing the radioactivity of the electrophoregram. Autoradiographs were made at the same time to locate the different radioactive compounds. Table III gives the percentages of free iodine for the different experi­ mental conditions. Light is another important factor which affects the stability of the compound. An ampoule containing the radioactive substance and exposed to the light of a 60-W lamp for 15 d at 20°С showed a free iodine concentration of 7%.'

3 .4 . 198A u colloidal gold ;

The initial colloidal 19?Au was prepared according to Ref. [4] and had the following characteristics :

Radioactive concentration: 50 mCi/ml Chemical purity: 99. 99% Radiochemical purity: over 98% Particle size: 200-300 Â pH: 5-7 ; Isotonic, sterile and pyrogen-free

198Au+++ was determined by chromatography using acetone-water- concentrated hydrochloric acid (70:20:10) as solvent. Whatman No. 3 MM chromatographic paper was used and the development time varied between 1 and 3 h [2]. The method of determining the radioactivity of the chromatograms was the same as for 131I-hippuran and 131I-Rose Bengal. For this radiopharmaceutical we also took into consideration possible changes in particle size resulting from the action of the physical agents used. Electron microscopy was employed to determine particle size. Table IV gives the percentages of free ionic gold for the different ex­ perimental conditions. Figure 2 shows a photograph obtained in the electron microscope (magnification X 100 000).

TABLE IV. DEGRADATION OF 198Au COLLOIDAL GOLD AS A FUNCTION OF TEMPERATURE AND LIGHT

Percentage of free 198Au Time elapsed since preparation 0°C 20°C 40 е С Light Cd) C a .l.)a ( a .i .) ( a .i .) (40 W - 10 cm)

0 - 1 .1 - -

1 1 .2 1 .2 0 .8 0 .6

3 * 0 .9 0 .8 0 .8 0 .8

6 0 .8 0 .9 0 .7

10 0 .9 0 .9 • - 0 .8

3 a .i. = in the absence of light 186 CASAS MEDINA et al.

FIG. 2. Electron microphotograph (x 100 000) of the colloidal particles of 198Au. 10 d storage at 40°C.

4. CONCLUSIONS

The conclusions derived from the above results, which should be con­ sidered as preliminary owing to the incompleteness of the data obtained, may be summarized as follows. The storage life of the radiopharmaceuticals in question may be estab­ lished by fixing an upper limit for the percentage of the isotope in its ionic form and only in respect of this percentage. The results given are only valid for the radioactive concentrations and other variables assigned in this paper. IAEA-PL-336/14 187

(1) The physical agents used have a substantial influence on the deterioration of all the radiopharmaceuticals studied except colloidal gold. (2) 131I-hippuran: An ¡increase in the concentration of free iodine is observed when the substance is kept at temperatures below and above ambient and when it is exposed to light. It is recommended that 131I-hippuran be kept away from light. The increase in the concentration of free iodine at 0°C seem s to be due to a greater radiolytic effect at the bottom of the container, as a consequence of a density gradient in the substance which occurs at this temperature. (3) 131I~Rose Bengal: The effect of temperature and light, especially the latter, is evident. It is recommended that the product be stored away from light and at a low temperature. (4) 131I-lipiodol: The effect of temperature and light on this product is also evident. Storage of the product at a low temperature away from light is recommended. (5) 198Au colloidal gold: Stabilization of the colloid is observed when it is subjected to the temperatures of the experiment and to light. These factors have no manifest influence on the size of the colloid.

: REFERENCES

[1] REBOLLO GARRIDO, D. V ., del VAL COB, M ., Synthesis of orthoiodohippuric acid (Hippuran) with 131I, JEN Rep. IS-1211/I-3 (1962) (in Spanish). [2] del VAL COB, M ., REBOLLO GARRIDO, D .V ., CASAS MEDINA, F ., RADICELLA, R ., RECCHI, N ., Specifications and Standards for Radiopharmaceuticals, JEN (Spain) and CNEA (Argentina), JEN (1969) (in Spanish). [3] REBOLLO GARRIDO, D. V ., Synthesis of 131I rose bengal (tetrachlorotetraiodofluorescein), JEN Rep. IS-1211/1-2 (1962) (in Spanish). [4] HENRY, R ., HERCZEG, C ., CEA Rep. N 0.733 (1957) (in French). [5] REBOLLO GARRIDO, D . V ., Preparation of lipiodol F and UF labelled with iod ine-131, JEN Rep. IS-1211/I-6 (1965) (in Spanish).

IAEA-PL-336/L5

POSSIBLE ARTIFACTS IN THE CHROMATOGRAPHICAL DETERMINATION OF RADIOCHEMICAL PURITY OF 35S- AND 75Se-LABELLED METHIONINE*

I. GALATZEANU Institute of Atomic Physics, Laboratory of Radiochemistry, Bucharest, Romania

Abstract

POSSIBLE ARTIFACTS IN THE CHROMATOGRAPHIC AL DETERMINATION OF RADIOCHEMICAL PURITY OF 35 S- AND TCSe-LABELLED METHIONINE. The introduction of artifacts during paper chromatography of selenomethionine in the presence of air is discussed.

INTRODUCTION - LITERATURE DATA

It is mentioned in the literature that during the chromatographic sepa­ ration of individual amino acids, a considerable oxidation and loss occurs [1-3]. A sample of methionine pipetted on the filter paper and stored in air for a period of 1-3 d suffers oxidation with formation of methionine sulphoxide. This process of oxidation is significantly accelerated during the solvent development on paper [ 5 ] It was observed by repeating the chromato­ graphic development two to three times on paper, with the solvent system n-butanol-acetic acid-water = 4 :1 :5 , that pure methionine was almost completely oxidized to methionine sulphoxide. Selenium analogues of the common sulphur amino acids are more unstable and, therefore, separation methods involving the use of mild solvent systems must be employed [4] . It was shown that 75Se-labelled compounds of high activity from plant extracts suffer a decomposition during the chromatographic development on paper. The following solvent systems were investigated [4, 6]:

A. n-butanol — pyridine' — water (1:1:1, v/v); B. n-butanol — acetic acid — water (25:6:25, v/v); C. n-butanol — ethanol — water (2:2:1, v/v); D. tert-butanol — formic acid — water (14:3:1, v/v).

Solvent D caused a severe decomposition although it has been used by previous workers [7]. When 75Se- and 35S-labelled amino acids were chromatographed on paper with solvent В only slight decomposition was observed and no evidence of decomposition in solvents A and С [4].

This work was performed under the auspices of the IAEA in its Laboratory at Seibersdorf, near Vienna.

189 190 GALATZEANU

EXPERIMENTAL

A series of experiments were carried out using the following methods: (1) Paper chromatography of L- and DL-methionine (Whatman No. 1) with the solvent system:

(a) n-butanol - acetic acid - water = 60:15:25 (ascending). Volumes of 10, 20 and 30 ц! of labelled amino acid solutions were pipetted; in some cases ca rrier was also added.

(2) Thin-layer chromatography on silica gel plates with the solvent systems:

(a) ethanol — water = 70:30 (b) isopropanol — butanol — water = 20:60:20 (c) n-butanol — acetic acid — water = 60:20:20

(3) Gamma spectrometry for determination of radionuclidic purity

RESULTS AND DISCUSSION

Our investigations on the chromatographic behaviour of non-radioactive DL- and L-methionine indicated that oxidation of these substances on the start line to methionine sulphoxide occurred during the pipetting and drying in air. Samples from a freshly prepared solution of non-radioactive DL- and L-methionine were pipetted on thin-layer chromatograms and dried in air . for 10 min. Only one spot (with solvent 1 (a) and 2(b)) of Rf 0. 62 and 0. 30, respectively, was detected. When samples from the same solution were pipetted two days after preparation of the- solution, and dried for 20 min in air, two spots were detected (reaction with ninhydrin) with Rf of 0. 62 and 0.27 for solvent 2(a) and Rf of 0.30 and 0.06 for solvent 2(b). The second spot with a lower mobility in both cases is due to oxidation products (sulphoxide and/or sulphone). The addition of small amounts of H2O2 to the freshly prepared solutions of non-radioactive DL- and L-methionine produced an oxidation of these substances to sulphoxide and/or sulphone. The Rf values in this case were the same as observed with the two-day-old solution as above. Investigations with radioactive L-selenomethionine-'75Se were carried out using the method described in the experimental part. All operations (pipetting, drying, developing) for paper and thin-layer chromatography were performed in an inert atmosphere of nitrogen (Fig. 1). In par.ellel, the same samples of L-selenomethionine-75Se were handled and developed in air. To prevent any oxidation due to the presence of air, only solvents re-distilled in vacuum and stored under nitrogen atmosphere were used. The experiments performed in an inert atmosphere showed that no difference in quality was observed between the paper chromatograms of L-selenomethionine-75Se developed with "c a r rie r" (Fig. 2) and those without "carrier" (Fig. 3) using re-distilled solvents. A similar behaviour of L-selenomethionine-75Se was observed when paper chromatograms were handled and developed in air (Figs 4 and 5). 1AEA-PL-336A5 191

GLOVEBOX

OUTLET

FIG .l. Working scheme in inert atmosphere.

S e M 4 2

RfXioo

FIG.2. SeM42 paper chromatogram of L-selenomethionine-75 Se, first supplier, with carrier (40 ^ig), developed in nitrogen atmosphere, solvent 1(a) redistilled. 192 GALATZEANU

FIG.3. SeM44 paper chromatogram of L-selenomethionine-75 Se, first supplier, without carrier, developed in nitrogen atmosphere, solvent 1(a) re-distilled.

FIG.4. SeM48 paper chromatogram of L-selenomethionine-75 Se, first supplier, without carrier, developed in air, solvent 1(a) re-distilled. IAEA-PL-336/15 193

FIG. 5. SeM49 paper chromatogram of L-selenornethionine-75 Se, first supplier, with carrier (40 ¿ig), developed in air, solvent 1(a) re-distilled.

FIG.6. SeM22 paper chromatogram of L-selenomethionine-75Se, first supplier, developed in air, 18 h, solvent 1(a) non-redistilled. 194 GALATZEANU

FIG.7. SeM24 paper chromatogram of L-selenomethionine-75Se, first supplier, developed in air, 28 h, solvent 1(a) non-iredistilled.

FIG. 8. SeM29 thin-layer chromatogram of L-selenomethionine-75 Se, first producer, with carrier, developed in nitrogen atmosphere, solvent 2(a). IAEA-PL-336A5 195

FIG. 9. SeM32 thin-layer cnromatogram of L-selenomethionine-^Se, first producer, without carrier, developed in nitrogen atmosphere, solvent 2(a).

Increased oxidation was observed during the development on paper in air of the same samples, With the same solvent (non-redistilled) for 18 and 28 h (Figs 6 and 7). The role played by the "carrier" in avoiding the "double spotting" on TLC plates in air (Figs 8 and9) can be explained by its partial oxidation, thus diminishing the oxidation of the radioactive substance and acting as protecting agent. The diminution or the elimination of the selenomethionine oxidation during the chromatographic development in an inert or air atmosphere by addition of carrier or without carrier, is shown in Table I. The results represent an average of three runs. As shown in Figs 10 and 11, the. amount of oxidized L-selenomethionine on the spot of Rf value 0.2 5 is increased when it is developed in nitrogen atmosphere without ca rrier. The same dependency was observed in the case of thin-layer chromatograms developed in air without and with carrier (Figs 12 and 13). To compare the oxidation process during the development on silica gel plates, a series of experiments was carried out with samples of L-selenomethionine-75Se-supplied by another producer. Figures 14 and 15 illustrate the oxidation of L-selenomethionine-7SSe from this supplier, handled and developed on thin-'layer plates in an inert atmosphere and in air (solvent 2 (a)). The behaviour of L-selenomethionine developed with solvent 2 (b) in an inert atmosphere and in air is shown in (Figs 16 and 17). Concerning the radiochemical purity of examined samples of L-seleno- methionine-75Se from two different producers, it was determined that no essential self-decomposition occurred during storage in a pencillin bottle (in the absence of air) for a period of from a few weeks to 11 months. It may be concluded that the accuracy in the determination of the radiochemical purity of L-selenomethionine-75Se by paper- or thin-layer chromatography depends to a great extent on storage and handling in the absence or presence of air because its oxidation leads1 to the "double spotting". 196

TABLE I. RADIOCHEMICAL PURITY CONTROL OF L-SELENOMETHIONINE-75Se FROM PRODUCER В BY TLC AND PAPER CHROMATOGRAPHY

Mean spot Spots of decomposition products L-Seleno- Carrier added Solvent Adsorbent Performed in: methionine-^Se C^g) Rf X 100 m Rf X 100 (°!°) Rf X 100 (°Io) Rf X 100 (.%)

Stored 11 2 (a ) Silica gel Nitrogen 0 82 79 0 1 .5 25 10.0 93 9 months at 2 (a ) 15 82 76 0 2 .0 25 8 .5 93 13 room 2 (b ) 0 40 85 3 9 .8 72 5.3 - - 40 86 3 9.1 72 5 .3 temperature 2 (b ) 15 " GALATZEANU Stored 10 2 (a ) 0 82 71 0 2 .0 25 12.2 93 15 months at 2 (a ) 15 82 81 0 2 .2 25 8 .3 93 7 room 2 (b ) 0 40 79 3 1 4 .5 72 6 .5 - - 15 40 80 3 14.0 72 5 .4 temperature 2 (b ) '

2 (a ) 0 82 98.0 0 .0 .5 25 1 .5 _ _ 2 (a ) 15 82 9 8 .5 0 0 .5 25 1.0 - - 2 (b ) 0 40 95.0 3 3 .5 72 1. 5 - - 40 96.0 3 2 .5 72 1 .5 2 (b ) 15 ' “

Stored 3 2 (a) 0 58 83.0 0 2 .3 18 8.1 78 4 .7 weeks at 2 (a ) 15 68 96.0 0 1 .2 18 2 .5 78 0 .3 - 30 *C

1 (a) Paper Nitrogen 0 78 9 8 .6 95 1 .4 1 (a ) 40 78 9 8 .5 95 1 .5 1 (a) Air 0 68 91.0 32 1 .0 80 4 .0 90 3 .5 1 (a )- 40 68 95.0 32 1 .0 80 2.0 90 2 .0 IAEA-PL-336/15 197

FIG.10. SeM30 thin-layer chromatogram of FIG. .11. SeM33 thin-layer chromatogram of L-selenomethionine-75Se, first producer, without L-selenomethionine-75Se, first producer, with carrier carrier, developed in nitrogen atmosphere, solvent 2(a). (15 pg), developed in nitrogen atmospnere, solvent 2 (a) Rf 0 .2 5 (6%) and Rf 0.82 (92. 5$). Rf 0 .2 5 (2 % ) and Rf 0.82 (95.6%).

FIG. 12. SeM47 thin-layer chromatogram of FIG.13. SeM46 thin-layer chromatogram of L-selenomethionine-75 Se, first producer, without L-selenomethionine-75 Se, first producer, with carrier carrier, handled and developed in air, solvent 2 (a). (15 jjg), handled and developed in air, solvent 2 (a). Rf 0.18(8% ), Rf 0.68 (83%) and Rf 0. 78 (4. 7%). Rf 0.18(2.5% ) and Rf 0.68 (96%). 198 GALATZEANU

FIG.14. Se M3 thin-layer chromatogram of FIG- 15. SeM13 thin-layer chromatogram L-selenomethionine- 75 Se, second producer, of L-selenomethionine- 75 Se, third producer, handled and developed in nitrogen atmosphere, handled and developed in air, solvent 2 ( a ) . solvent 2 ( a ) .

FIG. 16. SeM5 thin-layer chromatogram FIG. 17. SeM15 thin-layer chromatogram of L-selenomethionine- 75 Se, third producer, of L-selenomethionine- 75 Se, third producer, handled and developed handled and developed in nitrogen atmosphere, solvent 2 (b ). in air, solvent 2 (b ). IAEA-PL-336/15 199

ACKNOWLEDGEMENTS

The contribution of Dr. Doina Petrea (Department of Chemistry, University of Bucharest) to the experimental part and the discussions with Dr. G.B. Cook (IAEA Laboratory) are gratefully acknowledged.

REFERENCES

[1] SMITH, I., Chromatographic and Electrophoretic Techniques, (2nd Ed.), Heinemann, London (1960) 82. [2] DENT, C .E ., Biochem.J. 43 (1948) 169. [3] ARONOFF, S., Techniques of Radiobiochemistry, The Iowa State College Press, Iowa (1957) 172. [4] PETERSON, P. J., BUTLER, G. W ., J.Chromatog. 8 (1962) 70. [5] HARLAMOV, V .T ., SHUBNYAKOVA, L.P., Metody analiza radioactivenyh preparatov, Atomizdat, Moscow (1965) 58. [ 6] PETERSON, P.J., BUTLER, G .W ., Austr.J.biol. Sei. jj j (1962) 126. [7] TRELEASE, S .F ., DI SOM M A, A . A . , JACOBS, A .L ., Science 132 (1960) 3427.

IAEA-PL-336/16

RADIOMETRIC TITRATION OF INACTIVE AND RADIO-PHARMACEUTICALS

J. TÖLGYESSY Department of Radiochemistry and Radiation Chemistry, Slovak Technical University Bratislava, CSSR and T . BRAUN Eötvös Loránd University, Institute of Inorganic and Analytical Chemistry, Budapest, Hungary

Abstract

RADIOMETRIC TITRATION OF INACTIVE AND RADIO-PHARMACEUTICALS. Radiometric end-point detection‘can be used in the analysis of both inactive and radio-pharmaceuticals. Papers published so far mainly describe the determination of inactive preparations with a radioactive standard solution. Naturally, the method can also be applied to the analysis of radiopharmaceuticals. In most cases precipitate formation was used but recently procedures have been recommended in which the very sensitive complex formation reactions and ion-exchange separátions are used. The latter method was used by the authors for determining cyanocobalamin preparations by titrating the cobalt content with standard EDTA solution after decomposition with ozone. The iron content of Tlnctura Ferri aromatica preparation was determined in the same way. Radiometric titrations may also be applied to the determi­ nation of the specific activity and carrier content of radiopharmaceuticals. In this paper the principles, methods, and the practical aspects of radiometric titrations are treated, together with their applicability to the analysis of pharmaceuticals. The methods developed by the authors are described and some new suggestions given for the analysis of radiopharmaceuticals by radiometric titration.

INTRODUCTION

The authors have been dealing with the analysis of pharmaceuticals by radiometric titration since 1955. The joint research work was done by J.M ajer and P. Schiller in the Institute for Analytical Chemistry of the Pharmaceutical Faculty of the Comenius University in Bratislava; by M. SarSúnová in the County Control Laboratory for Pharmaceuticals, Bratislava; by J . Tölgyessy and J . Kone&iÿ, in the Department of Radio­ chemistry and Radiation Chemistry of the Technical University, Bratislava; and by T. Braun in the Institute of Inorganic and Analytical Chemistry of the L. Eötvös University, Budapest. The theme of the work was the analysis of inactive pharmaceuticals by radiometric titration. According to the experience of the authors, the methods elaborated are also applicable to a series of radiopharmaceuticals. The analysis of radiopharmaceuticals by titration methods worked out for inactive preparations offers even more advantages. To be determined radiopharmaceuticals need not be labelled by radioactive tracers since they are themselves labellëd and the desired component of the sample

201 202 TÖ LG Y E SSY and BRAUN can be readily determined by means of an inactive standard solution. In serial analyses simplified titrations can be used, and the entire titration curve need not be measured since, the end-point can be determined by means of monograms or calibration graphs on the basis of one or two activity measurements. The theory, fields of application, and the instruments of radiometric titrations will not be treated here. For these the reader may consult our monographs [1,2] and a revies [3]. However, the principle of the method will be outlined.

RADIOMETRIC TITRATIONS

Radiometric titration methods can be used in cases where the product of the reaction occurring during titration is easily separable from the . reacting species. They can be used in connection with precipitation reactions (the product of reaction precipitates) and also when the product is separable from the reactants by means of extraction, ion exchange or paper chromato­ graphy etc. The activity of one of the components of the system is measured during the titration as function of the volume of titrant added and the results plotted in a graph. The break point of the curve yields the end-point. Either the titrated sample (solution) or the titrant or both may be labelled. When inactive preparations have to be determined the titrant is usually labelled. It is usual to use an inactive titrating solution for the analysis of'radiopharmaceuticals since it is not necessary for both the titrated solution and titrant to be labelled.

RADIOMETRIC TITRATION BASED ON PRECIPITATE FORMATION

Precipitation reactions are most often used for the purposes of radiometric titration although the sensitivity of the determinations is rather low, typically of the order of milligrams. The sensitivity is related to the solubility product of the precipitate formed during the reaction. Inactive pharmaceuticals are most easily determined by this method. The sensitivity and accuracy of the determi­ nations are similar to those attained by the official methods applied so far but they surpass the latter in their rapidity. In radiometric titrations based on precipitate formation, the function 1 = f(v) is followed, where I is the activity of the supernatant liquid over the precipitate and v is the volume of titrant added. On plotting the titration curve, the end-point can be determined graphically. The determination of the end-point from a titration curve constructed from six points requires 40 to 50 min. The speed of determination depends on the nature and sedimentation rate of the precipitate formed. In the analysis of pharmaceuticals very often several samples have to be analysed within a short time. The method described is not suitable for this task owing to the relatively long time necessary to obtain the entire titration curve. Therefore, simplified methods were developed mainly for determining radioactive solutions with inactive titrants. The end-point can be determined on the basis of two points of the titration curve, viz. the initial activity of the radioactive solution, I0, and IAEA-PL-336/16 203 the activity, I, of the supernatant liquid over the precipitate formed when a certain amount of titrant, v, (less than that required for attaining the end-point) has been added.; The volume of titrant necessary for attaining the end-point, vex , can be calculated from

This simple method is also applicable to the analysis of radiopharmaceuticals. Determinations can be made more accurately and rapidly by other methods elaborated by the ¡authors. In these, nomograms were constructed on the basis of theoretical equations. By means of these nomograms a determination can be made within some minutes, from one activity measurement. These methods are dealt with in detail in the monographs of Refs [1, 2, 5]. 1 The components of pharmaceuticals, which were determined by radio- metric titration (mainly based on precipitate formation), are listed in Table I, together with details of the standard solutions and labelling radionuclides used and the references. By the methods mentioned, other radiopharmaceuticals (e.g. bromine-82-, calcium -47-, sodium phosphate ( 3 2 P)-, sulphur-35-injections, iodine-131 etc.) can be readily analysed by means of inactive standard solutions.

RADIOMETRIC TITRATIONS BASED ON COMPLEX FORMATION

In the radiometric detection of the end-point of complex-forming reactions, the separation of.the components is the most difficult operation. In these reactions, the reacting species and the reaction products are in the same phase, before and after the reaction. Thus, titration of this type can only be carried out by using an auxiliary method of separation: the success of the titration thus depends on the success of the separation. Several methods of separation have been described. Apart from solvent extraction, the method first applied, there are methods that are based on solid indicators, on paper chromatography, on ion-exchange and on focussed electrophoresis. The sensitivity of radiometric titrations based on complex formation depends on the factors known to operate in complexometry. However, in the present instance, sensitivity is also affected by the method of separation. Determinations can be carried out successfully even in 10 " 5 to 10“6 M solutions. In the analysis of inactive pharmaceuticals only ion exchange has been used so far for separation [13, 27-29]. This is also the most promising method for analysing radiopharmaceuticals by radiometric titration, and the application of ion-exchange membranes is very promising [29]. The method will be described by means of two determinations made by the authors. Iron was determined in pharmaceutical iron tincture (Tinctura aromatica) in amounts of tenths of per cent. The method developed can also be used for determining iron in submicro quantities. The determi­ nation of greater quantities of iron is also possible, mostly in routine 204 T Ö LG Y E SSY and BRAUN

TABLE I. ANALYSIS OF PHARMACEUTICALS BY RADIOMETRIC TITRATION

Radionuclide used .Component for labelling Titrant (substance) Notes Refs solution determined Test Titrant solution

Be" 0.1N_ A gN 0 3 - 110mAg Precipitation [6 ,7 ,8 ]

Ca2+ 0.01N oxalic 45 Ca Precipitation: [9,10,113 acid NaOH medium ' pH 12.0

nom. C l" 0.1N A gN 0 3 - • Ag Precipitation: [7 ] acidic medium

c n " 0.1-0.01N - I10mAg Precipitation: [ 6 , 8 , 12] AgN 0 3 neutral or slightly alkaline medium

3 + Fe 5 X 1(T3M EDTA 55 - 5 9 p e Radiochelat. titr. [13] separat, by ion- exchange membrane; 0.2°jo Fe in Tinct. ferri aromatica

I + Ij 0.1N T1ZS04 2 0 4 - j- j Precipitation [9 ,1 0 ,1 4 ]

POJ- 0. IN A gN 0 3 “ 110mAg Precipitation: [7 ] borate buffer, pH 9.0

S O 2" 0.1-0.01N' “ Precipitation: [9 ,1 1 ,1 5 ] 4 Ba01¿ acetone-water medium

nomA s 2o r 0. IN A gN 0 3 - Ag Precipitation: [7 ] acetate buffer

6 5 , Zn2 + 0.025M Zn Precipitation: [9 ,1 5-17 ] K 4[F e(C N )6] HC1 or H2S04 medium (NH4) 2S04 added; at 50 С

131J amidopyrine 0 .1 -0 .0 5M Precipitation: [18,19] KBiI4 acetate buffer, pH 4.0; at 0°C, KI and K2S04 present

nom A barbital, 0. IN AgN03 Ag Precipitation: [ 2 0 , 21] allobarbital, Na2CÛ3 present; phénobarbital, pyridine medium and their Na salts IAEA-PL-336/16 205

nomAg bromadal, 0.1N A gN 0 3 - Precipitation [ 22] bromisoval H2S04 medium

110m . Citrates 0 .IN A gN 0 3 - Ag Precipitation: [ 22]

Ephedrine 0.01N A gN 0 3 ¡ “ 110mAg Precipitation: [2 3 ] hydrochloride 0 .7 -0 .8N H N 03 Ï medium

Emetine 0.1Ñ A gN 0 3 ' - UOmAg Precipitation [ 22] hydrochloride Í Ну oscy amine 0.1 M K B iI4 - 1 3 1 j Precipitation [6 ,1 5 ,2 4 ]

Quinine 0.1-0.05M ' “ 1 3 1 j Precipitation: [6,18,19, KBiI4 HC1 medium; 24,25] KI and K2S04 present

0.01M ! - 32p Precipitation: [6,18.19, H, [P(W20 7)6] HC1 medium 24,25]

Quinine O .l-O .O IN 110mAg ‘ Precipitation: [22,23] hydrochloride, A g N 0 3 neutral or slightly quinine acidic medium sulphate

Codein 0 .1 -0 .0 1 N uomAg Precipitation: [22,23]

phosphate, A gN 03 : NaOH medium codein hydrochloride

Cocaine 0. 01N AgN03 - uomAg Precipitation: [2 3 ] hydrochloride HN03 medium

Lobeline O .IN A g N O j - 110mAg Precipitation [ 22] hydrochloride

131 j Morphine 0 .1 M KBiI4 " Precipitation: [6,24] cf. determination of quinine

0 .0 1 M 32 p Precipitation: [6 ,2 4 ]

h ,[ p( w 2 o 7)6 ] cf. determination of quinine

11 0 m . Morphine 0. IN AgN03 A g Precipitation [ 22] hydrochloride, ethylmorphine hydrochloride

11 0 m . Sodium O .IN A g N O j A g Precipitation: [ 2 2 ] p-am ino- ethanolic medium, salicylate pH 7.0

Oxalie acid, 0. IN AgN03 - I10mAg Precipitation: [ 1 . 22 ] oxalates ethanolic medium

0 . 025M - 59Fe Precipitation: [26] K 4[F e (C N )6] indirect titration 206 TÖ LG Y E SSY and BRAUN

TABLE I (cont.)

Radionuclide used Component for labelling Titrant (substance) ‘ Notes Refs. solution determined Test Titrant solution

u°mAg Papaverine 0.01N AgN03 - Precipitation [22,23] hydrochloride

Pilocarpine 0.01N A gN 0 3 - 110mAg Precipitation [ 2 2 ] hydrochloride

Procaine 0. IN A gN 0 3 - 110mAg Precipitation: [ 22 ] hydrochloride acidic medium

Scopolamine 0. IN A gN 0 3 - UOmAg Precipitation [ 2 2 ] hydrochloride

Strychnine 0.1M KBiI4 131 j Precipitation: [6 ,2 4 ] cf. determination ' of quinine

0.01M 32p Precipitation: [6 .2 4 ] H, [P (W 20 7)6 ] cf. determination ■ of quinine

K2HgI4 - Z03Hg Precipitation [23]

Strychnine 0.01N A g N 0 3 - lloraAg NaOH medium [23] sulphate precipitation

nomAg Theobromine O .IN A g N O j - Precipitation; [ 20, 21] NH4OH medium

Theophylline 0. IN AgNOj - UOmAg Precipitation: [ 20 , 21] aminophylline NH 4OH medium

Thiopental 0. IN A g N 0 3 - 110mAg Precipitation: [ 20 ] IM KNO3 medium

Vitamin B12 10" 5 M EDTA 60Co Radiochelato metric [27,28] titration, separation by ion- exchange membrane IAEA-PL-336/16 207 analyses (as in our case, in the serial analysis of pharmaceutical products), where calibration curves are mainly used. In the titration of Fe3+ ions (labelled with 55 + 59Fe) by EDTA titrant, a negatively charged chelate complex is formed

Fe3++ H2Y2^---- 4 FeY' + 2H+ (2)

Non-reacting Fe3 + . ions are separated from the chelate complex formed during titration by an anion: exchanger. The radioactivity of the chelate complex (solution) is measured.. Although in the determination of Fe3+ ions present in concentrations as in the pharmaceutical product Tinctura Ferri aromatica the titration curves can be evaluated and extrapolated up to the end-point, it is more advantageous to use a calibration curve. To understand this method we refer to Figs 1 and 2, where titration and calibration curves obtained in our experiments are shown. Figure 1 indicates the relation between the activity of different quanti­ ties of labelled chelate f'F eY ) and the amount of EDTA added to the investi­ gated solution. Figure 2 indicates the relation between the activity of labelled chelate and the quantity of iron at constant EDTA titrant volume.

FIG. 1. The relation between different'quantities of labelled chelate and EDTA titrant (Curve 1; indicator: curve 2: indicator +0.28 mg Fe; curve 3: indicator +0.56 mg Fe; curve 4: indicator + 0. 84 mg Fe.

The amount of iron can be determined by the calibration curves shown in F ig .2 as follows: always . exactly the same amount of radioactive indi­ cator ( 55 + 5 9 Fe) is added to the solution under investigation. After the addition of definite quantities of EDTA titrant, the separation is done by means of a cation exchanger and the radioactivity of the labelled chelate (solution) is measured. This measured activity is then marked on the у-axis and from its intercept with the calibration curve constructed for the corresponding (added) quantity of titrant a line perpendicular to the x-axis is drawn which gives the sought amount of Fe3+ [13]. Naturally the method is applicable to iron-containing radiopharma­ ceuticals as well (iron-55 injection — ferric citrate in isotonic solution — iron-59 injection — ferric citrate, ferrous ascorbate). 208 T Ö LG Y E SSY and BRAUN

mg Fe

F IG .2. Calibration curves constructed by using the values in F ig .l. (Curve 1: 4 ml; curve 2:3 ml; curve 3: 2 ml; curve 4: 1 ml, 5 x l0 "3 M EDTA)

FIG. 3. Calibration curves for the determination of cyanocobalamin.

The method elaborated for determining cyanocobalamin (vitamin B 1 2 ) by radiometric titration combined with ion exchange is of outstanding practical importance [27, 28]. The cyanocobalamin to be determined is decomposed with ozone and the cobalt liberated from the complex is then titrated with EDTA solution

Co2+ + H2y 1= — ^ CoY2~+ 2H+ (3)

The amount of cyanocobalamin originally present is calculated from the result of titration. The chelate complex CoY2" formed during titration can be separated from unreacted Co2+ by either of the following two ways:

(1) Separation of the unreacted Co2+ ions using cation exchangers (cation- exchange membranes) (2) Separation of the chelate complex CoY2' formed during titration, using anion exchangers (anion-exchange membranes). IAEA-PL-336/16 209

During the titration process the radioactivity of either the solution or the ion exchanger may be measured. Utilizing the measured activity values, a titration curve is plotted from which the end-point may be read. In the case of serial analyses, it is favourable to use calibration curves. The calibration curves constructed for our experiments are shown in Fig.3. This method might be used successfully for the analysis of radioactive cyanocobalamin preparations (vitamin B12 - 57Co; vitamin В^ - 5 8 Co; vitamin Bi2 - 60Co). Here it is enough to decompose the molecule with ozone and titrate the librated radioactive cobalt with EDTA standard solution.

DETERMINATION OF THE SPECIFIC ACTIVITY OF RADIO - PHARMACEUTICALS AND THE QUANTITY OF CARRIERS

Radiometric titrations are applicable to the determination of the specific activity of radiopharmaceuticals and the quantity of carriers they contain. It is surprising that the method has not yet been used for the investigation of either radiopharmaceuticals or other radioactive preparations although, according to the experience of the authors and to the few published papers, radiometric titration methods were very valuable aids in this field. The use of radiometric titrations in the determination of the specific activity of radioactive preparations has recently been suggested by Kametani, Kumira and Kataoka [16]. This possibility is favourable because the labelling of the test solution is not required. The radioactivity of the substance to be determined serves as its own indicator. The titration is carried out with an inactive titrant. The starting point of the titration curve gives the actual radioactivity of the preparation whereas the concentration of the preparation can be calcu­ lated from the end-point. For example, the specific activity of a 65Zn preparation was deter­ mined by precipitation radiometric titration, using a solution of potassium hexacyanoferrate (II) as titrant and also by radiometric titration based on solvent extraction using dithizone as titrant and CC14 as extractant. The measuring instrument was calibrated with standard 65 Zn preparation. The method, used for "the determination of the quantity of carriers present in radioactive preparations, is based on the combined use of radiometric titrations and isotope dilution analysis [30]. Two titrations are needed for the evaluation: in the first the solution containing the carrier to be determined and the radioactive preparation is titrated and extracted. The second is a similar titration of a similar aliquot, to which a known amount of inactive carrier has been added. The curves for the titration of a solution containing 203Hg and mercury carrier with sodium • diethyldithiocarbamate are shown in Fig.4. The determination can also be carried out in the same solution by adding a known amount of carrier to the solution during the titration process. Mechanical shaking is used to equilibrate the two phases; and the titration is continued. Curves for such a titration of a solution con­ taining 203Hg and mercury carrier with sodium diethyldithiocarbamate are presented in Fig.5. 210 TÖ LG Y E SSY and BRAUN

NUMBER OF DROPS TITRANT

FIG .4. Titration of mercury with sodium diethyldithiocarbamate using two aliquots (a) original solution; (b ) original solution plus known amount of carrier.

F IG .5. Titration of mercury with sodium diethyldithiocarbamate using only one aliquot, (a) original solution; (b) addition of known amount of carrier; and (c) further titration of original solution.

On evaluating the titration curves, the amount of carrier in question (mx) can be determined as follows:

(a) With the aid of end-points determined from the titration curves.

Let mx denote the amount of carrier in question, and m the known amount of carrier added to the solution. Then

where v2 and vx are the volumes of titrant used at the two end-points.

(b) Besides the end-points, two other arbitrarily chosen points on the titration curves corresponding to the same activity value may be used for the calculation which is carried out with the aid of Eq.(4). IAEA-PL-336/16 211

(с) Similarly, by using values of activities in both titration curves at the same titrant consumption, mx can be calculated from

I2 - В т х = т т^Г" ■ч." 2 where ^ and I2 are the activities measured in the two titrations at the same titrant consumption, whereas В is the background activity of the aqueous solution prior to the titration. In this method, in contrast to conventional titrim etry, the result is not necessarily calculated from the titrant concentration, but rather by utilizing the possibilities offered by isotope dilution analysis. In this way, the accurate knowledge of the concentration of. the titrant is not required because, as has been shown earlier, it is necessary only to maintain the concentration of titrant at a constant level throughout the determination. This is an appreciable advantage, especially for extraction titrations. In these, the solutions of organic reagents which are applied as titrant often decompose relatively quickly, particularly at high dilutions. Isotope dilution technique avoids this disadvantage. . The method outlined can be used for the determination of the amount of carrier in radiopharmaceuticals. The determination given above for mercury can be applied to the determination of carrier in m ercury-197 injection (mercuric chloride in isotonic saline). ■ The examples and possibilities of application presented in this paper show that radiometric titrations can also be used in the analysis of radio­ pharmaceuticals. The purpose of the paper has been to show these prospects of the method.

REFERENCES

[1 ] BRAUN, T ., TÖLGYESSY, J ., Radiometric Titrations, Pergamon Press, Oxford (1967); and Akadémiai Kiadó, Budapest ( 1967). [2 ] BRAUN, T ., TÖLGYESSY, J., Radiometrische Titrationen, S. Hirzel Verlag, Stuttgart (1968); and Akadémiai Kiadó, Budapest (1968). [3 ] BRAUN, T ., TÖLGYESSY, J., Talanta 11 (1964) 1277.' [4 ] (This Ref. No. was accidentally omitted when re-numbering the references in the text.) [5] TÖLGYESSY, J., KONECNY, J., BRAUN, T., Nucl. Applications 3 (1967) 383. [ 6 ] MAJER, J., SARSUNOVA, M ., TÖLGYESSY, J ., Pharmazie 14 (1959) 218. [7] TÖLGYESSY, J., SARSÚNOVÁ, M.. Z. analyt. Chem. 195 (1963) 429. [ 8 ] TÖLGYESSY, J ., SARSUNOVA, M ., MAJER, J ., 18th Congr. int. sei. pharm., Bruxelles, 6-15 Sept. 1958. ' [9 ] MAJER, J., SARSÚNOVÁ, M ., TÖLGYESSY, J ., 19th Int. Kongr. pharm. W iss., Zürich, 6-10 Sept. 1969. [1 0 ] MAJER, J., SARSÚNOVÁ, M ., TÖLGYESSY, J ., Schweiz. ApothZtg. 98 (1960) 631. [ 11] SARSÚNOVÁ, M ., TÖLGYESSY,, J., MAJER, J ., Cslká. Farm. 9 (1960) 68. [1 2 ] BEBESEL, P ., SIRBU, I . , Rev. Chim. (Roumania) 11 (1960) 288. [1 3 ] KONEÖNY, J ., TÖLGYESSY, J. „ BRAUN, T ., A ctachim , hung. 51 (1967) 245. [1 4 ] SARSÚNOVÁ, M ., MAJER, J ., TÖLGYESSY, J ., Cslká. Farm. 8 (1959) 567. [1 5 ] TÖLGYESSY, J., Dissertation, Bratislava (1961). [1 6 ] KAMETANI, F ., KIMURA, K ., KATAOKA, A ., Nippon Genshiryoku Gakkaishi (j. Atom. Energy Soc. Japan) 4(1962) 373. [1 7 ] TÖLGYESSY, J ., SARSUNOVA, M ., MAJER, J ., Cslká Farm. 8 (1959) 565. 212 TÖ LG Y E SSY and BRAUN

[1 8 ] MAJER, J., TÖLGYESSY, J ., Acta Fac. Pharm. Brunen, et Bratisl. Ш (1960) 77. [1 9 ] MAJER, J ., TÖLGYESSY, J ., SCHILLER, P ., Sb. chem. Fak. SVST (Rep. Trans. Technical University, Dept, of Chemistry) Bratislava 21 (1960). [20] SARálÍNOVÁ, M., TÖLGYESSY, J., J. analyt. Chem. 196 (1963) 107. [2 1 ] TÖLGYESSY, J ., JESENAK, V ., SARSÚNOVA, M ., Dept. Chemistry, Technical University Rep. 24/03.04d, Bratislava (1963) v [22] TÖLGYESSY, J., SARSUNOVA, M., Z. analyt. Chem. 196 (1963) 192. [2 3 ] SIRBU, I., BEBESEL, P ., Rev. chim. (Romania) 10 (1959) 641. [2 4 ] MAJER, J ., SARSÚNOVA, M ., TÖLGYESSY, J ., 18th Congr. int. sei. pharm., Bruxelles, Rep. 10 6-15 Sept. 1958. [2 5 ] MAJER, J., TÖLGYESSY, J ., 18th Congr. int. sei. pharm., Bruxelles, Rep. 9, 6-15 Sept. 1958. [2 6 ] TÖLGYESSY, J ., Magy. kem. Foly. 65 (1959) 149. * [27] KONECNY, J., TÖLGYESSY, J., Chem. Zvest. 20(1966) 692. [2 8 ] KONECNY, J., TÖLGYESSY, J., SARSÚNOVA, M ., Z . analyt. Chem. 232 (1967) 343. [29] TÖLGYESSY, J., KONECNY, J. , BRAUN, T., Nucl. Applications 3 (1967) 383. [30] KUKULA, F., KRIVÁNEK, M., Chem. Zvest. 20 (1966) 188. [3 1 ] TÖLGYESSY, J ., DSC. Dissertation, Moscow (1968). ГАЕА-РЬ-336/17

SUMMARY OF THE ACTIVITIES OF COMECON WITH REGARD TO THE CONTROL OF RADIOACTIVE MEDICAL PRODUCTS

D. OSTROVSKI Deputy Secretary, Council for Mutual Economic Aid (COMECON)

Short contribution

For several years now the Council's Permanent Committee on the Utilization of Atomic Energy for Peaceful Purposes has been devoting its attention to the promotion of co-operation between the Council's Member States with regard to the use of isotopes in medicine, especially in medical research, diagnostics and therapy. The Committee is concerned with equip­ ment problems involving instruments and apparatus used for these purposes (development, standardization and specialized production of instruments and equipment for medical purposes — scintigraphy units, scintiscanners, gamma-therapy equipment; etc. ). The Committee is also concerned with ensuring that the requirements in respect of radioactive preparations for medical uses can be satisfied (dévelopment of methods of production, stan­ dardization of characteristics), with the study and introduction of radiation methods of sterilizing materials useti in medicine, and with the construction of equipment for this purpose. Radioactive isotopes and labelled compounds of about 200 different types are produced in the Member States of the Council for Mutual Economic Aid for medical purposes. Most of these products are specially manufactured in individual Member States in accordance with the recommendations of the Permanent Committee on the Utilization of Atomic Energy for Peaceful Pur­ poses. The Permanent Committee is constantly seeking to widen the range of radioactive products for medical purposes, trying to raise the quality of the products and to standardize the characteristics of the more important products so as to facilitate1 interchangeability between the Council's Member States. So far, 46 products manufactured in these States have been stan­ dardized (see Annex I). The Committee has worked out and recommended various techniques for controlling the parameters of many of these standardized products: e.g. chromatographic, electrophoretic, potentiometric, spectrophotometric and other methods of determining the content of the basic substance or element, the chemical and radiochemical impurities, and the content of free-iodine ions. The establishment of these methods permits a qualitative comparison between products from the Council's different Member States and the manu­ facture of standardized products of uniform high quality. In accordance with a plan of work established by the Permanent Commit­ tee, the Council's Member States perform comparative measurements of ■ certain parameters of the different products using both preferential and routine control methods.

213 214 OSTROVSKI

ANNEX I

LIST OF STANDARDIZED RADIOACTIVE PREPARATIONS FOR MEDICAL PURPOSES (1969)

No. Isotope Product description

1 24Na Solution of sodium chloride, isotonic, sterile

2 32 P Solution of sodium phosphate, di-substituted

3 32P Solution of sodium phosphate, isotonic, sterile

4 35S Solution of sodium sulphate, carrier-free, isotonic, sterile

42 5 К Solution of potassium chloride, isotonic, sterile

6 51Cr Solution of sodium chromate, isotonic, sterile

7 51Cr Solution of chromium chloride (III), isotonic, sterile

8 58Co Vitamin Bi2 , for peroral administration

9 59Fe Solution of ferrous citrate, isotonic, sterile

10 59Fe Solution of ferrous ascorbinate, isotonic, sterile

11 wCu Solution of copper ethylenediamine tetraacetate isotonic, sterile

12 64Cu Solution of copper glycinate, isotonic, sterile

13 85 Kr Solution of sodium chloride containing krypton-85, isotonic, sterile

14 90Y Granules of yttrium oxide, sterile

15 90Y Granules of m etallic yttrium

16 "mTc Solution of technetium-99m, carrier-free, obtainable from a generator

17 99Mo Solution of sodium molybdate, isotonic, sterile

18 115mln Solution of indium-115m, carrier-free, obtained from a generator, isotonic, sterile

19 125I Solution of Rose Bengal, isotonic, sterile

20 125I Solution of diiodofluorescein, isotonic, sterile

21 125I Solution of sodium iodide, isotonic, sterile

22 125I Oleic acid, for peroral administration

23 l25l Triolein, for peroral administration

24 1S1'I Solution of sodium iodide, carrier-free, containing sulphite and thiosulphate

25 131I Solution of sodium iodide, carrier-free, isotonic, sterile IAEA-PL-336/17 215

ANNEX I(cont.)

No. Isotope Product description

131 T 26 Sodium iodide, carrier-free, in gelatine capsules

131т 27 Solution of human serum albumin, isotonic,sterile

131 t 28 Human serum albumin, (macroaggregates) in isotonic solution, sterile

131-r 29 Solution of Rose Bengal, isotonic, sterile

131J 30 Solution of Bilignost, sterile

131 T 31 Solution of Hippuran, (sodium salt of o-iodohippuric acid), sterile

131T 32 Solution of diiodofluorescein, isotonic, sterile

131, 33 Solution of insulin, for use in vitro

Ш т 34 Solution of cardiotrast, isotonic, sterile

131- 35 Oleic acid

36 I31I Solution of propylinulin chloride, sterile

131т I 37 Glycerine trioleate

38 131j Solution of L-thyroxine

131j 39 Solution of L-triiodothyronine

132j Solution of «sodium iodide, carrier-free, obtained from a generator

41 Ta Metallic tantalum, sterile

42 Au Colloidal solution of gold, isotonic, sterile

43 “Au Colloidal solution of gold, containing silver, isotonic, sterile »

44 Gold pins in platinum filters

45 "Hg Solution of Neohydrin (chlormerodrin), sterile

46 *Hg Solution of mersalyl, sterile

CONCLUSIONS AND RECOMMENDATIONS OF THE PANEL

INTRODUCTION

In terms of its Statute [1] , the International Atomic Energy Agency "shall seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world" (article II). The Agency is authorized "to establish or adopt, in consultation and, where appropriate, in collaboration with the competent organs of the United Nations and with'the specialized agencies concerned, standards of safety for protection of health and minimization of danger to life and property" (article IIIA6 ). In accordance with these principles the Agency has encouraged by various means the production of radioisotopes in its Member States. Two regional Study Group Meetings on Radioisotope Production were organized by the Agency, one at Lucas Heights, Australia in June 1968 and the other at Sao Paulo, Brazil in October 1969. At Study Group Meetings on the Utilization of Research Reactors (Bogota, December 1967; Tokai, October 1967) the production of radioisotopes always constituted an important sec­ tion of the meetings. With developing nuclear centres in mind, in 1966 the Agency published in the Technical Reports Series the Manual of Radioisotope Production [2] . This well-known manual, based on procedures submitted by various Member States, has enjoyed a remarkable success. A new, expanded and revised edition [3] is scheduled to be published. In addition to the inclusion of new chapters on accelerator-produced nuclides and isotope generators, this new edition will include some recipes for the preparation of radiopharmaceuti­ cals. Only compounds labelled with relatively short-lived radioisotopes, such as Hippuran-131I or chlormerodrin-197Hg are included. Although the production of radioisotopes, including those for human use as radiopharmaceuticals-, was given much support by the Agency, less attention has hitherto been given to the quality control aspect-. Taking into account the increasing number of research reactors being used for radioisotope pro­ duction in developing countries, the Agency considers it has a duty towards its Member States to emphasize the necessity for a rigorous quality control of radioisotopes, especially these intended for human use. The present panel, the first of its kind ,tç be held by the IAEA, has been the outcome of this consideration. The following points were discussed:

(1) Role of international organizations in the field of radiopharmaceuticals (2) Organizational aspects of the analytical control of radiopharmaceuticals in production centres (3) Storage and decomposition of radiopharmaceuticals (4) Radiopharmaceuticals and their purity control (5) Data of interest for producers and users

217 218 CONCLUSIONS AND RECOMMENDATIONS

ROLE OF INTERNATIONAL ORGANIZATIONS IN THE FIELD OF RADIOPHARMACEUTICALS

Being both radioactive and pharmaceutical products, radiopharmaceuti­ cals are of interest both to the International Atomic Energy Agency and the World Health Organization. After several discussions with consultants and with WHO officials, an agreement was reached for co-operation between IAEA and WHO in the field of radiopharmaceuticals. To avoid duplication and to make best use of available facilities, manpower and funds, the WHO will be the only organization responsible for drafting specifications to be included in the International Pharmacopoeia [4] as recommendations to Member States. The IAEA will provide technical data (obtained in its laboratories or through research contracts) to the WHO and will contribute more actively to the dissemination and exchange of information in the radio­ pharmaceutical field through meetings, symposia or training courses. Several other multi-national bodies have manifested their interests in this field. The European Pharmacopoeian Commission has prepared mono­ graphs for several radiopharmaceuticals which, when published in the Euro­ pean Pharmacopoeia, will be mandatory for the Member States of the Council of Europe. In the World Health Organization the appendix "Radioactivity" [4] is being reviewed and ten new monographs for widely used radiopharmaceuti­ cals are being prepared. The Council for Mutual Economic Aid has made available a summary of its activity in this field (cf. these Proceedings, LAEA-PL-336/17). The Scandinavian Pharmacopoeia, which includes several radiopharmaceuticals [5] , also represents a co-operative multi­ national effort.

Recommendations

The IAEA should promote exchange of information concerning the quality control of radiopharmaceuticals in producing centres. For instance, electrophoresis is currently employed in some producers' laboratories and is seldom included in pharmacopoeias for checking radiochemical and chemical purities. The IAEA should stimulate exchange of views on new trends in radio- pharmaceuticals. For example, radiopharmaceuticals labelled with very short-lived radioisotopes obtained in the hospital, either from radionuclide generators or from accelerators, are increasingly entering into the medical practice.. It is proposed that the IAEA organize panels and symposia on the problems raised by these developments. Intercomparisons of methods for determining the radionuclidic and, radiochemical purity of radiopharmaceuticals should be continued in the IAEA laboratories on the basis of priority lists submitted by WHO. Since the IAEA laboratory has limited means, it should avoid embarking on pro­ jects which are, or can be, done in national laboratories. An important role of the IAEA resides in its educational activities. It is proposed that the IAEA organize training courses for the production and quality control of radiopharmaceuticals. Also, a participant made the pro­ posal to explore the possibility of a control kit to be distributed among stu­ dents attending these training courses. Another proposal, which was made by the panel, concerned the possi­ bility that the IAEA, which has already drafted recommendations for the CONCLUSIONS AND RECOMMENDATIONS 219 transport of radioactive materials, should also consider the transport of radio pharmaceuticals. Special problems arise, especially in the case of air transport to tropical countries (e.g. refrigerators for radiopharma­ ceuticals on air carriers, customs formalities, etc. ).

RADIOPHARMACEUTICALS AND THEIR PURITY CONTROL

It is recognized that, unlike ordinary pharmaceuticals, radioactive medical products have a-shelf-life determined on one hand by the decay of the radioactive nuclide itself, and on the other hand by the radiation damage. Because of their radioactivity, such products should be care­ fully controlled and should, lead to closer contacts between producer and users. ! It was therefore gratifying to hear from the WHO representative that the necessity of an expiry date to be specified on every radiopharmaceutical [ 6 ] *had been recognized and was to be included in the next revision of the International Pharmacopoeia.

Recommendations 1

While the organization of an analytical control laboratory may differ appreciably from one country to another, the panel urged producing centres to treat the quality control of radiopharmaceuticals with the utmost care. Especially at the beginning of a production program, extensive checks must be made before releasing a product for human administration. A sound practice for established centres is to have about 80-90% staff for production and development, and 1 0 - 2 0 % staff for quality control. The panel underlined the important role of a radiopharmacist in the hospitals where radiopharmaceuticals are administered to patients for diagnostic and therapeutical purposes.

DATA OF INTEREST FOR, PRODUCERS AND USERS

It is recognized that specifications of radiopharmaceuticals must comply with national or international pharmacopoeias and the labelling of radiopharmaceuticals should comply both with pharmacopoeias and with IAEA regulations for transport of radioactive materials. A compromise must bë found between the limited area of the label (which in the ideal case should be able to resist steam sterilization without losing its quality, inscription and adherence) and the amount of information to be supplied. Besides specifications which are being prepared by the WHO (name of product, radioactive, name and location of manufacturer, batch number, total radioactivity at a specified time, expiry date; for solutions, total volume and radioactive concentration; for injections, requirements implied by this word, namely the bacteriostatic agent present and its con­ centration, sterile, pyrogen-free) the following information was recommended to be supplied to the user.

If the radiopharmaceutical is intended for therapy, this should be stated on the label because several accidents (some of them fatal) were recorded 220 CONCLUSIONS AND RECOMMENDATIONS when the physician had mistakenly read mCi for pCi and applied for diagnostic purposes a radiopharmaceutical a thousand times more radioactive than prescribed. Storage conditions (temperature, absence of light) should be indicated when; as in the case of several radio-iodinated products, the shelf-life depends very much on these conditions. Some of this plus further information, such a s impurities present, particle size for colloids, recommended use, etc. could be supplied on an accompany­ ing certificate when the space on the label is insufficient.

REFERENCES

[1 ] INTERNATIONAL ATOM IC ENERGY AGENCY, Statute as amended up to 31 January 1963, IAEA, Vienna (1967) 5-7, Articles II and IIIA . [2 ] INTERNATIONAL ATOM IC ENERGY AGENCY, Manual of Radioisotope Production, Technical Reports Series No. 63, IAEA, Vienna, (1966). [3 ] INTERNATIONAL ATOM IC ENERGY AGENCY, Radioisotope Production and Control", IAEA, Vienna, 1970 (to be published). [4 ] WORLD HEALTH ORGANIZATION, Specifications for the Quality Control of Pharmaceutical Reparations, 2nd Edition of the International Pharmacopoeia, Geneva (1967). [5 ] Nordiska Farmakopenämnden, Addenda, 1 April 1968- 1 April 1969. [6 ] BALABAN, A. T ., "Specifications of radiopharmaceuticals. Possible international aspects of labelled compounds programmes", paper presented at the 2nd Interamerican Conf. Radiochemistry, Mexico City, 22-25 April 1968. LIST OF PARTICIPANTS

The participants whose names are marked by an asterisk acted as chairmen of the Panel Sessions

T. BRAUN Eötvös Loránd University, Institute for Inorganic and Analytical Chemistry, Museum körüt, Budapest, Hungary

J . CIPKA Nuclear Research Institute of the Czechoslovak Academy of Sciences, Rez, Czechoslovak Socialist Republic

* Y. COHEN Commissariat è 1' énergie atomique, CEN de Saclay, B.P. n°2, Gif-sur-Yvette (Seine et Oise) France

G.B. COOK Division of Research and Laboratories, International Atomic Energy Agency, Vienna, Austria

C. FALLAIS Département des radioisotopes, Centre d'étude de 1' énergie nucléaire, Mol-Donk, Belgium

K. FRÜHAUF, Radiochemisches Laboratorium der Farbwerke Hoechst AG, 623 Frankfurt/Main - Hoechst, Federal Republic of Germany

I. GALATZEANU Institutul de Fizica Atómica, Casuta Póstala 35, Bucharest, Romania

* N. G. S. GOPAL Quality Control Section, Bhabha Atomic Research Centre, Trombay, Bombay 74, India

221 222 LIST OF PARTICIPANTS

A. JÁSZ Institute of Isotopes, Hungarian Academy of Sciences, Budapest, Hungary

J . MA 1ER Chair for Analytical Chemistry, Faculty of Pharmacy, Comenius University, Bratislava, Czechoslovak Socialist Republic

*C.E. MELLISH Quality Control Department, The Radiochemical Centre, Amersham, Bucks, United Kingdom

M. MIKHAILOV Reactor Centre, Sofia, Bulgaria (at present: IAEA Fellow, Osterreischische Studiengesellschaft für Atomenergie, Seibersdorf, Austria)

* A. E . MITTA Comisión Nacional de Energía Atómica, Dirección de Investigaciones, Dep. de Química, Av. Libertador 8250, Buenos Aires, Argentina

T. NAGAI Division of Life Sciences, International Atomic Energy Agency, Vienna, Austria

L. ONICIU Division of Research and Laboratories, International Atomic Energy Agency, Vienna, Austria

L. PENTEK Ministry of Health, 5 Akademia u. 10, Budapest, Hungary

J . QUINN III, Division of Nuclear Medicine, Chicago Wesley Memorial Hospital, 250 East Superior Street, Chicago, 111. 60611, United States of America

P. SCHILLER Division of Research and Laboratories, International Atomic Energy Agency, Vienna, Austria LIST OF PARTICIPANTS 223

*H . SORANTIN österreichische Studiengesellschaft für Atomenergie, Reaktorzentrum Seibersdorf, 2444 Seibersdorf; Austria

E . STEINNES Institutt for Atomenergi, P.O. Box 40, Kjeller, Norway

J . TÖLG.YESSY Department of Radiochemistry and Radiation Chemistry Slovak Technical University, Bratislava, Czechoslovak Socialist Republic

*M. DEL VAL COB Sección de Isótopos, Dirección de Química e Isótopos, Junta de Energía Nuclear, Ciudad Universitaria, Madrid-3, Spain

* A. P . WOLF Department of Chemistry, Brookhaven National Laboratory, Upton, L. I. N. Y. 11973, United States of America

REPRESENTATIVES

H.S. GRAINGER Secretary to the European Pharmacopoeia Commission, Council of Europe, Avenue de 1! Europe, Strasbourg, France

J . VACEK Medical Officer, Pharmaceuticals, World Health Organization, 1211 Geneva 27, Switzerland

SCIENTIFIC SECRETARY

A. T. BALABAN Division of Research and Laboratories, IAEA

EDITOR

Monica KRIPPNER Division of Publications, IAEA

IAEA SALES AGENTS AND BOOKSELLERS

Orders for Agency publications can be placed with your bookseller or any of the addresses listed below :

ARGENTINA ■ r HUNGARY Comisión Nacional de Kultura Energía Atómica Hungarian Trading Company Avenida del Libertador 8250 for Books and Newspapers Buenos Aires P .O . Box 149 Budapest 62

AUSTRALIA Hunter Publications INDIA 23 McK illop Street Oxford Book & Stationery Comp. Melbourne, С. 1 17, Park Street Calcutta 16

AUSTRIA

Publishing Section ISRAEL International Atomic Energy Agency Heiliger & Co. Kärntner Ring 11 3, Nathan Strauss Str. P.O. Box 590 Jerusalem A-1011 Vienna

ITALY BELGIUM . Agenzia Editoriale Commissionaria Office International A.E. I.O.U. de Librairie Via Meravigli 16 30, Avenue Marnix 1-20123 M ilan Brussels 5

JAPAN CANADA Maruzen Company, Ltd. Queen’s Printer for Canada i P.O. Box 5050, International Publications 100-31 Tokyo International Ottawa, Ontario

MEXICO C.S.S.R. Librerfa Internacional, S.A. S.N.T.L. A v. Sonora 206 Spolena 51 M éxico 11, D. F. Nové Mesto Prague 1 NETHERLANDS Martinus Nijhoff N. V. DENMARK Lange Voorhout 9 Ejnar Munksgaard Ltd. P .O . Box 269 6 Norregade The Hague DK-1165 Copenhagen К

PAKISTAN FRANCE Mirza'Book Agency Office International de 65, Shahrah Quaid-E-Azam Documentation et Librairie P .O . Box 729 48, Rue Gay-Lussac Lahore - 3 F-75 Paris 5e POLAND U . S . S.R. Ars Polona Mezhdunarodnaya Kniga Céntrala Handlu Zagranicznego ■ Smolenskaya-Sennaya 32-34 Krakowskie Przedmiescie 7 Moscow G-200 Warsaw

U . K. ROMANIA Her Majesty’s Stationery Office Cartim ex P .O . Box 569 3-5 13 Decembrie Street London S. E .1 P.O. Box 134-135 Bucarest U . S. A . SPAIN UNIPUB, Inc. Librería Bosch P.O. Box 433 Ronda Universidad 11 New York, N.Y. 10016 Barcelona - 7

SWEDEN YUGOSLAVIA C. E. Fritzes Kungl. Hovbokhandel Jugoslovenska Knjiga Fredsgatan 2 T eraz ije 27 Stockholm 16 Belgrade

SWITZERLAND Librairie Payot Rue Grenus 6 CH-1211 Geneva 11

IAEA Publications can also be purchased retail at the United Nations Bookshop at United Nations Headquarters, New York, from the news-stand at the Agency's Headquarters, Vienna, and at most conferences, symposia and seminars organized by the Agency.

In order to facilitate the distribution of its publications, the Agency is prepared to accept payment in UNESCO coupons or in local currencies.

Orders and inquiries from countries not listed above may be sent toi '

Publishing Section International Atomic Energy Agency Kärntner Ring I I P .O . Box 590 A-1011 Vienna, Austria

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1970

PRICE: US $6.00 SUBJECT GROUP: IV Austrion Schillings 155,- Chemistry, Geology and Raw Materials/ (£2.10.0; F.Fr. 33,30; DM22,-) Labelled Compounds and Radiopharmaceuticals