Research Collection

Doctoral Thesis

Stability studies of the parental solutions of sympatol and noradrenaline

Author(s): Agrawal, Devendra Kumar

Publication Date: 1960

Permanent Link: https://doi.org/10.3929/ethz-a-000131813

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STABILITY STUDIES OF THE PARENTAL SOLUTIONS

of

SYMPATOL AND NORADRENALINE

THESIS

PRESENTED TO

THE SWISS FEDERAL INSTITUTE

of

TECHNOLOGY, ZURICH

for

THE DEGREE OF

DOCTOR OF NATURAL SCIENCES

by

DEVENDRA KUMAR AGRAWAL Citizen of India

Accepted on the recommendation of

Prof. Dr. K. Steiger—Trippi

and

Prof. Dr. J. BUchi

Job Printers, Allahabad 1960 Printed by Job Printers, 99, HewettRoad; Allahabad

1959—60 PREFACE

Physiological action may be achieved by the administration of drugs in various forms. One of the most rapid means of obtaining such an action is by administering the drug in the form of an injection. The inclusion of various monographs in different Pharmacopoeias relating to in¬ jections points to the significant advantages of such a form of medica¬ ment.

However one of the most intriguing problems before the pharmacist has been that of the stabilization of the galenicals in general and injec¬ tions in particular. In the words of Schou (1); "How is it that a question long known to be of great importance is still characterised by being insufficiently considered if not at all touched."

The present work has been carried out to determine the con¬ ditions necessary for the stability of the parental solutions of two of the sympathomimetic amines : Sympatol and Noradrenaline. ACKNOWLEDGEMENTS

The experimental work in connection with these investigations has been carried out at the School of Pharmacy of the Swiss Federal Institute of Technology, Zurich, under the supervision and guidance of Prof. Dr. K. Steiger. I have feelings of deep gratitude towards my teacher, Prof. Dr. K. Steiger for taking a keen inrerest in me, and giving his valu¬ able advice and guidance throughout the course of this work.

My sincerest thanks are due to Messrs Volkart Foundation, Winter- thur, for their scholarship to assist me to complete my research, which was a material financial help to me throughout the course of my work.

I am also thankful to Dr. Hippenmeier of the Kantons Apotheke, Zurich, for allowing me to work in his laboratories with the Zeiss Spectrophotometer, to Messrs Boehringer Sohn, Ingelheim am Rhein for supplying their original product of Sympatol and to Messrs Hoechst Farb- werke, Frankfurt for supplying Noradrenaline. DEDICATED TO MY DEAR PARENTS ERRATA

Page Errata Correct

2 Aquoeus Aqueous

18 Stndarad Standard

+ + 33 NH3 NH2 1 1

39 apporximate approximate

55 ehl the

77 SB as

119 fig. 15 fig 16

120 fig. 16 fig. 15 fig. 16 (in Table) fig. 15

124 ZUSA M VIE MFASSUNG ZUSAMMEMFA&

124 siehe page siehe seite

125 abbau Abbau

127 Sprtzein Spritzen Hohlandeln Hohlnadeln CONTENTS

Some considerations of Injections and Sympathomimetic amine s

CHAPTER I Page

Injections

.. .. 1. Requirements .. . . 1

of .. 2. Manufacture injections .. 2

3. Stability of injections . . 13

CHAPTER II

Sympathomimetic amines

1. General considerations ...... 16

of 2. Therapeutic uses sympathomimetic amines .. 18 3. Relation between chemical structure and pharmacological activity of sympathomimetic amines 19

4. Classification of sympathomimetic amines .. 20

CHAPTER III

Stability of Sympatol solutions

1. Problem 29 2. Working procedure 29

CHAPTER IV

Sympatol

31 1. Synonyms 31 2. Synthesis 31 3. Tests 33 4. Methods of quantitative analysis described in the literature

.. 34 5. Pharmacological actions

.. 35 6. Clinical uses ..36 •• •• 7. Dosage ..

CHAPTER V

Testing of the materials and apparatus

..37 •• •• 1. Sympatol •• 39 2. Distilled water V1U

Page

3. Ampules .. • • .. .. 39

4. Other materials .. .. 41

5. Apparatus ...... 41

CHAPTER VI

Methods of analysis for Sympatol

1. Quantitative methods ...... 42 for of 2. Colour standards comparison the colour .. 57

3. pH determination .. .. 61

CHAPTER VII

Determination of the stability of Sympatol solutions

1. Principles .. 62 of the solution 2. Preparation .. 62 3. of the Testing ampules .. 64 4. Discussion and conclusions of the results and of colour pH determination .. 70

5. Injection of Sympatol .. 71

CHAPTER VIII

Stability studies of Noradrenaline (NA) solutions with respect to race mization

1. Introduction ...... 72

2. Problem ...... 72

CHAPTER IX

Noradrenaline

1. names Synonyms and proprietary .. 73 2. Synthesis .. 73 3. Physical properties .. 74

4. tests Qualitative .. 74 5. Methods of quantitative analysis described .. 75 in the literature

6. actions Pharmacological .. 78

7. Clinical uses .. 80 8. Dosage .. 80

CHAPTER X

Testing of the material

1. Noradrenaline hydrochloride .. 81 IX

Page CHAPTER XI

Determination of fresh and deteriorated Noradrenaline in solution 1. Introduction .. 83 ......

2. UV. method 84 Spectrophotometric .. ..

3. methods .. .. 87 Colorimetric ..

' 4. Summary ...... 94

CHAPTER XII

Racemizations studies of the dilute solution of Noradrenaline

1. Method for concentrating the solution and for deter¬ mining the optical rotation 96 2. Preparation of the solution 97 3. Results 98 4. Summary of the results of racemization studies 102 5. Discussion and conclusions 105

CHAPTER XIII

Prediction of the stability of the solutions of Noradrenaline with respect to racemization

1. Introduction 106

2. 106 Order of reaction ... 3. Temperature coefficient 108 4. Experimental 109 5. Racemization studies at 50°, 60° and 70° 117 121 6. Summary ..

CHAPTER XIV

Summary 122 References 127

The following abbreviations have been used in the text

° scale = Temperature has been given in centigrade

g = Gramme

ml = Millilitre

AR = Analytical reagent

NA = Noradrenaline Some Considerations of Injections and Sympathomimetic amines

CHEATER I

INJECTIONS .

Injections are sterile aqueous or nonaqueous solutions or suspen¬ sions intended for administration under, or through one or more layers of skin or mucous membrane. This form of medicament has several advantages (2) :—

(a) Instantaneous physiological action is achieved especially if the drug is given intravenously.

(b) Drugs which may be useless or may lose their potency if given orally can be easily administered.

(c) Drugs can be administered even when the patient is unable to take anything orally.

(d) The physician controls the amount of medicament given, and so there is no possibility of over dosage.

1. Reqnirenjents

General requirements for the injections may be summarised as follows :-—

(a) They should be clear, free from dust particles and homogeneous if the substance is insoluble. (b) They should be sterile.

(c) They should be isotonic with blood serum.

(d) They should have the same pH value as that of the blood serum, if the conditions permit.

when (e) They should not give rise to any undesirable effects injected.

(f) They should be stable.

In order to comply with the above requirements it is important the manufacture of that every care is being taken during injections. 2

2. Manufacture of Injections

Butikofer (3) has discussed the different aspects of the manu¬ facture of injections. The following scheme summarises the steps invol¬ ved for their manufacture :

Solvent Active ingredient Additive Container I I ' I test test test test (physical & (physical & .(physical & (alkali) chemical) chemical) chemical) ( I Chromic-acid-bath Pyrogen test Pyrogen test I cleaning I Solution sterilization

Filtration I Filling

Closing

Sterilization I Tests

Sterility Pyrogen pH checking Visual (dust particles, colouration)

Labelling I Packing

Expedition

we shall discuss In the following pages the important points con¬ cerning the requirements of injections. 2.1. Solvent

2. 1. 1. Aquoeus :ol\ents Water for injection is used for the preparation of parental solutions for which most of the pharmacopoeias give a separate monograph. It is prepared by distilling potable or distilled water from a neutral glass still, fitted with an efficient device to prevent the entrainment of droplets.

It is important that water for injection is free:from dissolved gases like carbon dioxide as it could precipitate dissolved 'substances like phenobarbitone sodium, sulphadiazine sodium, theophylline with ethylenediamine, pentobarbitone sodium, sodium dihydrocholic etc., while the presence of oxygen could oxidize such substances as adrenaline, noradrenaline, ascorbic acid, para-amino-salicylic-acid, apomorphine, mersalyl etc. Further it should be free from metallic ions especially iron and copper as they considerably accelerate the processes of oxidation. Schou Gredsted and (4) have investigated the oxygen content of differ¬ ent types of water which is reproduced in table I.

Table I

Oxygen content of water at 20°.

1. Collected directly from the apparatus 1-2 to 2-34 ml/litre

2. Distilled water stored in ordinary containers 6-35 ml/litre

3. Boiled and rapidly cooled 4-0 ml/litre

4. Sterilized by autoclaving in Erlenmeyer flasks, 2-46 to

plugged with nonabsorbent cotton wool. (120*20 min). 3-58 ml/litre

5. Water freed of oxygen through liberation of COa 0-45 ml/litre

by added bicarbonate.

From the above table' it is clear that the oxygen content of water varies considerably under different conditions of storage. In the case of those substances which are easily oxidized it is essential that the water which is used for their solution should contain the minimum amount of dissolved oxygen, and therefore it is recommended to give in the monographs ofinjections of such substances a limit for the oxygen content of the solvent.

2.1. 2. Non aqueous solvents

Fixed oils or isolated esters of the higher fatty acids are generally used as non-aqueous solvents.' They should have no odour or taste suggesting rancidity and should comply with the following tests : (a) Cooling test showing the absence of solid paraffin. (b) Iodine value. (c) Saponification value.

(d) Test for the presence of peroxides. Acid . (e) value.

(f) Test for mineral oils.

(g) Viscosity test.

Beside the above solvents ether, ethyl alcohol, propylene glycol, glycerin, benzyl benzoate and different polyethylene glycols are also used .as non-aqueous vehicles. Recently glycofurol (tetrahydrofurfuryl alcohol-polyethyleneglycolether, containing an average of two ethylene has been glycol groups) reported by Spiegelberg et al. (5) as a very suitable non-aqueous solvent.

2. 2. Isotonicity of the solution

A solution is said to be isotonic with respect to blood serum, when the two show the same osmotic pressure against water. In the following we consider isotonicity only with respect to blood serum. In 4

order to prevent haemolysis it is important that the injections are isotonic, especially when great quantities are to be injected directly into the blood stream.

A o.9% solution of sodium chloride is isotonic with blood serum since it has the same osmotic pressure as that of the blood serum cor¬ responding to a freezing point depession of 0*56° (against water),' though Lund el al. (6) found this value to be 0-52°. The solutions can be made isotonic in one of the following ways :

2. 2. 1. By calculation of the freezing point depression. 2. 2. 2. By sodium chloride equivalent method. 2. 2. 3. By tables of the freezing point depression. 2. 2. 4. By graphical method.

While making calculations by any of the above methods it should be noted that in the case of a solution containing more than one substance, the calculations are valid only when there is no chemical reac¬ tion. Szekely and Goyan (7) have given a critical reveiw of the com¬ monly used methods for making solutions isotonic. 2.2. 1. By calculation of the freezing point depression

Depression of the freezing point follows Raoult's law which can be expressed as :

k. W. 1000. n

t =- where M. L

t = freezing point depression of the solution in relation to the solvent (°C)

k = molar freezing point depression.

W = weight of the dissolved substance (g).

n = number of ions in which the molecule is dissociated.

M = molecular weight of the dissolved substance.

L = weight of the dissolved substance and solvent (g).

Now if it is required to prepare solutions which are to be isotonic with blood serum by means of the above equation, the freezing point depres¬ sion of the blood serum is introduced instead of t, and the molar freezing point depression of a substance which does not dissociate when dissolved in water instead of k, this value being —1-86° when one mole of the substance is dissolved in lOOg of water and then by introducing the values of M and L in the above equation one will get the value of W. The above law is however valid only in case of dilute solutions.

2.2. 2- Sodium chloride equivalent method

Wells (8) and Goyan et al. (9) have described a method based on the fact that the molal* lowering of the freezing point for substances of the

*moles of a dissolved substance per lOOOg of water. same ionic type is fairly constant. They divided the substances in different groups having approximately the same value of molal lowering of the freezing point.

Molal lowering of the freezing point is defined by the equation : A t L = where " C

L - molal lowering of the freezing point (°C).

A t = depression of the freezing point produced by the solution in question (°C).

C = concentration of the solution (moles per 1000 g of water).

The calculations are made by finding out the value of sodium chlo¬ ride equivalent E for a substance by the following equation :

L X 58-45

E = or M X 3-44

E = 17. (L/M) where

M = molecular weight of the active substance.

of active substance L = molal freezing point depression the (°C)

58-54 = molecular weight of sodium chloride. for sodium 3-44 = molal freezing point depression chloride (CC).

The sodium chloride equivalent E for a substance means the amount of sodium chloride which shall give the same osmotic pressure as 1 g of the substance in question when dissolved in an equal volume of water.

The practical application of the above method may be seen from the following example :

Ephedrine sulphate 0*60 g

Chlorobutanol 0*12 g Aqua 30 ml

to make the above solution isotonic.

Amount of sodium chloride required to make the above solution isotonic = 0-9 x 30/100 = 0-27 g.

Sodium chloride equivalent for ephedrine sulphate = 0*60 X E for ephedrine sulphate = 0-60 x 0-17 = 0*10 g.

Sodium chloride equivalent for chlorobutanol = 0*12 X E for chlorobutanol = 0-12 X 0*18 = 0-02 g. 6

Amount of sodum chloride required to make the solution isotonic = 0-27 — (0.10 + 0-02) = 0-15 g.

2.2.3. By tables of freezing point depression

Ph. Helv. V. Suppl. II (10) has given a table of the depression of the freezing point of a 1 % w/v solution of the adjusting substances commonly used in practice. The weight of the substance required to make the solution isotonic is then calculated from the follwoing formula :—

0-56 — a

W = where b

W * weight of the adjusting substance (g).

of blood serum 0-56 = the depression offreezing point (°C).

the a = depression of the freezing point of unadjusted solution (°C).

b = depression of the freezing point of a solution of the ad¬ justing substance (1 g +water to make 100 ml)

The values of a and b can be read directly from the table. In case of solutions having more than one substance, the value of a is obtained by adding the depression of the freezing point of different substances.

The utility of the above method is limited for substances having a linear.relationship between the depression of the freezing point with in¬ creasing concentration of the substance in solution.

2.2.4. Graphical method

Lund et al. (6) have worked out a graphical method for making the solutions isotonic. The method has been adopted in the Ph. Dan. IX. They plotted the experimental values of the depression of the freezing point first in a system where the ordinate is the log of the concentration and the abscissa is the log of the freezing point depression. The straight line thus formed is then moved into a corresponding arithme¬ tical system, where the ordinate is the concentration and the abscissa the freezing point depression.

Now by drawing a "mirrored" sodium chloride curve, starting from the point on abscissa which corresponds to the freezing point depression at which the prepared solution is to be fixed, it is possible to read directly the amount of sodium chloride to be added (see fig. I).

In the following figure the amount of sodium chloride required to make a 2 % w/v solution of resorcinol isotonic with blood serum is is on the ordinate at d. 0-325 g, which directly obtained point ,

Cone, in % . 30 —

^^Resorcinol a i 20

10 '

Sodium Chloride 1 ~"

"~ |i-^_. c

' 'oi 02 '0-3 0-4 05 0-6

Fig. I. 2.2.5. Test for the isotonicity of the solution This may be simply done by determining the depression of the freezing point of the solution by means of Beckmann's thermometer, or indirectly by the thermoelectric method oi Hill (11), in which the de¬ pression of the freezing point is indirectly measured by the difference of vapour pressure of a 0'9 % solution of sodium chloride and that of the solution in question. Lund et al. (6) have used the apparatus oi Hill with slight modifications to determine the depression of the freez¬ ing point of several substances. 2.3. Clarity of the solution

A perfectly clear solution may be obtained by the use of sintered glass funnels, of which several makes are available in the market, (Jenaer glass filter, Pyrex glass filter). In table 2, a summary is given of the different tvpes of filters available, as well as their practical applications (12)- Table 2

Summary of the different types of Pyrex and Jenaer glass filters with their porosity no., diameter of the pores and applications.

Porosity number Jenaer or French English pyrex Practical application! pyrex filters filters

Calcuculated diameter of the pores in /i

1 90 — 150 100 — 120 for heavy precipitates, 2 40—90 40—50 for preparative work with medium sized crystals or precipitates.

15 — 40 20 — 30 for preparative work with fine ppt. or analytical work with medium fine ppt.

5 — 15 '5 — 10 for preparative and ana¬ lytical work with very fine precipitates.

1 — 1-5 1-2 — 1-3 for bacteria-proof filtration. 8

Beside the above filters the filters mentioned below are also often used when large quantities of the solution have to be filtered :

(a) Asbestos filters : In this category different types of Seitz fil¬ ters, Filtrox filters and many others are used.

(b) Candles of kieselguhr and porcelain, of which different makes are available, are also extensively used.

(c) There are also many synthetic filters available of which the following may be mentioned : polyvinyl chloride filters, polytetrafluorethylene filters etc.

(d) Metal filters : some types of metal filters are also availa¬ ble which can be used when the solution does not react with the metal.

Ordinary filter paper is not suitable as it usually gives solutions with fine fibres. Further for clarity of the solution it is essential that the container is properly cleaned and freed from dust and glass particles which we shall discuss under the next heading.

2.4. pH of the solution

Solutions for injection should maintain their optimum pH value during heat sterilization and on storage as it plays a very important role with respect to the following :

(a) precipitation of the free base from the alkaloidal solutions, for example : solutions of morphine, strychnine, etc.

(b) hydrolysis of ester alkaloids and glycosides, e.g. atropine, scopolamine etc.

(c) oxidation of such important drugs like adrenaline, apomor- phine, eserine etc.

(d) change in. optical rotation. i Beside the above changes, pH can also effect the stability of collo¬ idal substances and emulsions.

Since most of the parental solutions are dispensed in glass containers -it is important that they should not impart any alkalinity to their contents. Pharmaceutical preparations of varying composition are stored in glass containers under different conditions of storage, so the containers are required to fulfil several specifications which we shall discuss below :

2.4.1. Tiie glass containers should be chemically resistant

Chemical resistance is the most important requirement for glass con¬ tainers. To fulfil this condition glass is required to have certain specific compositions. In table 3 compositions of typical glass containers, glass tubing and some chemically resistant glasses is given. Table 3 after Dimbleby (13) and Fahrig (14)

Percentage composition of typical container glasses, tubing and some chemically resistant glasses.

Constituent Colourless containers Soft soda Colourless Pyrcx Jenaer oxides British U.S.A. tubing neutral glass apparat¬ ampule us glass-20

SiOa 73-4 73-3 7012 670 80-7 74-5 Ba03 ~ —— 0-78 7.5 120 4-6 to 12-6

Ti03 004 1 ] U.S. n.s. 005 _ A120, 0-75 > 0-48 }• 2-58 8-5 2-2-30 8-5 Fe2o;r 0045 J J n.s. 008 —

MnO — — n.s. — CaO 8-9 5-3 5-4 40 0-20 0-8 MgO 0-1 39 3-6 0-3 — 01 Na20 15-9 16-31 16-82 8-7 3-9-41 7-6 K20 0-4 1 0-35 40 — —

— As2Or 001 — — — Z. 001 S

Coefficient of — _ 9-6 7-3 3-3 linear expansionXlO^

n.s. ;= not stated, n.d. = not determined

2.4.1.1. Test for chemical resistance of glass continers

All glass containers used for injections are required to comply with the tests for the limit of alkalinity of glass as given in different pharmaco¬ the crushed poeias. There are generally two types of tests, one being Brit. glass test, U. S. P. XV (15), and the other being the surface test, Ph. 1958 (16), Ph. Helv. V. (17).

In the crushed glass test a certain amount of powdered glass is au- amount of re¬ toclaved for a definite period together with a specified distilled water of a defined pH. Then the water is titrated aginst stan¬ used is a measure of dard acid for any given alkali. The amount of acid the alkalinity of the glass. Thus the quality of the whole galss is tested. The principle of the surface test as given in Brit. Ph. 1958 and in Ph. Helv. V. is the same, i.e. the neutralization of a limited amount of acid. However Ph. Helv. V. directs that the interior surface of the container should be calculated and a quantity of the standard acid + indicator thus that the of the per unit area of the surface added, ensuring quality glass is tested and the size eliminated as a factor. But by this method large containers are subjected to a much more rigorous test than small con¬ tainers.

On the other hand Brit. Ph. 1958 directs that the container should be filled to its prescribed capacity with the standard acid + indicator.. In this case the volume of the container is the deciding factor of the result.' with the volume of the For if one compares the ratio of the surface area 10

standard acid, it is obvious that the smaller containers will be subjected to a much more rigorous test than the larger contianers of the same quality of glass. In table 4 the approximate surface area of different sizes of containers is given together with the quantity of 0.1 N acid per 100 ml of the test solution for different pharmacopoeias.

Table 4

Surface area and the amount of acid per ioo ml of the test solution according to different pharmacopoeias

Nachtrag;. DAB6 Capacity Inner Ph. Helv Ph. Dan . U. S.P. Codex. Brit. surface V. bottles ampules IX. XV Gall. 7. Ph. 1958.

cm2 ml of 0-01 N acid „

1 ml 5-9 1-18 (2-1) (0-42) 0-9 1-4 1-5 1-66

10 ml 28-8 0-57 2-1 0-42 0-42 1-4 1-5 1-66

100 ml 98-2 0-196 0-95 019 019 1-4 1-5 1-66

500 ml 306-2 0-122 0-55 0-11 011 1-4 1-5 1-66

1000 ml 511-1 0-102 0-43 0086 0086 1-4 1-5 1-66

By looking at the above table it is interesting to note that the amount of acid per 100 ml of the test solution for 1 litre containers is about 15 times less compairing the Ph. Helv. V. or the Ph. Dan. IX. methods with those of U. S. P. XV, Brit. Ph. 1958 or Codex Gall. 7.

Some work has been done in the laboratories of this institute on this problem and on the basis of these results the following quantities of acid to be used for different containers is being recommended :

Table 5.

Recommended acid for alkali test of different size of containers.

Sterilization Capacity of ml of 0.01 N ml of water neutralized time at 120° container H2S04 with methyl red

20 min. up to 2 ml 1-0 99 0

20 min. 2 to 10 ml 0-9 991

30 min. 10 to 100 ml 0-8 99-2

40 min. 100 to 500 ml 0-5 99-5

50 min- 500 to 1000 ml 0.4 99-6

Beside the above official methods, the method of Mylius (18) is worth mentioning in which the quality of the glass is tested by determining the '• the container. amount of [Na201 per 100 cm of the surface area of Berry (19) and Fahrig (14) have discussed the methods which are used for testing the quality of glass containers. 11

2.4.2. The glass containers should be mechanically strong

Containers for parental solutions should be strong enough so as not to give way on sterilization or during handling. Christensen (20) has worked out a formula by which the pressure exerted on the container during ster¬ ilization can be calculated. Miinzel (21) has used his method to test the infusion bottles which are used in Switzerland.

2.4.3. The glass containers should resist thermal shocks

The thermal coefficient of expansion of glass containers should be such that on heating for sterilization and on cooling the container does not break. In table 3 on page 9 the coefficient of linear expansion of a few types of glass is given.

2.4.4. The glass should be easy to melt and seal and should not ' splinter on opening

The glass for containers should be such that it can be easily melted and sealed. This property mainly depends on the silica content of the glass. From table 3 on page 9, one may see that the silica content of soft soda tubing or ampules is much less than that of Pyrex or Jenaer glass. In order to avoid glass splinters in the container care should be taken while opening and cleaning. In an investigation by Dreweny (22) in which he examined the finished ampules from different firms, he found that in some cases glass splinters were present. Further he showed that the amount of splinters was reduced to a minimum when the ampules "were opened by downward pressure instead of horizontal or vertical pressure. He also proposes a method in which the ampules are opened after heating at 250°. By this method he showed that the amount of splinters in the containers become negligible.

2 4.4.1. Test for splinters and otl.er foreign particles

This is done by viewing the contents in incident light against a white and a black background. The details of the method are given in U. S. P. XII (23).

2. 5. Sterility of the solutions

Solutions for injection may be sterilized by one of the following methods. The choice of the method however depends on the properties of the medicament.

(a) Sterilization by dry heat in the hot air oven. This method is used for the sterilization of non-aqueous solutions, since the sterilization in an autoclave has the same effect as that of the dry heat which is not adequate to kill the spore- bearing bacteria due to the inability of the steam to penetrate below the non-aqueous solutions.

(b) Sterilization by moist heat, i.e,. by heating in an auto¬ clave in saturated steam under pressure. This method is commonly used for aqueous solutions of those substances which do not get decomposed by heating at high tempera¬ tures. 12

(c) Sterilization in presence of a bactericide. This method is for applied those substance which can not be heated above 100' without decomposition.

Sterilization the use of (d) by bacteria-proof filters or by the use of aseptic methods. These two methods are used for those substances which can not be heated sufficiently to kill micro-organisms without undergoing decomposition.

In table 6 the time is given for different methods of sterilization as mentioned in different pharmacopoeias .

Table 6

Table showing the different methods cf sterilization together with the temperature and duration as given in different pharmacopoeias.

Pharmacopoeia Method of sterilization

Dry heat Saturated steam In presence of a under pressure bactericide temp •C time tempt "C time tempt"C time

Brit. Ph. 1958 150 1 h 115-116 30 min 98-100 30 min

Ph. Int. I 150 2h 115-116 30 mm 98-100 30 min

Ph. Dan. IX 140 3h 120 20 min 160 2h

Ph. Helv. V 160 1-5 h 110-120 15-20 min 100 30 min 120 2h

Codex Gall. 7 120 1 h no 20 min 100 30 min

Ph. U.S.S.R. 8 120 12-15 min

Nachtr. DAD 6 130 1 h 120 15 min 140 45 min 180 20 min 200 10 min

For a detailed study of the subject we refer to the works of Baumann (24), Stick (25), Cazzani (26) and that of Lesure and Lavagne (27).

2.5.1. Tests for Sterility

Injections sterilized by method number c, b or c are generally not required to be tested for sterility, however, when the injection has been prepared by filtration or by means of aseptic techniques it must be tested for sterility. Most of the pharmacopoeias give the details of the sterility test. The principle of testing the sterility is the creation of optimum conditions for bacterial growth. The sample passes the test if the growth of microorganisms does not occur in any of the incubated tubes. 13

3 Stability of injections

One of the important obligations of the pharmacist is to market phar¬ maceutical preparations that will maintain their label therapeutic value and initial appearance for the duration of their shelf life.

Different destructive processes which affect the stability of injections into three broad : may be divided categories 3.1. Biological (due to the development of micro-organisms)

3. 2. Chemical, which may be due to any one of the following processes 3.2.1. Hydrolysis.

3.2.2. Oxidation.

3.2.2. Racemization.

The above processes are mostly dependent on light, temperature, pH, oxygen and enzymes.

3.3. Physicl factors :

3.1. Biological

The development of micro-organisms can be checked very easily by sterilization or by the addition of a suitable bacteriostatic agent.

3.2. Chemical

the 3.2.1. Hydrolysis : It may be explained by following general equation : R-C-O-R+H 0-> R—COOH+R-OH " u O

It is an important destructive factor in the case of all esters, many alkaloids like atropine, scopolamine etc. and local anesthetics like procaine etc. The process is dependent on pH and temperature.

3.2.2. Oxidation

Oxidation plays an important role in the case of all oxidizable substances like adrenaline, noradrenaline, ascobic acid, ergot alkaloids, apomorphine, physostigmine, eserine etc. The process is dependent on oxygen pressure or on the presence of 02 donors, temperature, pH and

is easily catalysed by different ions like Cu+ ,Fe+^ . It may be checked by the replacement of oxygen with an inert gas, the use of anti¬ oxidants in the form of reducing agents and by inactivating the above mentioned ions.

3.2.2.1. Inert gases. In many instances replacement of air with an inert gas like nitrogen or carbon dioxide helps to check the oxidation. For example: Whittel (28) reported that the injections of sulphonamides can be protected from colouration if air is replaced by nitrogen. Foster and Stewart (29) showed that injections of ergometrine when filled with 14

nitrogen retained 60 % of the activity after a period of about five years while those filled with air were completely inactive. Brunnhofer and Steiger (30) showed that indigocarmine solutions in ampules filled with oxygen lose 63 % of their chemical activity while those filled with nitrogen lost only 6 %.

3.2.2.2. Antioxidants. So far the most commonly used antioxidants are sodium and potassium metabisulphite. They act by being more readily oxidized than the material, having the following reaction :

NaaS.,04 + 0, + H30 = Na3S04 + HaS04

Berry and West (31), West (32) showed that in the pesence of 0.1 % of potassium metabisulphite and at pH 4.2 the solutions of adrenaline are least oxidized. West (33) further found that for solutions of noradrenaline 0.1 °/„ of sodium metabisulphite and a pH of. 3.5 fo 3'9 are essential to check the oxidation. In table 7 a comparative list is given of the antioxidants used as mentioned in different pharmacopoeias.

Table 7

Injection of Antioxidant Ph. Int. I Brit. Ph. U.S.P. Ph. Dan. 1958 XV IX

Adrenaline Na metabisulphite 0.1% 0.1 % none 0 05 •>/„

Adrenaline & Na metabisulphite — 0.1 % none 0.1 % Procaine

Hydromorphine Na metabisulphite — — — 0.05 %

Morphine Na metabisulphite none 0.1 % none —

Metaoxedrine Na metabisulphite — — — 0.05 %

Menadioni Na metabisulphite — — — ;0.l %

Noradrenaline Na — — metabisulphite none 0.05 %

Oxedrine Na metabisulphite — — — 0.6 %

Na — Physostigrainc metabisulphite 0.05% — —

Procainamide — Na — metabisulphite 0.1 % —

Na — — Stibophen metabisulphite 0.1 % none *

Vitamin A & D Nordihydroguajaretic acid DAK 7th ed.

+ + 3.2.2.3. 'Effect of different ions. Schou (34) reported that Cu acts as a catalyst on autooxidative processes and thus can affect the of stability parental solutions to a large extent. Girard and Kerny (35) that the of reported injections adrenaline deteriorated in the presence of metallic ions. Pfeiffer and Offermann (36) reported that ethylenediamine 15

tetraacetate retards the oxidation by forming a complex with metallic ions, + + + e.g. Cu "*"+, Fe etc, which in turn does not catalyse the reaction. Solutions of ascorbic acid (37) and of para-amino-salicylic-acid (38) have been reported to be stable in presence of ethylene-diamine-tetra acetate.

3.2.3. Racemization

The process of converting an optically active compound into its optically inactive modification, containing an equal amount of the op¬ tically active components, is called racemization.

500S*

Optically inactive mixture

The mechanism which by racemization takes place is not always clear, but where tautomerism is it possible may be explained on the basis (39):

G — c - 0

The laevorotatory form of such important natural occuring alkaloids like hyoscyamine, alkaloids, ergot ephedrine etc. and of adrenaline, no¬ radrenaline etc. is physiologically much more active than the racemic form. Racemization takes place as a function of pH and temperature. Kisbye and,Schou found that the (40) solutions of adrenaline are most stable at 4.2 with pH regard to racemization. Kisbye (41), Kisbye and Boh have (42) investigated the relation between pH and racemization of many sympathomimetic amines. Their results show that the specific rotation is on highly dependent hydrogen ion concentration, as the rotation shows a marked change in alkaline solutions.

3.3. Physical factors Separation of suspensions, emulsions, colloidal solutions, change in size of the suspended crystals, e. g. of hormone preparations, in of change viscosity nonaqeous solutions etc. are a few of the physicl factors which can affect the appreciably stability of some parental soulutions and make them unfit for dispensing. CHAPTER II

SYMPATHOMIMETIC AMINES

1. General considerations

Sympathomimetic amines are a class of basic compounds the general characteristics of which are to mimic or act like the effects of stimulating the sympathetic nerves to the organs of the body.

The human body is controlled by two types of nerves : sensory and motor. Sensoiy nerves carry impulses away from the surface or organ while motor nerves carry impluses towards the surface or organ. Motor nerves are of two types: voluntary and autonomic (vegetative or involun¬ tary). Voluntary nerves control the limbs which are directly operated by the the will, while autonomic nerves control organs of the body, e.g. heart, liver etc. Autonomic nerves have been further divided into two types of nerves: sympathetic and parasympathetic. Most organs which are innervated by autonomic nerves possess both sympathetic and parasympathetic nerves which are almost antagonistic to one another in function. The division is as follows :

NERVES

SENSORY MOTOR Nerves carrying impulses Nerves carrying impulses towards the surface or from the surface or away organ organ I |

VOLUNTARY . AUTONOMIC Nerves controlling the limbs Nerves controlling the organs of the body

SYMPATHETIC PARASYM- PATHIC

nerves have their in the Sympathetic origin thoracolumbar segments of the spinal cord. The effects of stimulation of the sympathetic and parasympathetic nerves on the-chief organs of the body are given in table 8 17

Table 8

The effects of stlmnlation of the Sympathetic and Parasympathetic nerves on the chief organs of the body

Organ Sympathetic Parasympathetic

in Blood-vessels Constriction, except coronary Nil (except certain special vessels which arc dilated. cases where dilation occurs, and iti the coronary vessels which are constricteJ.

Heart-rate Acceleration and augmenta- Inhibition, tion.

Eye: Iris Contraction of radial musck Contraction of circular muscle (Mydriasis). (Miosis).

Ciliary muscle Nil. Contraction.

Skin : Sweat secretion Augmentation Nil.

Erection of hairs Increased Nil.

Salivary glands Slight viscid secretion Free secretion and vasodila¬ tion.

Stomach : Contractions Inhibition Augmentation.

Secretions •• Increase.

Sphincters Contraction'or relaxation Relaxation or contraction.

Intestinal movements Inhibition Augmentation.

Gall bladder Relaxation Contraction.

Liver Glycogenosis Nil.

Spleen Contraction Nil.

Pancreatic secretion •• Increase.

Bronchial muscles Relaxation Contraction.

Bronchial secretion Nil increase.

Suprarenal glands Secretion Nil.

Ureter Relaxation Contraction.

Bladder: Fundus Relaxation Contraction.

Sphincter Contraction Relaxation.

Uterus Contraction and relaxation

Wihon and Schild (43). 18

Stimulation of sympathetic or parasympathetic nerves results in the liberation of acetylcholine at the synapse and the transmission of the nerve impulse from pre to post ganglionic fibres is achieved by the inter¬ vention of acetylcholine. However in case of parasympathetic division acetylcholine is liberated at the post-ganglionic endings, while in case of sympathetic division sympathin is liberated at the post-ganglionic endings. The liberation of acetylcholine and sympathin in the autonomic nervous system is illustrated below (44).

Sympathin Sympathetic Q^Zj^Von 1st neuron i f£3T reacting cell Acetylcholine

1st neuron

-&V-"' Parasympathetic Acetylcholine

The concept of chemical mediation of nerve impulses to various or¬ gans was developed by Loewi (45). Cannon and Rosenblueth (46) suggested that Sympathin was more than one substance, or a mixture of substances. (Excitatory effect was attributed to Sympathin E, and Inhibitory effect to Sympathin I). Later the experiments of Euler (47), (48), Bulbring and Burn (49), Gaddum and Goodwin (50), West (51) and others confirmed that Sympathin was a mixture of Adrenaline and Noradrenaline. Sympathin E being pure 1-Nor-adrenaline and Sympathin I being pure 1-Adrenaline.

2. Therapeutic uses of Sympathomimetic amines

Sympathomimetic amines have got a wide use in therapy. Below we shall mention only their mam clinical uses (52), (53) based on the effects : following ,

' 2.1. Vascular effects -:

In control of haemorrhage of skin and mucous membranes for congestion of nasal mucosa in cases of chronic and vasomotor rhinitis, si¬ nusitis, acute coryza, etc. and in conjugation with local anesthetics, etc.

2.2. Cardiac effects

In cases of cardiac arrest, acute cardiac failure, spinal anesthesia and in post operative shocks, etc. 2 3. Allergic disorders

In bronchial asthma, miscellaneous, allergic disorders like serum reactions, serum sickness, hayfever, etc.

2.4. Miscellaneous uses

In severe hypoglycemia, chronic urticaria, milder forms of whooping cough, mydriatic uses, in cases of narcolepsy, depressions, etc. Besides the above mentioned uses, sympathomimetic amines have a wide field of clinical got application for which the stndarad works on and Pharmacology Therapeutics may be referred to. 19

3. Relation between chemical structure and pharmacological acti¬ vity of sympathomimetic amines

No other known group of chemically related and pharmacologically similar compounds reveals such a high degree'of positive corelation between structure and activity as is revealed in the case of sympathomi¬ metic amines. Barger and Dale (54) were the pioneers to investigate the relationship between chemical structure and activity of sympathomi¬ metic amines.

will . In the following pages only the main points be metioned The the literature on the subjct is very large, however, following authors have given excellent reviews on the subject : (55) (56), (57), (58).

18 « 3.1. Alkyl amines CHS— (CH2)n—CH-CH3 ( ' 1 1 N A

(a) In alkyl amines a minimum of a four carbon atom structure is required for sympathomimetic activity ; maximum activity is achieved with a carbon chain of six, while on further increasing the carbon chain, activity decreases with increasing toxicity.

in (b) The optimum position of the amino group seems to be this is not the case. the o< or fi position, though always

loss of (c) Substitution in the amino group results in the activity.

/3 '«

3.2. Phenylalkylamines ^—G—G-N<

of the side (a) An alcoholic group on the ft position aliphatic chain generally increases the pressor activity, e.g. propa- benzidrine. drine is seven times more active than

reduces the pressor (b) The presence of an << methyl group with an increase activity, however, it confers great stability in the case oi in duration of action and oral activity as chain to more than ephedrine. By increasing the carbon is affected three carbon atoms pressor activity adversely with increased toxicity.

activity is some¬ (c) By saturating the phenyl ring pressor of action is increased. what diminished, though the duration 20

1 amines HO—/==\_ci 3.3. Monohydrox> alky c N< ^ I I

(a) By introduction of a phenolic group pressor activity is very much enhanced. Meta and para compounds being equally active, though ortho compounds are feebler.

(b) Substitution on the N atom results in the loss of pressor activity.

3. 4. Dihydroxyphenylalkylamines

OH I jB «

HO—<\ A-O-C—N< I I

(a) By the introduction of two OH groups in the benzene ring in meta and para positions in relation to the aliphatic side chain, the compound becomes truely sympathomimetic and the activity is very much increased, as in the case of adrena¬ line. But these compounds are very unstable due to the presence of catechol nucleus.

(b) By methylating the amino group pressor activity is reduced. For example : noradrenaline is 5/3 times as potent as ad¬ renaline when tested on the blood pressure of the cat. However the methyl group seems to confer bronchodilator property which is well marked in adrenaline.

(c) If the methyl group attached to the amino group is replaced by higher aliphatic groups, the activity is changed from pressor to depressor, though the compounds retain the peri¬ pheral properties Of adrenaline, e.g. isoprenaline, aleudrine, neo-epinipe, etc. art powerful bronchodilators.

4. Classification of Sympathomimetic amines

In the following table (no. 9) those sympathomimetic amines which are commonly used (59) are listed together with their chemical name, formula and poprietary names in the following order of classification : 4.1. Alkylamines 4.2. Cycloalkylamines 4.3. Arylalkylamines 4.3.1. Phenylalkylamines 4.3.2. Monohydroxy or methoxyphenylalkylamines 4.3.3. . Dihydroxyphenylalkylamines 4.4. Miscellaneous TABLE 9

having 4.1. Alkylaimnes a Carbon chain of from 4-8 atoms

General formula : CH3-(CH2)n-CH-CH3 NH„

Chemical name and other commonly used names Formula Proprietarynames

2—Amino-4-methyl-hexane-carbonate CH3—CH2—CH—CH2—CH - NH3 .C03 Forthane (Lilly)

I I CH, CH, r + 2-Methylamino-heptane-hydrochloride CH3-(CH,)4- CH-NH2-CH8 — Oenethyl (BilhuberKnoll). .CI CH, 4- 2-Amino-heptane-sulfate CH3-(CHa)4-CH-NH3 .so. Tuamine sulfate (Lilly) Tuamineheptane sulfate Heptedrine (Roger Bellon) CH, Pacamine (Lab. Hue Paris)

4.2. , . Cycloalkylamines

General formula : • CH,2 — CH — CH„ o 3 I /nh-ch,

\ 1-Cyclohexyl-2-methylamino-propane / — CH2-CH-NH-CH,

I Benzedrex (S.K. F.) Propylhexedrine CH, Chemical name and other commonly used names Formula Proprietarynames

4- Q-CH9-CH-NH9- CH, Gyclohexyl-isopropyl-methylamine-hydro- .CI Eventin (Minden) chloride. Di- (cyclohexylethyl)methylamine-hydro- 0-CH9-CH2 + chloride NH-CH, .CI Gyverine hydrochloride o -CH.-CH,/ 4.3. Arylalkylamines 4.3.1. Phenylalkylamines dl-1 Benzedrine K. -Phenyl-2-aminopropanesulfate ^J-CH2-CH-NH3 so4 (S. F.).,Elas- tonon (Nordmark Werke, Amphetamine CH3 Ortedrine (Specia) + . 1 dl-1 -Phenyl-2-aminoethanol-sulfate Q-CH-CH,-] NH, so4 1 Phenylethanolamine OH Apophcdrin (RiedeldeHaen)

d-l-Phenyl-2-amino-propyl-sulfate Q-CHa-CH-NH3 . so4 Dexedrine (S.K.F.) d-Amphetamine Maxiton (Delagrange) d-Benzedrine CH 3 J 2 Phenedrine (Grimault) + 1-1 -Phenyl-2-methylamino-propanol-hyd- Q—CH—CH—NH3—CH CI rochloride I I 1-Ephedrine OH CH, Chemical name and other commonly used names Formula- Proprietarynames

dl-l-Phenyl-2-methylamino-propanol- hydrochloride o-? CH—CH—NH2—CH, CI Ephetonin (Merck) Racephedrine hydrochloride 1 Racedrin (Hoechst) OH CH, l-l-Phenyl-2-methyl-ethyl-amino-pro-',P-— + panol-hydrochloride. 6^)—CH—CH—NH—C3HB CI Nethamine *—^ (Merell) I 1 I 1-N-Ethylephedrine-hydrochloride|

I. OH CH. CH3 l-Phenyl-2-methylamino-propane-hyd- ' rochloride ^ y)~CH2—CH—NH2—CH3 CI Pervitin '(Temmler),''Desoxyne d-Desoxy-ephedrine (Abbott),Isophen (Knoll) K5 CH3 Methedrin (BurroughsWellcome)

' dl-l-Phenyl-2-amino-propanol-hydro- 4- chloride -CH—CH—NH3 CI Propadrine (Merck, Sharp & dl-Nor-Ephedrine 1 1 Dohme) Phenylpropanolamine OH CH,

dl-l-Phenyl-2-methylamino-propane-* ^-CH-CH^NH^CHg. CI Vonedrine (Merrell) hydrochloride CH, CH3 1 -Phenyl-2-methyl-2-methylamino- " ' propane-sulfate Q_CH2-C-NH.-CH3 S04 Wyamine (Wyeth) " Mephentermine Mephine (Wyeth Engl.) CH„ Monohydroxy 4.3.2. or methoxy phenylalkylamines

Chemical and other commonly used names Formula Proprietarynames OH

1-1 -(3-HydroxyphenyI)-2-methylami- ^_GH—CHa—NHa—GH3 CI Adrianol (Boehringer) no-ethanol-hydrochloride Neo-synephrine)Winthrop- OH Stearns).Phenylephrine(Boots) OH

1 l-l-(3-Hydroxyphenyl)-2-aminopro- —CH—CH—NH, GHOH-COOH ~ Aramine (Merck Sharp & I CHOH-COO panol-bitartrate ! Dohme). Metaraminol I OH CH, 4» dl-1 -(4-Hydroxyphenyl)-2-[phenyI- HO—Q—CH—CH—NH2—CH,—CH2—CH2—^ CI Dilatol (Tropon- (3-methyl)propyl-aminopropanol werke) hydrochloride OH CH, CH OH I + dl-l-(3-Hydroxyphenyl)-2-ethyl CI Effortil (x)—CH—CH9—NH_—CUS (Boehringer) amino-ethanol-hydrochloride OH OH dl-l-(3-hydroxyphenyl)-2-amino- £>>—CH-CH2—NH3 . CI Novadral (Diwag) ethanolhydrochloride OH Chemical and other commonly used names Formula Proprietarynames

dl-1 - Q—CH—CH—NHa—CH3 I (4-hydroxyphenyl)-2-methylamino- HO— . CI Suprifen(Hoechst) propanol-hydrochloride OH CH J + dl-l-(4-Hydroxyphenyl)-2-amino- HO — O—CH2—CH—NH . Br Paredrin (S.K.F.) propane-hydrobromide 2-Aminopropylphenol CHS j dl-1 - (4-Hydroxyphenyl)-2-methyl- HO— {J—CH—CH2—NH2 CHOH-COO Sympatol (Boehringer). aminoethanol-tartrate 1 1 CHOH-COO- Synephrine tartrate Oxedrinetartrate OH CH, (Winthrop-Steams) to en dl-1 - (4-Hydroxyphenyl)-2-methylamino- HO— Q—CH,—CH—NH2—CH3 . S04 Veritol (Knoll) propane-sulfate Paredrinol CH, Pholetone (Boots) rocH3 \ + dl-l-(2-Methoxyphenyl)-2-methylamino- CI Orthoxin(Upjohn) propane-hydrochloride Q>-CH,—CH—NH2—CH3 Methoxyphenamine hydrochloride CH3 OCH3A dl-l-(2,5-Dimethoxyphenyl)-2-amino- CI Vasoxyl (Burroughs propanol-hydrochloride ^_J>—CH—CH—NH3 Wellcome) Vasoxine Methoxyamine hydrochloride OH CHJ (Burroughs OCH3 Wellcome) 4.3.3. Dihydroxyphenylalkylamines

Chemical name and other commonly used names Formula Proprietarynames

dl-1 -(3,4-Dihydroxyphenyl)-2-isopro- HO—t^s— CH—CHa—NH,—GH(GH3)2 S04 Aludrin (Boehringer). pylamino-ethanol-sulfate HO—^JJ 1 Aludrin (Lilly), OH Isuprel(Winthrop Stearns), 3 Neo-Epinine (Burroughs- Wellcome) Norisodrine (Abbott) OH

l-l-(3,4-Dihydroxyphenyl)-2-amino- . CHOH-COO ethanol-bitartrate N3 HO'\J—CH_CH3—NH3 1 Arterenol,Noradrenaline OH CHOH-COOH Aktamin (Schering) Levarterenolum (DGI),Levophed OH (Winthrop Stearns),1-Arterenol dl--l-(3,4-Dihydroxyphenyl)-2-amino- CI (Hoechst) butanol-hydrochloride HO"W~CH—CH—NH3 Ethyl-noradrenaline Butanephrine(WinthropStearns) OH C2HS OH 1 Corbasil (Hoechst) dl-l-(3,4-Dihydroxyphenyl)-2-amino-HO- propanol i}—CH—CH—NH2 OH CH, Chemical and other commonly used names Formula Proprietarynames OH 2-(3,4-Dihydroxyphenyl)ethyl- 1 methylamine-hydrochloride HO— 6 }—CH,—CH3—NHa—CH, CI. Epinin (BurroughsWellcome) OH

I _ " + l-l-(3"4-Dihydroxyphenyl)-2-isopro-HO— CHOH-COO Isolevin (Cilag) pylamino-ethanol-bitartrate ^—CH-CHa—NHa—CH(CH3), OH CHOH-COOH

f OH

1 dl 1 -(3,4-Dihydroxyphenyl)-2-dimer Methadren (Lakesdie) thylamino-ethanol HO- Q-CH-CH2-N(CH3)9 N-Methylepinephrine OH OH

1 -1 - + (3,4-Dihydroxyphenyl)-2-methyl- HO— k CI. Suprarenin(Hoechst) aminoethanol-hydrochloride (or \_J—CH—CH,—NH2—CH3 tartrate) OH Adrenaline, Epinephrine OH l_ 1 -(3,4-Dihydroxyphenyl)-1-oxo-2-me- + CI. HO— C\- CH3 Kephrine (Winthrop Stearns) thylamino-ethane-hydrochloride v—' C—CH,-^NH2— Stryphnon II (Chemosan) Adrenalon O 4.4. Miscellaneous

Chemical and other commonly used names Formula Proprietarynames

CH, 4-Methyl-2-amino-pyridine %j>—NH, Ascensil (Raschig) N l-Cyclopentyl-2-methylaminopropane- \-CH2—CH—NHa—CH3 .CI Clopane (Lilly) hydrochloride I 1 + Cyclopentaminehydrochloride CH, O N3 CO 2-Phenyl-3-methyl-morpholine-hydrc-f'S—© CI. Preludin (Boehringer) chloride k^-CH3 NH9 +

2—(1—NapthyImethyl)-imidazoline- nitrate .N" "CHo NOs Privine (Ciba) Naphazoline nitrate Q_CHa-C^ NH9 CH,

2-(l-23-4-Tetrahydro-l-napthyl)- ~ Tyzine (Pfizer) imidazoline hydrochloride o y ;N CH, Tetrahydrazolinehydrochloride CI "NH2. CH,

I STABILITY OF SYMPATOL SOLUTIONS

Chapter III

PROBLEM AND WORKING PROCEDURE

1. Problem

Aqueous solutions of sympatol are used in therapy by parental and oral administration. On heat sterilization and after storage, the above solutions become coloured and may loose their potency. Stockton et al. (60) observed that the solutions of sympatol turned pink in about three weeks time after sterilization by boiling, however, without any loss of biological activity.

The purpose of this investigation was to evaluate the conditions under which the solutions ofsympatol used parentally, could be heat sterilized and stored without developing any unpleasant colour or loss in sympatol content. However due to the costly and time consuming biological methods of analysis it was not possible to evaluate the biological activity under different conditions of storage.

2. Working Procedure

In order to ascertain the stability of sympatol solutions, the following investigations were carried out :

2.1. Quantitative estimation of sympatol content

2.1.1. Colorimetric method.

2.1.2. Ultra-violet spectrophotometric method. 2.1.3. Chromatographic method.

2.2. pH value : Determination by potentiometric method.

2.3. Colour estimation : By comparison with standard colour solu¬ tions.

2.4. Material used in the following experiments were of the following standards : 2.4.1. Sympatol (Boehringer) Nachtr. DAB 6 (61) standard. 2.4.2. Ampules conforming with the "Powdered glass test" of U.S. P. XV (15).

2.4.3. Water conforming with the requirements of "water for injec¬ tion", Ph. Int. I (62).

2.4.4. Other chemicals were of A. R. standard.

2.5. Preparation of the solution

distilled water A 6 % w/v solution of sympatol was prepared in and the following conditions were made to obtain : 30

(a) pH values : 3, 4, 5, 6 and 7

(b) each containing : rongalit 0.2 % sodium metabisulphite 0.1 % filled with nitrogen filled with air.

2.6. Storage i The ampules were stored at 4°,. 20° (room temp.) and 37°.

2.7. Testing : The above prepared solutions were- analysed: after 20, 40, 80, 160 and 350 days. CHAPTER IV

SYMPATOL: SYNONYMS, SYNTHESIS, TESTS (PHYSICAL AND CHEMICAL), METHODS OF QUANTITATIVE ANALYSIS, PHARMACOLOGY, CLINICAL USES AND DOSAGE.

1. Synonyms

Sympatol Boehringer Oxedrine tartrate Ph. Dan. IX (63) Synephrine tartrate NNR. 1934 (64) Aethaphenum (65) Vasocordin Leo (Copenhagen)

(C9H1303N)2.C4HaOe Mol. Wt. 484. 49

0

11 - CHOH —C-O HO-f~^—CH—CHa—NH,-CH3 1 I — CHOH —C-O OH 11 0

Chemical name: Sympatol is dl-l-(4-Hydroxyphenyl)-2-mcthylamino- ethanol-d-tartrate.

2. Synthesis

Sympatol was first synthesised in the year 1927. Several methods are available for its synthesis under the patents of different countries ; (66), (67), (68), (69), (70), (71),. One of the best methods for the preparation ofsympatol is achieved by condensation of w-Chloro-p-ben- zoyloxy-acetophenone with methylbenzyl amine, followed by hydrolysis and subsequent hydrogenation, (66).

Sympatol can also be prepared by treating w-Methylamino-p- hydroxy-acetophenone hydrochloride with hydrogen under ordinary or raised pressure in the presence of a hydrogen carrying catalyst such as Pd, Pt or their oxides. After addition of ammonia p-Oxyphenyl-ethanol- methylamine crystallizes, (67).

Lately some new methods of synthesis have also been described by Bergmann and Sulzbacher (72), Fodor and Kovacs (73) and Asscher (74).

3. Tests

The following tests are described in Nachtr. DAB 6 -(61) and in Ph. Dan. IX (63). 32

3. 1. Physical

3.1.1. Description. Sympatol is a white crystalline powder ; odourless ; tastes bitter.

3.1.2. Melting 'range, for tartrate 188° to 192° Ph. Dan. IX

for base 185° to 189" Ph. Dan. IX

for base 176° to 180° Nachtr. DAB 6.

3.1.3 Solubility. Easily soluble in water ; sparingly soluble in alcohol; insoluble in solvent ether and in chloroform."

3.1.4. Specific rotation. The specific rotation is 4-10° 'to+15'!, corresponding to a rotation of+0*40° to +0*60" when 0'50 g of sympatol is dissolved in 25 ml of water and a 20 cm long polarimeter tube at 20J is used, Ph. Dan. IX.

3. 2. Chemical

3.2.1. Identification tests

3.2.1.1. To 2 ml of aqueous solution of sympatol (14-49 ) add 1 ml of copper sulphate solution (10 %) and 1 ml of sodium hydroxide solution (15%); a deep ultramarine blue colour is formed ; shake with 2 ml of solvent ether; the ether layer must remain colourless.

3.2.1.2. Add a drop of ferric chloride solution (14-9) to 1ml of the aqueous solution of sympatol (1+49); an intense yellow coulor is formed which changes to greenish brown on further addition of two drops of ferric chloride solution. *

3.2.1.3. To 2 ml of the aqueous solution of sympatol (1+49) add 2 drops of glacial acetic acid, 1 drop of ferrous sulphate solution (1+9) followed by 3 drops of hydorgen peroxide (3%); a green colour is fomed which turns brown by adding 4 ml of 2 N sodium hydroxide solution.

3.2.1.4. Moisten a few milligrams of sympatol with a drop of water, add to 2 ml of sulphuric acid, followed by a crystal of rcsorcinol; on care¬ fully warming a red violet colour* is formed.

3.2.2. Parity tests

3.2.2.1. On dissolving 0.01 g of the sympatol in 1 ml of sulphuric acid; the colour of the solution should not be more than very slightly yellowish (foreign organic matter).

3.2.2.2. The aqueous solution of sympatol (1+9) must be clear and colourless ; it should not change the colour of litmus paper to more than slight red (pH 5.8-6.6).

3.2.2.3. The sympatol should be free from chloride and sulphate- ions. 33

3.2.2.4. To the aqueous solution of sympatol (1+49) add 3 drops of sodium sulphide solution (10%); there should be no change in the solution (salts of heavy metals).

3.2.2.5. To the aqueous solution of sympatol (1 +49) add 1 drop of ammonia solution (10%) and 1 ml of dimethylglyoxime solution (1% in C,HgOH),; after keeping for several hours no red colour or turbidity should develop (nickel compounds).

3.2.2.6. To 2 ml of the aqueous solution of sympatol (1 +49) add 1 drop of phenyl hydrazine ; there must not be any change after keeping the solution for 5 minutes (test for methylaminomethyl-(4-oxyphenyl)- ketone).

3.2.2.7. 0.20 g of the sympatol on drying at 105° must not loose more than 0*0010 g in weight.

3.2.2.8. 0"50 g of the sympatol on ignition must not give a residue of more than 0"0005 g.

4. Methods of quantitative analysis described in the literature

Methods available in the literature for the quantitative estimation of sympatol may be divided into two broad categories :

4.1. Volumetirc methods

4.2. Colorimetric methods

We shall discuss them in detail on the following pages.

4.1. Volumetric methods

4.1.1. Bromometric methd. This method is based on the quan¬ titative formation ofdibromosympatol with bromine. The excess ofbromine is then determined iodometrically. Thus, from the amount of bromine used up in the reaction, the sympatol content is calculated. KSllstram (75) found that the accuracy of the method depends on the bromine con¬ centration and bromination time. Awe and Stohlmann (76) further showed that the acid concentration and sympatol content used for the bromina¬ tion are also equally important.

The reaction may be formulated as follows :

CHOH.COO H0~0~CH—ch»~nh» + 4Br, = CHOH.COO OH CH„ J 2

Br CHOH.COO

HO-Q-Cf-^-1*o CHOH.COO +4HBr Br OH CH, 2 34

4.1.2. By estimation of nitrogen content. Here the nitrogen content is determined by the Kjeldahl method bv which the sympatol content is calculated. The method is official in Ph.' Dan. IX (63) and NNR. 1934 (64).

4.1.3. By precipitation as tetraphfenyl borate. Flaschka et al. (77) and Worell and Ebert (78) have used the reaction of tetraphenyl borates with organic bases for the estimation of sympathomimetic amines. Aklin (79) has applied the method to assay the opium alkaloids. Sodium-tetra- phenyl-borate gives an insoluble precipitate with organic bases which reacts with mercuric chloride with the liberation of free hydrochloric acid according to the following equation :

+ — + — RNH3C1 +Na (BPh4) -> RNH3 (BPh4) + NaCl

RNH3 (BPh4) + 4 HgCl2 + 3 HaO -» RNH3C1 + 4 PhHgCl + 3 HC1+ HaBOa

The acid content is determined by titration against standard alkali. The method can be applied for the quantitative determination of sympatol.

4..2. Colorimetric methods

4.2.1. Sinodinos and VuillUume (80) have described a colour reaction which can be used for the estimation of sympatol content. Diazotized p-nitro-aniline couples with sympatol and gives a rose colour in an alkaline medium. The colour can be concentrated by extracting with a mixture of ethanol and butanol; then the intensity can be measured in a suitable photometer. Jindra et al. (81) reports that the colour formed after diazotization gives a peak at 425 mju and that it does not exactly confirm with Lambert-Beer's law and therefore it is necessary to draw calibration curves.

None of the methods described above permit an unambiguous determination of the undecomposed sympatol content in a solution because they are all based on the presence of either an amino group or a hydroxy phenyl nucleus, whereas the decomposed product of sympatol can give the same results by the above methods like the pure sympatol, as the amino group or the hydroxy phenyl nucleus may remain intact in the decomposed product.

So we had to find out some other method which could be used for determining the undecomposed as well as the decomposed sympatol ' in a solution.

5. Pharmacological actions

The action of sympatol is,dn general, qualitatively similar to that of adrenaline, exhibiting more prolonged but less intense action. It raises the blood pressure, constricts the peripheral vessels, dilates the pupil, re¬ laxes the smooth muscles of the intestines and increases the muscular tone of the uterus as reported by Ehrismann (82), Lasch (83), Ehrismann and Malqff (84) and Kusehinsky (85). 35

Ehrismann (82), Kuschinsky (85) and Schuntermann (86) reported that sympatol does not cause cardiac irregularities, or arrhythymia as re¬ ported by Oberdisse (87), which are observed in the.case of adrenaline injec¬ tions. Ergotamine diminished or abolished but did not reverse the pres¬ sor action of sympatol (84), (85), (88), but it reverses the vasoconstrictor action in perfused vessels as reported by Schretzenmayr (89), Barkan et al. (90), and Behrens and Taeger (91). Tainter and Seidenfeld (88) showed that the cause of the pressor action ofsympatol was due to the direct stimulation of the vascular muscles as indicated by the results of analyses in experi¬ ments on ergotaminised and cocanised cats. Pressor action is unaffected (88) or possibly slightly increased by cocaine (85), though Gurd (92) reported that pressor action is reduced by cocaine.

The median pressor dose of 1-sympatol is 0.5 mg/k.g. intravenously as reported by Tainter and Seidenfeld (88).. On comparing the pressor acti¬ vity of optical isomers they found that racemic form was \ and d-isomer only 1//60 as active as the 1-sympatol.

5.1. Potency ratio of sympatol to adrenaline

There is a wide variation in the results of various workers for the potency of sympatol when compared to adrenaline. The following figures will illustrate the results which have been obtained for 1-sympatol and adrenaline by different workers.

Table 10

Poteney ratio of 1-sympatol to adrenaline

Activity Reference

Pressor Constrictor Intestines Uterustcrus

1/25 — — Ehrismann (82)

1/25 — — — Lasch (83)

1/25 1/100 — — Ehrismann and (84) Maloff

1/58 — — — Tainter and (88) Seidenfeld

1/60 to 1/100 1/100 1/70 to Kuschinsky (85) 1/100

1/375 1/700 — — Gurd (92)

5.2. Toxicity Kuschinsky (85) reported that the toxicity of 1-sympatol compared with 1-adrenaline is 150 times less as against the findings of Ehrismann (82) who reported it to be 200 to 400 times less toxic.

6. Clinical uses

Sympatol is used as a vasoconstrictor in mild collapse due to failing perepheral blood flow and in conjugation with procaine in local anesthesia. 36

It is used to shrink the mucous membrance in hay fever and in asthma (64), (93). It is superior to ephedrine for topical application in the nose as reported by Stockton et al. (60). It has been reported to be useful in chronic urticaria (94), in combination with quinine for the treatment of arrhythymia perpetua (95), in cases of circulatory disorders in dip- theria (96), etc.

Besides the above mentioned uses syihpatol has many other clinical applications which have been summarised by Boehringer and Sohn (97),

7. Dosage

Orally : 0.1 to 0.3 g daily.

Intravenously : 0.03 to 0.06 g. CHAPTER V

TESTING OF THE MATERIALS AND APPARATUS

1. Syrnpatol The sample of sympatol used gave the following results when tested as decribed previously on page 32.

1.1. Physical tests

1.1.1. Melting range of sympatol : between 189°-I91°.

To 2.5 ml of the Melting range of the base : (1 +9 ) solution of sympatol 1*5 ml of ammonia solution (10 %) was added. On rubbing the sides of the test tube free base was precipitated. It was washed with distilled water and dried at 105°. The melting range of the base was between 176°-178°. Heating was so maintained that the rise in temperature of the bath was 5° per minute.

1.1.2. Specific rotation : (test as described on page32) + 13'5° at20°.

1.2. Chemical tests

1.2.1. Identification tests : the sample of sympatol was tested for identity tests numbers 3.2.1.1., 3.2.1.2. and 3.2.1.4. as described on page 32, to which it complied.

1.2.2, Parity test*

Sympatol complied with the following purity tests which have been described on page 32.

1.2.2.1. Foreign organic matter : 0.01 g of sympatol were dissolved in 1 ml of sulphuric acid. The solution was practically colourless.

1.2.2.2. Acidity : pH of (1+9) solution of sympatol was 6.2.

1.2.2.3. Chloride and sulphate ions : the sample of sympatol did not give any positive tests for chloride or sulphate ions.

: the of 1.2.2.4. Heavy metals sample sympatol did not give any positive tests for heavy metals.

(a) Copper : For testing the presence of Cu"*" "Hons a 5 mg % solution of dithizone (diphenylthiocarbazone) in carbon tetrachloride was used. To 5 ml of the 6% solution of sympatol 0*1 ml of the above solution of dithizone were added. The colour of the carbon tetrachloride layer turned pink after shaking for 60 seconds, while on the addition of more than 0*5 ml of the dithizone reagent the colour of the carbon tetrachloride layer remains unchanged showing that the amount of + Cu 'present is less than 10 /igper 100 mlof the sympatol solution. (1 ml of dithizone reagent =l'6/*g of Cu '"•"). 38

(b) Nickel : the sample of sympatol did not give any positive tests for nickel.

1.2.2.5. Methylaminomethyl-{4'oxyphenyl)-ketone: the sample of sympatol did not give any positive tests for methylaminomethyl-(4-oxy- phenyl)-ketone.

1.2.2.6. Residue on ignition : residue of the sample of sympatol on ignition was within limits.

1,3, Quantitative estimation of sympatol For quantitative determination of sympatol the following procedure, as given in Analysmetoder (98), was used.

1.3.1 Method : weigh accurately 50 to 80 mg of sympatol in a 200 ml glass stoppered flask. Add 30 ml of water, 20 ml of 0.1 N bromide- bromate reagent and 10 ml of2N hydrochloric acid. Close the stopper and keep it in the dark for 15 minutes. Add lgof potassium iodide and keep in the dark for five minutes. Titrate the liberated iodine with 0.1 N sodium thiosulphate solution, using mucilage of starch as indicator. Each ml of 0*1 N bromide-bromate solution = 0*006053 g of sympatol.

1.3.2. Results

The results of the quantitative estimation of sympatol are given in table U.

Table 11

Table showing the volume of 0.1 N bromide-bromate solution used for different amounts of the sample of sympatol.

Weight of Volume of 0.1 N bromide- Volume of 0.1 N Volume of 0.1 N bromate solution added sol. used bromide- sympatol NaaSOj . bromate sol.

ml 0.0880 g 20 ml 5.55 ml 14.45

0.0656 g 20 ml 9.2 ml 10.8 ml

blank 20 ml 20.0 ml 20.0 ml

14.45 . 0.6053 (a) Sympatol content = = 99*4 0.0880

10.8 . 0.6053 ' (b) Sympatol content = = 99-7 0.0656

Sympatol content as found by bromometric method = 99*5%.

In the following experiment the above sample ofsympatol was always used. 39

2. Distilled water

Double distilled water was prepard as follows, and it complied with the requirements of Ph. Int. I (62) for "water for injection". Fresh distilled water was distilled from a neutral glass still (pyrex) which was fitted with an efficient device for preventing entrainment. The first portion of the distillate was rejected and the rest was collected in a neutral galss container leaving about 1/10 of the original volume in the still. + + Distilled water was free from Cu ions when tested with dithizon reagent as described on page 37.

The above prepared distilled water was used immediately.

3. Ampules

The ampules complied with the following test (powdered glass test of U.S.P. XV (15).

3 1. Method of U.S P. XV for powdered glass test

3.1.1. Apparatus:—

3.1.1.1. Autoclave—For these tests use an autoclave capable of maintaining a temperature of 121 i 0.5°, equipped with the thermometer, a pressure gauge and a vent cock.

3.1.1.2. Mortar—Use a steel mortar made according to the specifications of U.S.P. XV. 3.1.1.3. Other equipment—Also required are 8" sieves including of the No. 20, No. 40. and No. 50 sieves, 250 ml Erlenmeycr flasks made resistant glass aged as specified, a 2-lb hammer, a permanent magnet, volumetric a desiccator, tin foil, and adequate apparatus.

3.1.2. Reagents :

3.1.2.1. Special distilled water. The water to be used in these still constructed tests is distilled water, re-distilled from an all-glass In of chemically resistant glass and equipped with ground glass joints. operating the still, add 1 drop of phosphoric acid to each liter of water contained in the still. Reject the first 10 or 15% of the distillate and retain the next 75#%.

3.1.2.2. Methyl red solution—Dissolve 200 mg. of methyl red in to make 100 ml. 60 ml of alcohol, then add sufficient purified water, 0.02 N sodium so If necessary, neutralise the solution with hydroxide 5 that the titration of 100 ml of special distilled water, containing drops ml of 0.20 N sodium of indicator, does not require more than 0.20 hydroxide to effect the colour change of the indicator. 3.1.3. Powdered glass test. containers selected Rinse thoroughly with purified water six or more in a stream of clear air. Crush the at random and dry them dry divide about 100 of containers into fragments about 25 mm. in size, g into 3 and the coarsely crushed glass apporximately equal portions, 40

place one of the portions in the special raqrtar. With the pestle in the place, crush the glass further by striking 3 or 4 blows with the hammer. Nest the sieves and empty the mortar into the No. 20 sieve. Repeat the operation on each of the two remaining portions of glass, emptying the mortar each time into the No. 20 sieve. Shake the assembled nest of sieves for 5 minutes, preferably with a mechanical shaker and return the glass remaining on the No. 20 and No. 40 sieves to the mortar and repeat twice the crushing and sieving operations. Discard the contents of the receiving pan, reassemble the sieves, and continue the shaking for 5 minutes. Transfer the portion retained on the No. 50 sieve, which should weigh in excess of" 10 g, to a closed container and store in a desiccator until used for the test.

Spread the sample on a piece of glazed paper and pass a magnet through it to remove particles of iron that may be introduced during crushing. Transfer the sample to a 250 ml Erlenmeyer flask of resistant glass and wash it with six successive, 30 ml portions of acetone, swirling each time for about 50 seconds, and carefully decanting the acetone. After washing, the sample should be free from agglomerations of glass powder, and the surface of the grains should be practically free from adhering fine particles. Dry the flask and the contents for 20 minutes at 140°, transfer the grains to a weighing bottle and cool in a desiccator. Use the test sample within 48 hours after drying.

3.1.4. Procedure

250 ml Transfer exactly 10 g of the prepared sample to a Erlenmeyer flask that has been digested (aged previously) with special distilled water for at least 24 hours in a bath at 90°or for 1 hour at 121°. Add exactly 50 ml of special distilled water. Prepare a blank by adding 50 ml of special distilled water to a similarly prepared flask and cap with all flasks with crimped new tin foil that has been rinced twice acetone. Place the containers on the rack in the autoclave and close issues it securely, leaving the vent cock open. Heat until steam vigourously from the vent cock and continue heating for 10 minutes. Close the vent cock and adjust the temperature so that it rises 1° per minute until it reaches 121°, taking 19 to 21 minutes to reach the desired temperature. Hold the temperature at 121±0.5° for 30 minutes, counting from the time when this temperature is reached. Then release the steam and cool at the rate of 0.5° per minute, venting to prevent the formation of a vacuum and allowing 38 to 46 minutes to drop to atmospheric pressure. Cool the flask at once in running water, add 5 drops of methyl red solution, and titrate immediately with 0.02 N sulphuric acid. If the volume of the titrating solutions is expected to be less than 10 ml; use^a microburet. Record the volume of 0.02 N sulphuric acid used to neutralize the extract from 10 g of the prepared sample of glass, corrected for a blank on the flask.

Powdered glass test requires no more than 1.0 ml. of 0.02 N acid per 10 g of glass.

3.1.5. Results

Two sets of observations were taken for each type of ampule. The results are tabulated in table 12. 41

Table 12

Table showing the amount of 0'02 N acid used for glass.

of 0.02 N Size of ampule Weight of the Initial reading Final reading Amount used ml powder ml ml H2S04

0.05 0.05 Blank — 0.0

0.55 0.05 Blank — 0.5

0.35 0.35 5 ml 10.300 g 0.0

0.35 5 ml 10.310 g 0.0 0.35

0.60 25 ml 12.486 g 0.0 0.60

0.47 25 ml 10.21 g 0.0 0.47

Amount of acid used for :

— 10 (0.35 0.05) . (a) 5 ml ampules = 0.29 ml 10.30

— 10 (0.35 0.05) . (b) 5 ml ampules = 0.29 ml 10.31

— (0.60 0.05) . 10 = 0.44 ml 12.486

— 10 (0.47 0.05) . (d) 25 ml ampules = 0.40 ml 10.21

the amount Both typs of ampules passed the powdered glass test as of acid used was well within the prescribed limits.

It is to be noted that we have made the above tests with sieves mesh diameter of No. Ill, No. IV and No. IV a (Ph. Helv. V) with a instead of sieves No. 1.5 mm, 0.47 mm. and 0.32 mm. respectively 20, of No. 40 and No. 50. The sieves correspond very closely to the sieves U.S.P. XV. Further the mortar and pestle were also not of the same dimensions as described in U.S.P.XV.

4. Other materials

All the other materials used in the following experiments were of A. R. standard.

5. Apparatus

5.1. UV-Spectrophotometer

For spectrophotometric measurements the Zeiss Spectrophotometer model PMQ;II, with 1 cm quartz cuvettes to hold the solution and the solvent bank, was used throughout our work. CHAPTER VI

METHODS OF ANALYSIS FOR SYMPATOL

1. Quantitative methods

None of the methods which we have discussed on page 33 permit in solution of So an estimation of the deteriorated product a sympatol. which be useful to estimate we have investigated the following methods may the deteriorated product in a solution.

1 1. Colorimetric method

The use of Millon's reagent for the quantitative determination of phenols and phenolic derivatives was first described by Folin and Ciocalteau (99) Lugg (100) and Arnow (101). The formation of colour is based on the reaction of nitrous acid with phenols or phenolic derivatives in the pre¬ sence of mercury. The nitrous acid may react in a position orthotoOH formation due to their group. Mercury compunds accelerate the colour great reactivity towards organic compounds, However the actual mechani¬ sm of the reaction is not very clear.

Ellin and Kondritzer (102) have made use of the above reaction for determination of the phenylephrine content by measuring the absorbancy at 495 m/*. We have used their method with the following modifications:

(a) In the original method the solutions were diluted immediately after addition of sodium nitrite solution. However, we found that in order to obtain the full development of the colour, the solutions should be diluted 15 minutes after the addition of sodium nitrite solution. t

1.1.1. Method

1.1 1.1. Extinction wavelength curve

Initial experiments showed that a maximum peak is obtained at 500 m/t corresponding to filter no. 10 of Pulfrich's photometer. The readings are given in table 13 and the relationship between extinction and wavelength is graphically shown in fig. 2.

Table 13

l Relation between extinction and wavelength by using4mg% solution of sympatol for development of the colour.

Filter no. Corresponding wavelength Extinction values 4 rng.% E

1 cm.

1 570 m/t 0-280

2 590 m/* 0-22

3 610 ml* 0.10 43

Filter no. Corresponding wavelength Extinction values 4 mg% E

1 cm.

4 666 m/* 006

5 720 m/» 006

6 750 m/A 006

7 430 m/» 0-395

8 450 m/i 0-48

9 470 m/* 0-57

10 500 m M 0-62

The above readings are mean af 3 different sets of observations

E1cm

0/ /I

0.2 ^

„ . n,. ' ' 700 500 BOO 8fx mg

ABSORPTION-CURVE of SYMPAT0L after color reaction with HgSO^

Fig. 2

1.1.1.2. Stability of the colour. The colour is very stable and there is no change for at least 90 minutes after development of the colour.

1.1.1.3. Time of dilution.

It is very important that after the addition ofsodim nitrite solution, flasks are kept at room temperature for 15 minutes and then diluted. If the solution is diluted immediately after the addition of sodium nitrite 44

solution the colour does not develop fully, as is clear from the following readings. :

Table 14

Effect of time of dilution, on extinction values

Concentration of Extinction values at 500 m/n sympatol Immediately after dilution After 15 minutes dilution

After the addition of sodium nitrite solution

40 j".g / ml 0-57 0-63

' 20 f»g / ml 0-295 0-32

1.1.2. Final method

To a 50 ml volumetric flask add sympatol solution representing about 3 ml of mercuric solution 1.5 mg of sympatol. Add sulphate (15% w/v). minutes cool the flasks to room Heat on a boiling water bath for 10 ; of solution of sodium nitrite temperature ; add 3 ml freshly prepared (0.1 %). Keep the flasks at room temperature for 15 minutes then make the volume to 50 ml by the addition of" distilled water; mix. Then read the extinction at 500 imi in the Pulfrich's photometer using 1 cm cuvettes as the coloured solution but against a blank prepared in the same way without sympatol.

1.1.2.1. Reagents used

1.1.2.1.1. Standard solution of sympatol. Sympatol which complied with the tests described on page 38 was used to prepare the standard solution. A 1 mg per ml solution in water was prepared.

1.1.2.1.2. Mercuric sulphate solution. A 15 % w/v solution of HgS04 in 5 N H2 S04.

1.1.2.1.3. Sodium nitrite solution. 0.1% solution of sodium nitrite in cold water (freshly prepared).

1.1.2.2 Apparatus

Pulfrichs photometer with 1 cm cuvettes was used.

1.1.2.3. Standard curve of sympatol at 500 m/(,

Sympatol solutions representing 400, 700, 1000, 1500 and 2000 ^g of sympatol were accurately measured in 50 nil volumetric flasks. Then the colour was developed as described above. At least 3 sets of obser¬ vations were taken for each solution. The results are given in table 15. ^ The maxium deviation of readings was 1.5 to 2%. 45

Table 15

Extinction values at 500 mji, for different cone, of sympatol

Concentration of Mean extinction values of 3 different sets of sympatol readings, at 500 m/l.

40 fg / ml 0-625

30 /*g / ml 0-470

20 /*g / ml 0-320

14 fig / ml 0-220

8 fig / ml 0-125

A graph showing the relationship between the extinction and the concentration of sympatol was drawn (See fig. 3).

06

0.4 -

0.2 -

10 20 30 » . . . 40 sympatol content vg/ml.

STANDARD CURVE of SYMPATOL at 500 mji.

Fig. 3

The relationship between the extinction and the concentration of sympatol is linear as is clear from the above Fig. Thus it is possible to use the method for quantitative estimation of sympatol.

1.1.3. Application of color i metric method for estimation ofdestroyed sympatol in solution

For this investigation a 6% w/v solution of sympatol in water was heated at 120° for 10 hours. After heating the solution turned yellow with an orange precipitate, showing that some of the sympatol had d eteriorated. 46

The precipitate was filtered and the extinction readings for the fil¬ tered solution were taken at 500 imi after developing the colour as des¬ cribed on page 44. The results are shown in table 16.

Table 16

Extinction readings of the freshly prepared and of the heated solution of sympatol.

(a) freshly prepared solution 3 mg% E

1 cm . = 0.470

(b) heated solution (120°-10 h.) 3 nig % E

1 cm . = 0.455

1.1.4. Results

The results show that the colour formed after development with sodium nitrite in the presence of mercuric sulphate is vey stable and obeys Lambert-Beer's law for the range of concentration of sympatol studied. Thus it is possible to use the above method for the quantitative estimation of sympatol.

However a solution of which was heated at 120° for , sympatol 10 hours, did not give any appreciable difference in the extinction values (the maxium loss was found to be 3%), though the solution, after heating, turned yellow with an orange precipitate, showing that the product was deteriorated. As the reults of the fresh solution and that of the deteri¬ orated solution practically remain the same, we conclude that the method is not good to measure the amount of destroyed sympatol in solution.

1.2. U. V. Spectropliotomctric method

In the literature we did not find any ultraviolet absorption studies of sympatol, though similar studies were available for phenylephrine by Schou and Rhodes (103) and Ellin and Kondritzer (102). Since sympatol differes from phenylephrine only in respect to the position of the hydroxyl group in the benzene ring where it occupies a para position instead of meta position, it was expected that sympatol might also give absorption spectra which could be used as a basis for the estimation of oxidized and unoxidized sympatol content in aqueous solutions.

1.2.1. Experimental 1.2.1.1. Apparatus

U.V. Spectrophotometer which has been described on page 41 was used.

1.2.1.2. Method

Standard aqueous solutions containing 1.2 mg% of sympatol were prepared by diluting 0.01 ml of the 6% w/v solution of sympatol to 50 ml. The solution was measured by means of an Agla micro pipette. Spectral extinction curves between the wavelengths of 200 and 290 tap. were drawn. The results are tabulated in table 20. They are the mean values of at least 3 different sets of observations. The maxi¬ i mum difference of readings was 1%. 47

Sympatol shows absorption maxim at 223 nut and at 273 mp and a values at 223 and at minimum at 247 rap.. The ratio of the absorption m^ extinction and 273 m/* is 5-86 : 1. The graphical relatloship between the wavelength is shown in fig. 5.

. 1.2.1.3. Standard curve of sympatol at 223 m/x

: Solutions representing 2.4, 4.8, 7.2, 9.6, 12.0, 14.4, 16.8, 19.2, 21.6 and 24.0/tg/ml of sympatol, were prepared by accurately measuring different volumes of a 6% w/v solution of sympatol by means of an Agla pipette and then diluted to 50 ml. The extinction readings were taken at solvent blank. 223 m/i by using 1 cm cuvettes to hold the solution and the The results are given in table 17.

Table 17

Relationship between the extinction values and the sympato 1 content at 223mfi

Sympatol content p.g / ml 'Extinction values at 223 m/i using 1 cm cuvettes

2-4 0.091

,4-8 0.178

7-2 0.2725

9-6 0.3625

120 0.4450

14-4 0.538 .

16-8 0.608

19-2 0.750

21-6 0-805

240 0.890

* The above results are the mean values of 3 different sets of observations For ench 3 readings were taken. 48

A graph showing the relationship between the extinction values and the sympatol content at 223 mM is shown in fig. 4, which is a straight line.

*/

0.8 _

/• o.s- y

0A-

0.2-

-

s 0 5 20 l»a/ml.

STANDARD CURVE of SYMPATOL at 223 m>i.

Fig. 4

1.2.1.4. Effect of pH on the extinction wavelength curve of a 6 w/v solution of sympatol

For this study a 6% w/v solution of sympatol of pH values 3, 4, 5, 6 and 7 was prepared. The extinction wavelength curve between the wavelengths of 200 and 290 rmt was drawn, using a concentration of 1.2 mg% of sympatol, as described on page 44.

Results

The results of the extinction values for solutions of different pH values are given in table 18. They are the mean values of at least 3 different sets of observations. From the following extinction readings at different pH values it is clear that by varying the pH of the sympatol soultions from 3 to 7, the extinction wavelength curve remains unchanged. 49

Table 18

Effect of pH on the extinction wavelength curve of sympatol.

Wave -length in m/i Extinction values for 1.2 mg% solution 1.2 mg% E of sympatol with 1 cm cuvettes. 1 pm

7 6 5 pH 4 pH 3 pH . pH pH

200 0-67 0-67 0-665 0-67 0-665

0-26 205 0-27 0-265 0-27 0-27

0-285 210 0-285 0-28 0-235 0-28

215 0-34 0-3375 0-3375 0-335 0-335

220 0-425 0-425 0-425 0-42 0-415

221 0-44 0-4375 0-435 0-435 0-435

222 0-4425 0-445 0-445 0-44 0-445

223 0-445 0-445 0-445 0-4425 0-445

0-435 224 0-44 0-44 0-44 0-435 I 0-42 225 0-43 0-425 0-425 0-425

230 0-26 0-255 0-255 0-25 0-245

235 0-072 0072 0074 0072 0.074

240 0-0195 0018 0-0195 0-018 .0018

245 0-016 0015 0016 0-015 0015

250 0-018 002 0-02 0-02 0018

255 0-028 0026 0-026 0024 0-026

260 0-038 0038 0040 0-038 0-038

265 0-054 0054 0-053 0053 0054

270 0068 0068 0068 0064 0-066

272 0072 0072 0072 0-072 0-072

273 0074 0074 0074 0073 0 074

274 0072 0-072 0072 0072 0072

275 007 007 007 0-07 0068

280 0-06 006 0058 0059 006

285 0-024 0-026 0026 0024 0024

0-009 290 0008 0-012 002 001 50

1.2 1.5. Effect of heating on absorption spectra andpH of sympatol solution

A 6 % w/v solution of sympatol of pH 6.2 was filled to 2/3 capacity in 5 ml ampules. The ampules were sealed and heated in an autoclave at 120° for a period of 1 to 10 hours. The pH and extinction values at time intervals after the 223 mp were measured at different filtering solution. The readings are given in table 19.

Table 19

Effect of heat sterilization on extinction values at 223 rn/t, and pH of a 6% w/v solution of symxpatol.

Time of sterilization at Extinction values at 223 m/i 1.2 mg % E pH 120° with a cone, of 1.2 mg% l cm

Unsteril solution 0-445 6-2

30 min. 0-445 60

1 h. 0-445 5-7

0-4425 2 h. , 5-5

3 h. 0-44 5-4

4 h. 0-44 5-2

7 h. 0-1375 515

10 h. 0-435 50

Further the results of the extinction wavelength for a 6% w/v solution of sympatol which was heated at 120° for 10 hours are j-iven in table 20. The readings were taken after filtering the solution.

Table 20

Extinction wavelength curves of:

(1) freshly prepared solution of sympatol.

(2) heated solution (120°-10 h.). The ppt. was filtered off.

(3) The product which formed on heating a 6% w/v solution of sympatol. The product was recrystallized and then dissolved in 20% alcohol. 51

Concentration of each solution = 1.2 mg%

Extinction values 1.2 mg% E Wavelength in mf* 1 cm

solution solution solution (1) (2) (3)

200 0-700 0-627 0-720

205 0-390 0366 0-505

210 031 0295 0.315

215 035 0335 0-357

220 0-42 041 0-338

221 0-43 0-42 0-338

222 0-44 0-43 0-338

823 0-445 0-435 0338

0425 0-338 224 0-44 ,

225 0-43 0-42 0-338

230 0-285 0-27 0-298

240 0045 0037 0-159

245 0033 0029 0123

247 0025 0020

250 0034 0027 0114

260 005 0045 0121

270 007 007 0153

272 0075 0081 0-162

273 0076 00825 0-165

274 0075 0-082 0-165

275 0074 0080 0-174

280 0-065 0071 0192

290 0019 0-024 0-216

293 0-216

294 0-216

295 0015 0019 0-216

296 0-2145

300 0013 0-017 0-211

320 0180 •

340 0087

360 0-026 52

Results:

(a) After heating at 120° for 10 hours the pH of the 6 % w/v solution of sympatol drops down from 6.2 to 5.0.

(b) The maximum loss of sympatol content found by measuring the extinction at 223 mp, is about 2%.

(c) There is practically no change in the extinction wavelength curve of the freshly prepared solution and that of the heated solution (120°- 10 h.) of sympatol.

1.2.1.6. Absorption studies of the product formed on heating the sympatol solutions

On heating at high temperatures the sympatol solutions turn yellow with an orange precipitate, showing the deterioration of the product. However the extinction wavelength curve of the heated solution (120°- 10 h.) remains unchanged as is clear from fig. 5. So we have also studied the nature of the extinction wavelength curve of this orange product.

1.2.1.6,1. Experimental

20 ml of a 6% w/v solution of sympatol was heated at 120° for 10 hours. The solution was filtered. The orange precipitate was washed with cold water and recrystallized from 45% alchohol (3 times). The product was dissolved in 20 % alcohol so as to give a concentration of 1.2 mg % Extinction readings were taken between the wavelengths of 200 to 360 m^ as described on page 46. The results are given in table 20. A graph showing the relation between the extinction and the wavelength is given in fig. 5.

Results

(a) Melting range of the product = 202°—204°.

(b) Extinction wavelength curve. The curve of this product is completely different from that of the sympatol. There is no maximum either at 223 tap, or at 273 mp, instead another maximum at 290-295 ntyi is obtained. However, by referring to fig. 5, it is seen that the extinc¬ value of tion this product at 223 rap. is about 3/4 and at 273 mp. about 2 times that of the pure sympatol.

Thus it is not possible to estimate the amount of destroyed sympatol the extinction at the by measuring two. maxima i.e. either at 223 mp or at 273 imw 53

1.SYMPATOL-fr«sh solution , ertaxat x.223&273mu. 2.SYMPATOLSOLUTIONJhtatedat12u° lOh.E^,, at » = 223»273mu 3.THE PRODUCT formed on heating.Solution made m20°oalcohot £„,„ at X. 290-295 mp. Fig. 5

1.2 1.7. Summary of the results of U. V. Spectrophotomc'ric method 1 2.1 7.1. ^Extinction wavelength curve

Sympatol gives absorption maxima at 223 m/* and 273 mju. The ratio of the absorption values at 223 m/iand273m/tis5,86: 1. Further the extinction obeys Lambert Beer's law within the concentration range of sympatol studied. So the method can be used to assay the sample of sympatol.

1.2 1 7.2 Effect of pH oa extinction wavelength curve

There is no effect by varying the pH of the sympatol solution from 3 to 7 on the extinction wavelength curve.

1.2.1.7.3. Effect of heating on absorption curve and pH

(a) after 10 hours heating at 120° sympatol solutions turned yellow with an orange precipitate and the pH dropped from 6.2 to 5.0.

(b) The extinction wavelength curve of the heated solution (120°- 10 h.) remains practically unaffected.

(c) The maximum loss by measuring the extinction at 223 mju of a heated solution of sympatol (120°-10 h.) was 2.3%. 54

1.2.1.7.4. Absorption curve of the product formed on beating

The product does not show any maximum either at 223 nty or at 273 m/t further the melting range of the product is different from that of the pure sympatol, showing that it is some different product. However it gives another maximum at 290-295 rmi. So we conclude that this method can not be used for quantitative estimation of the deteriorated product in solution. 13. Quantitative chromatographic method For quantitative estimation of substances by paper chromatography, the spot area of an unknown substance must be compared with spots obtained from the same volume of a known concentration of the same substance developed on the same sheet of paper. Fisher and Holmes (104), Fisher et al. (105) showed that the spot area of round and ovoid spots increases proportionaly with the logarithm of the amount of substance forming the spot. For evaluation of the spots one of the following techniques can be employed : (a) Planimetric measurement. Fisher et al. (105) obtained an accuracy of db 2 % by this method.

(b) Counting of squares on a graph paper. The spot area can be traced out with a sharp pencil and copied on a graph paper and then the number of squares counted. Reid and Lederer (106) obtained an accuracy of i 5 % by this method. (c) By elution of the substance. In this method the piece of filter paper containing the substance is pinched between two pieces of glass res¬ ting in a petric dish. The liquid rises between the glass plates due to capillary attraction and after eluting the substance from the filter paper drops in a beaker. Then it is analysed by a suitable method. We have used methods (a) and (b) in our studies due to their relative simplicity and very small percentage of errors. 1.31. Experimental 1.3.1.1. Apparatus 1.3.1.1.1. A chromatographic chamber of 30x25x50 cm (L. B. H.) was used.

1.3.1.1.2. Paper. Whatman No. 1 paper cut into 46x20 cm strips was used. 1.3.1.1.3. Planimeter made by Amsler & Co., Schaffhausesn. 1.3.1.2. Reagents 1.3.1.2.1. Solvent system. The following solvent system described by Wickstrom and Salveson (107) has been used : Ethyl acetate : acetic acid (95%) : water: : 3:1:3.

The solvents were shaken and kept over night before use. 1.3.1.2.2. Developing reagent. As developing reagent diazotised p-nitro-aniline was used. 0.25 g of p-nitroaniline is dissolved by gentle in ml of N heating 25 HCl and diluted with ethanol to 50 ml. 0.01 g of sodium nitrite is added to every 10 ml of the above solution before spraying (cooling under running water). 55

Sprayed chromatograms were allowed to dry for 3-5 minutes and then passed through a 0.5 N ethanolic solution of sodium hydroxide. The excess sodium of hydroxide was removed with a clean filter paper.

1.3.13. Tectnique

The same technique for descending chromatograms as described by Consden et al. (108) has been used. 1.3.1.3.1. Application of eht substance. 0.01 ml of solutions of sympatol ofdifferent concentrations were applied on the starting line .by means of an Agla pipette so as to give spots of 200, 150, 100, 50 and 25 p.g of sympatol content.

1.3.1.3.2. 'Measurement of the spot area.. The spots were outlined by means of a sharp pencil. Then the area was measured by a planimeter and by counting the squares on the graph paper after tracing the spot area. The results are given in table 21.

Table 21

Relationship between the sympatol content and the spot area

No. of chromato¬ 1 2 3 4 5 6 Average grams

Quantity of sympatol Area in square centimeters

200 MS a 9.0 8.8 8.4 8.0 9.1 8.0 8.53 b 9.07 8.78 7.88 7.99 9.31 8.01

150/*3 a 7.0 6.6 7.0 6.8 6.7 7.0 6.86 b 7.1 6.56 6.89 • 6.98 6.75 6.91

100>g a 5.6 5.2 6.1 5.9 5.7 5.5 5.67 b 5.65 5.24 6.05 5.91 5.71 5.51

50 /»3 a 3.7 4.0 4.2 4.0 3.8 4.2 4.18 b 3.84 3.96 4.13 4.14 3.91 4.23

25 f-5 a 3.1 3.0 2.9 2.3 2.5 2.7 2.77 b 3.14 2.89 2.93 2.41 2.7 2.68

a — Measurement on the chromatogram by planimeter.

— Measurement b on graph by counting the number of squares.

1.3 2. Seperation of the oxidized product

A solution of sympatol was heated at 120° for 10 hours. The precipitate was filtered and the solution was chromatographed with the following solvent systems :

(a) Ethyl acetate : acetic acid 95%: water : : 3:1:3. 56

(b) N-butanol : acetic acid 95% : water :: 4 : 1 ; 5.

acid 95%: : 2:2:1:1: (c) N-butanol : toluene : water : acetic

However in no case two spots were obtained. Only one spot cor¬ responding to the pure product was obtained.

1.3.3. Results

1.3.3.1. On plotting the log of concentration against the spot of area, we got a linear relationship for spot contents of 25 to 150 /tg sy¬ mpatol. However, when the sympatol content was more than 150 jug 6. there was no more a linear relationship, as would be clear from fig.

/ •

_ / / •

/ s / 8- / I • / • / • / / ' I / f w _ w i a y

V tf 6 // / / S

- tf in Jd jf I4" - / 2 • _ i » i i 1.0 1'5 2.0 2,5 X xu log of tho cone.

RELATION BETWEEN THE LOG OF THE CONC

OF SYMPATOL & AREA.

Fig. 6

Thus the method can be used for quantitative estimation of sympatol content.

1.3.3.2. On chromatographing the heated • solution of sympatol 120°-10h.), there was neither a difference in the spot area when compared with that of the freshly prepared solution nor did we get two different spots, one for the pure product and another for the deteriorated product.

Thus by this method too, it is not possible to estimate the amount of deteriorated product in a solution. 57

1.4. Sommary of the analytical methods a colorimetric 1.4.1. Colorimetric method. From the foregoing as follows : To a 50 method for the estimation of sympatol may be stated 1.5 of ml volumetric flask add sympatol solution representing mg sympatol Heat in a water bath for 10 followed by 3 ml of HgS04 solution (15%). then add 3 ml of minutes, cool the flasks to room tepmerature, NaNO, and then make the solution (0.1%). Keep the flasks for 15 minutes extinction at 500 in a sutiable volume to 50 ml, mix and read the m^ pho¬ tometer.

The colour is stable for at least 60 minutes and obeys Lambert-Beer's studied. law within the range of cone, of sympatol maxima 223 1.4.2. Spectrophotometric method. Sympatol gives at the values at the two maxima is and 273 m/x. The ratio of absorption used to the solutions. 5.86 : 1. Both maxima can be assay sympatol eifect on the Change in pH from 3 to 7 does not have any spectral transmission curve. On heating the sympatol solutions for a long time, an orange product is formed which gives absorption maximum at 290-295 nip. 1.4.3. Paper chromatographic method. A linear relationship between of between 25 and 150 is spot area and the log of the cone, sympatol pg obtained when the solutions are chromatographed on the same sheet of Thus the method be used to paper by the descending technique. may estimate the sympatol. content quantitatively.

However all the 3 methods failed to give any difference in the results between fresh and deteriorated solution of sympatol. Since none of the foregoing methods could be used to estimate the stability of sympatol solu¬ of tions, we have based the results of the stability experiments sympatol on comparison with the standard colour solutions described in'the following pages.

2. Colour standards for comparison of the colour Solutions.of sympatol become coloured after heat sterilization and the colour increases with storage as mentioned before. The intensity of time. The coloured solutions do not give any maximum absorption in the visible range of light as would be clear from the extinction readings given in table 22. So it was not possible to express the intensity of the colour in terms of figures. Table 22

Relation between the extinction and wavelength for solutions of sympatol (6% w/v) which were coloured after heating

Wave length in mf* Extinction values for 6 % w/v solution of sympatol, using 1 on cuvettes.

Solution of pH 6 Solution of pH 5

375 2.00 1.90

400 1.05 0.95 58

Table 22

Wave lenghtmft Extinction values for 6 % w/vsolution of sympatol, using 1 cm cuvettes.

Solution of pH6 Solution of pH 5

425 070 062

450 045 042

475 0-30 6-27

500 0-215 018

525 . 0-15 013

550 0105 009

575 007 006

600 005 004

625 004 003

650 004 003

700 0025 0015

750 0015 00075

800 0015 00075

900 0005 00025

However in order to express the development of colour quantitatively and to impose a certain standard limit for the colouration of the injections of sympatol, the colour of the sympatol solutions was compared with the standard colours proposed by Buchi (109), in the following way. For comparison of the colour 2.0 ml of the solution are taken in a test tube with an inner diameter of 13 mm. The comparison is made with a standard in diffused daylight against a white background and viewing horizontally. 2.1. Stock coloured solutions

The following reagents are required to prepare the stock coloured solutions.

2.1.1. Ferric chloride standard solution. Dissolve 55g of FeCl3. 6HaO in a portion of a mixture of 25 ml of concentrated sulphuric acid RS and 975 ml of water and make the volume to 1000 ml with the above mixture. Mix 10 ml of the above solution with 15 ml of water, 5ml of cone acid sulphuric RS and 4g of potassium iodide in a glass stoppered Erlenmeyer flask and keep in the dark for 15 minutes. Dilute with 100 ml of water and titrate the liberated iodine with 0"1 N sodium thiosulphate solution using starch solution as indicator. 1 ml of 0.1 N NaaS2Os = 0.02703g FeCl3. 6H20. After the titration, dilute the solution with suphruic acid water mixture so that each ml contains 0.0450g of FeCl8. 6H20. 59

2.1.2. Cobalt chloride standard solution. Dissolve 65g of CoCl^. 6 H40 in a portion of a mixture of 25 ml of cone, sulphuric acid RS and 750 ml of water and make the volume to 1000 ml with the above mixture. Mix 5 ml of this solution in a 250 ml Erle'nmeyer glass stoppered flask with 5 ml of hydrogen peroxide RS (30% w/v) and 10 ml of cone, sodium hydroxide solution RS (10 N). Boil the mixture for 10 minutes, cool and then mix with 2 g of potassium iodide and 40 ml of dilute sul¬ phuric acid RS (10% w/v). As soon as the precipitate dissolves titrate the liberated iodine with 0.1 N sodium thiosulphate solution.

1 ml of 0.1 N NaaS203 = 0.0238 g CoCl2.6 HaO.

After the titration dilute the solution with sulphuric acid water mix¬ ture so that each ml contains 0.0595 g of CoCl^.OHgO.

2.1.3. Copper sulphate standard solution. Dissolve 65 g of CuS04. 5H20 in a portion of a mixture of 25 ml of cone, sulphuric acid and 750 ml of water and make the volume to 1000 ml with the above mixture. Dilute 10 ml of this solution with 50 ml of water and add 12 ml of dilute acetic acid RS (30 % w/v) and 3 g of potassium iodide. Titrate the liberated iodine with 0.1 N sodium-thiosulphate solution using starch as indicator.

1 ml of 0.1 N Na2SaOa = 0.02497 g of CuS04.5H20.

After the titration dilute the solution with sulphuric acid water mix¬ ture so that each ml contains 0.0624 g of GuS04.5 H sO.

coloured solutions the Then prepare the stock by mixing following quantities of standard solutions.

Table 23

Stock coloured solutions.

Stock coloured Standard solutions 1% HC1

FeCl, RS C0CI2 RS CuS04 RS ml ml ml ml

B Brown 30 3-0 2-4 1-6

BY Brownish-yellow 24 10 0-4 62

Y Yellow 24 06 00 70

R Red 06 1-2 00 82

2.2. Standard colour solutions the Standard colour solutions were prepared by mixing following acid. quantities of stock solutions and 1 % hydrochloric 60

2.2.1 Browrish-yellow solution BY

Solution ml of stock ml of 1 '/,i HC1 solution BY

BY 1 20 00

BY 2 1-5 05

BY 3 10 1-0

BY 4 05 1-5

BY 5 025 1-75

BY 6 01 1-9

2.2.2. YcUow solution Y

solution ml of stock ml of 1 % HC1 solution Y

Y 1 20 00

Y 2 1-5 05

Y 3 10 1-0

Y 4 05 1-5

Y 5 025 175

Y 6 01 1-9

2.2.3. Red solution R

Solution ml of stock ml of 1% HC1 solution R

R 1 20 00

R 2 1-5 0-5

R 3 10 10

R 4 05 1-5

R 5 025 1-75

R 6 01 1-9

2.2.4. Brown solution B

Solution ml of stock ml of 1% HC1 solution B

B 1 1-5 05

B 2 10 10

B 3 075 1-25

B 4 0-50 1-5

B 5 0-25 1-75

B 6 010 1-9 61

One more stock solution BY2 was prepared in the same way as that of stock solution BY (p. 59), however, twice more concentrated solutions of ferric chloride, cobalt chloride and copper sulphate were used. Stock solution BY2

FeCl3 solution (each ml contains 0.09 g of FeCl3. 6H20) .. 2*4 ml 1-0 CoCla solution (each ml contains 0.119 g ofCoCl2. 6Ha0) .. ml

CuS04 solution (each ml contains 0.1248 g of CuS04.5H20). 0-4 ml ml acid ...... 6-2 Hydrochloric 2% ..

From this stock solution the following standard colour solutions were prepared.

2.2 5. Standard colour solutions (Brownish-yellow solutionBV2)

Solution ml of stock ml of 2% HC1 solution BY2

BY2 1 2-0 00

BY2 2 15 0-5

BY2 3 10 10

BY2 4 05 1-5-

BY2 5 0-25 1-75

BY2 6 01 1-9

3. pH determination

All the pH determinations were done with Philips pH meter model GM 4491, with a glass electrode. Before making measurements the apparatus was always checked by means of a standard buffer solution of pH 4-632. CHAPTER VII

DETERMINATION OF STABILITY OF SYMPATOL SOLUTIONS 1. Principle

In order to examine the stability of autoclaved soslution sympatol ' the follwoing variables were used : (a) storage time : 20, 40, 80, 160 and 350 days. (b) storge temperature : 4°, 20° and 37°. (c) pH values : 3, 4, 5, 6 and 7. (d) antioxidants : sodium metabisulphite rongalit.

(e) gas nitrogen

air .

2. Preparation of the solution

Sympatol was accurately weighed and dissolved in freshly prepared double distilled water in a volumetric flask, so as to give a concentration of 6% w/v ofsympatol. The solution was divided into 5 parts and the pH was adjusted to 3, 4, 5, 6-25 and 7 by the addition of the following quantities of either tartaric acid or sodum bicarbonate : Table 24

PH Substance added

3-0 3-06% of tartaric acid

4-0 0'565% of tartaric acid

5-0 0-072% of trartaric acid

6'25 none

7*0 0-l % of sodium bicarbonate

In the case of solutions of pH 3 and 4, there was a slight increase in volume of the solution due to the addition of tartaric acid. The sympatol content was adjusted exactly to 6 % w/v by the addition of sympatol and the concentration rechecked by measuring the extinction at 223 me, The solutions were then filtered through a sintered glass funnel (Jenaer G 4).

Each of the solutions of different pH values was then divided into 4 equal parts. To one part 0.1 % ofsodium metabisulphite, to the 2 nd part 0.2 % of rongalit and to the other two parts no antioxidant was added. 2.1. Filling

Filling was done with a graduated burette with a large glass capillary so thai the volume in each ampule could be controlled. The solution was 63 exposed to air throughout the filling operation. Each of the four parts of each soultion was filled into 5 ml ampules approximately to 2/3 of their capacity (3.8 ml of the solution was filled in each ampule).

2.2. Sealing

Ampules containing the 1st part (namely 0-l% sodium metabisul- phite) of each solution of different pH values were sealed in the presence of air. Similarly, ampules containing the 2nd and 3rd parts (namely 0.2 % rongalit and no antioxidant respectively) were sealed in the presence of air. The 4th part of each solution containing no antioxidant was, however, sealed in the presence of nitrogen. For this, the ampules were placed under a bell-jar from which air was then removed by means of a vacuum pump. Nitrogen was then allowed to enter the bell-jar and the ampules were sealed immediately one by one.

2.3. Sterilization

in an The ampules were sterilized at 120° for 30 minutes Egro autoclave.

2 4. Storage

Sterilized ampules were stored at 4°, 20° (room temp.) and 37° in cardboard boxes, protected from light, for a period of 20, 40, 80, 160 and 350 days.

In all, the following sympatol solutions were prepared :

Table 25

Sympatol solution (6 % w/v)

pH Antioxidant present Gas Lot number

None Air 1 A Sodium metabisulphite 0.1 % Air 2 SA None Air 3 A

None Nitrogen 4 N2 Rongalit 0.2 % Air 5 RoA Sodium metabisulphite 0.1 % Air 6 SA None Air 7 A None Nitrogen 8 N2 Rongalit 0.2 % Air 9 RoA Sodium metabisulphite 0.1 % Air 10 SA None Air 11 A

None Nitrogen, 12 Na Rongalit 0.2 % Air 13 RoA Sodium metabisulphite 0.1 % Air 14 SA None Air 15 A

None Nitrogen 16 Na Rongalit 0.2% Air 17 RoA Sodium metabisulphite 0.1 % Air 18 SA 64

3. Testing of the ampules

Sterilized ampules were then analysed after different storage timings for development of colour, change in pH and sympatol content. 3.1. Colour test

For testing the development of colour the method as described on page 58 was applied. 3.1.1. Results

The results of the colour test for sterilized sympatol solutions which were performed immediately after sterilization, and after storage for 20, 40, 80, 160 and 350 days at 4°. 20° 37° are given in tables 26, 27 and 28 respectively.

Table 26

Effect of storage time on the COLOUR of the sterilized solution of sympatol (6 % w/v) at 4°.

Solution Time in days after which the colour was compared

pH Lot number

20 40 80 160 350

1 A BY3 BY3 BY3 BY3 DY3 BY2-BY3

2 SA R4 BY4 BY3 BY3 BY2 BY2

BY2 BY2 BY2 3 A B3 B3-BY3 BY1

BY5 BY5 BY4 4Na R6 BY6 BY4

Y3 BY3 BY2 5 RoA BY3 Y3 BY2

BY3 BY2 BY2 6 SA B5 BY3-BY4 BY1

Y4 Y3 7 A BY6 BY5 Y3 Y3

BY6 Y6 8N2 CL BY6 Y4 Y4

CL CL 9 RoA CL CL CL CL

CL CL 10 SA CL CL CL CL

BY4 BY4 BY4 11 A BY5 BY5 BY3

CL Y6 12 Na CL CL Y5 Y5

Y6 Y6 13 RoA CL Y6 Y6 Y6

CL CL 14 SA CL CL CL CL

Y6 Y6 15 A CL Y6 Y6 Y5

CL CL 16 N2 CL CL CL CL

CL CL 17 RoA CL CL CL CL

CL CL 18 SA CL CL CL CL 65

Tabic 27

Effect of storage time on the COLOUR of the sterilized solutions of Sympatol (6%w/v) at 20°.

Solution Time in days after which the colour was compared PH lot number

20 40 80 160 350

7 1 A BY3 BY2 BY2 BY2 BY1 C

2 SA B3-B4 BY3 BY3 BY2 BY2 BY1

3 A 133 BY2 B1-BY1 BY1 BY1 C

6 4 N2 R6 BY5 BY4 BY3-BY4 BY3 BY3

5 RoA BY3 Y3 Y3 Y3 Y3 Y3

6 SA B5 BY2-BY3 BY1 BY2 — C

7 A BY6 BY3 BY1 BY3Z BY2 C

5 8 N2 CL BY5 Y5 Y4 Y4 BY3

9 RoA CL CL Y6 Y6 Y5 Y4

10 SA CL CL Y6 Y5 Y5 C

11 A BY5 BY4 BY3 BY3 BY2 BY1

4 12 CL N2 CL Y6 Y5 Y4 Y4

13 RoA CL Y6 Y6 Y6 Y6 Y6

CL 14 SA CL CL CL CL CL

15 A CL Y6 Y5 Y5 Y4 Y4

16 CL CL N2 CL CL Y5 Y5

17 RoA CL Y6 Y6 Y5 Y5 Y4

18 SA CL CL CL CL CL CL

18 SA after storage for 24 months was colourless. 66

Table 28

Effect of storage time on the COLOUR of the sterilized solutions of Sympatol (6% w/v) at 37°

pH Solution Time: in days after which the colour was compared lot number

0 20 40 80 160 350

7 1 A DY3 BY1 BY32 BY22 BY1» C

2 SA R4 BY1 BY1 .BY1 BY1 C

3 A B3 BY1 BY22 BY22 BY22 C

6 4 N2 R6 BY4 BY3 BY3 BY3 BY3

5 RoA BY3 — Y3 Y3 Y3 Y2

6 SA B5 B BY22 BY22 — C

7 A BY6 BY1 BY12 BY12 BY12 C

5 8 N2 CL Y4 Y4 Y4 BY3 BY3

9 RoA CL Y6 — Y6 Y4 Y3

10 SA CL CL BY4 Y4 Y4 C

11 A BY5 Yl BY1 BY1 BY1 C

4 12 N2 CL Y5 Y5 Y4 Y3 Y3

13 RoA CL Y6 Y6 Y5 Y5 Y5

14 SA CL CL CL CL BY3 BY3

15 A CL Y5 Y4 Y3 BY4 BY1

3 16 N2 CL CL Y5 Y5 Y4 Y4

17 RoA CL Y5 Y4 Y4 Y4 BY3

18 SA CL CL CL CL CL CL

18 SA aftet storage for 15 months, CL; after 18 months Y6

CL • = colourless for colour standards

BY1 A \ = air

Yl N* «s nitrogen

RoA = rongalit 0'2%+air BY2 etc see 'page 60.

SA = sodium-metabisuphite 0'1 % +air.

' = deeply coloured beyond range of standards used. 1 67

3.12. Summary of the results of colour determinations 3.1 2 1 Sterilization at 120° for 30 minutes

Heating at 120° for 30 minutes is sufficient to colour the sympatol solutions of initial pH 6 and 7, irrespective of the antioxidant or gas content. Solutions of initial pH 5 and 4 containing neither antioxidant nor gas showed colouration on autoclaving, while those containing either of the two remained colourless. Further all the solutions of pH 3 remained colourless on autoclaving. 3.1.2 2. Storage at 4° For storage periods between 20 and 350 days, time .factor in general had no qualitative and indeed little quantitative influence as a factor in the colouration. Without exception all solutions of 7 and 4 and initial pH and 6, of pH 5, 3 not containing any antioxidant, became more coloured. The colour variation were between CL to Y5 or from B5 to BY1. Further solutions of pH 5 and 4 with nitrogen also developed a colour to the extent of Y4. Solutions of pH5 with rongalit or sodium metabisulphite, solutions of pH 4 with sodium metabisulphite and all the solutions of pH 3 with antioxidant or nitrogen remained colourless even after 350 days.

3 1.2.3. Storage at 20° between 20 and 350 days.

On storage at 20° the development of colour is more prominent than at 4°. All solutions of initial pH 7, 6 and 5 show colouration to the extent of colour standard BY3 or brown. The Solution of pH 5 with rongalit however shows colouration only to the extent of Y4. Solutions of pH 4 and 3 with sodium metabisulphite remain fully colourless after 350 days while solutions of pH 3 containing nitrogen remain colourless till 80 days. The rest of the solutions of pH 4 and 3 develop colour to the ex¬ tent of Y4 to Y6.

3.1.2.4. Storage at 370 between 20 and 350 days

On storage at 37° the development of colour reacnes tne maximum. Solutions of initial pH 7, 6, 5, 4 and 3 become coloured to the extent of Y5 to brown, with the exception of the solution of pH 4 with sodium metabisulphite which remained colourless up to 80 days and the solution of pH 3 with sodium metabisulphite which remained colourless for the entire period.

3.1.2.5. Solutions of pH 3 with sodium metabisulphite remain at least for completely colourless 24 months when stored at 4° or 20°, while at 37° a yellow colour to the extent of Y6 developed after storage for 18 months.

32. Determination of pH

The value of pH the sterilized sympatol solutions was determined as described on page. 61.

32.1. Results

The results of the pH determinations on the various solutions of sympatol are given in table 29. 68

Table 29

stor time at 4° 20° and 37° on the of the sterilized solutions of Effect of age , pH sympatol

pH of Sympatol solutions

before immedi- autoclaved solutions after storage of ately 350 pH Solution Storage steril- after Auto-20 days 40 days 80 days 160 days days temp.in "C ization claving

4 7-2 71 69 69 69 69 6-9

6-6 1 A 20 7-2 7-1 68 68 67 67

37 72 7-1 6-8 6-7 67 65 66

4 71 69 62 6-2 62 6-2 6,2

62 2 SA 20 7-1 6-9 6-2 6-2 62 62

37 7-1 6-9 61 6-2 62 62 60

58 4 62 5-9 5-8 '5-8 58 58

5-7 5-7 56 3 A 20 62 5-9 58 5-7

56 37 62 5-9 5-7 5-7 55 56

60 4 62 60 5-9 60 60 60

60 60 60 4 N2 20 62 60 60 60

37 62 60 60 60 60 60 5-9

5-5 55 4 57 55 5-5 5-5 5-5

55 55 20 57 55 55 55 5-5

5-5 55 55 55 6 RoA 37 57 55 5-5

5-3 5-3 53 4 5-3 53 5-3 5-3

52 5-2 5-2 5-2 6 SA 53 53 53

5-2 5-1 53 53 52 52 5-3 .

50 50 50 4 50 50 49 4-9

4-9 49 50 4-9 7 A 20 50 50 49

4-9 49 4-9 37 50 50 49 4-9

50 50 50 4 50 50 49 49

4-9 50 50 50 8 N3 20 50 50 49

50 50 5-0 37 50 50 50 50 69

pH of Sympatol solutions

pH Solution Storage before immediately/ autodaved solutions after storage of ' temp. in 'C steril¬ after auto- — ization claving 20 days 40 days 80 days 160 days 350 days

5 4 49 4-9 48 48 49 4-9 49

• 9 RoAl 20 49 4-9 4-8 48 4-9 4-9 4-9

37 4-9 49 48 48 48 4-9 49

4 49 4-9 49 4-9 49 49 49

10 SA 20 4-9 4-9 4-9 4-9 4-9 4-9 4-9

37 4-9 49 49 4-9 49 4-9 4-9

11 A 4 4-0 40 40 40 40 40 40

' 20 40 40 40 40 40 40 40

4 37 40 4-0 40 40 40 40 40

12 N2 4 40 40 40 40 40 40 40

20 40 4-0 40 40 40 40 40

37 40 40 40 40 40 40 40

4 40 40 40 40 40 4-0 40

13 Ro A 20 4-0 40 40 40 40 4-0 . . 4-0

37 40 40 4-0 40 40 40 40

4 40 40 40 40 40 40 4-0

4 14 SA 20 40 40 40 40 40 40 40

37 40 40 40 40 40 40 4-0

4 30 30 2-9 30 30 30 3-0

3 15 A 20 30 30 30 30 30 30 30

37 3-0 30 30 29 30 30 30

16 N2 4 30 3-0 29 3-0 30 30 3-0

20 30 30 2-9 30 30 30 3-0

37 30 30 2-9 30 30 30 3-0 70

pH of Sympatol solutions

autoclaved before immediately solution after storage of pH Solution Storage ' temp, in °C steril- after auto ization claving 20 days 40 days 80 days 160 days 350 days

4 30 3-0 30 30 30 30 3 0

" 1 RoA 20 30 30 30 30 30 30 30

37 3-0 30 30 30 30 30 30

4 30 3-0 30 30 30 30 30

B SA 20 30 30 30 30 30 3-1 30

37 3 0 30 30 30 30 30 30

A = Air = , N2 Nitrogen

RoA = SA = Sodium Rongalit 0.2 %-fair , metabisulphite 0.1%+air.

3.2.2. Summary of the results of pH determination of sympatol solutions

3.2.2.1. Immediately after sterilization at 120° for 30 minutes in an autoclave, the pH of sympatol solutions no. 1 to 5 slightly dropped down towards the acidic side, while the pH of all other solutions remained unchanged.

3.2.2.2. After storage at 4°, the pH of autoclaved sympatol solu¬ tions no. 1, 2 and 3 dropped down after 20 days (change of pH 0.1 to 0.7 pH units.) after which there was no change, while the pH of all of the other solutions remained unchanged after 350 days.

3.2.2.3. After storage at 20° the pH of the autoclaved sympatol solutions no. I, 2 and 3 tends towards the acidic side even after 350 days (change of pH 0.6 to 0.9 pH units) while the pH of all other solutions remained unaffected.

3.2.2.4. After storage at 37°, the effect on the autoclaved sym¬ patol solutions no. 1, 2 and 3 is the same as at 20° (change of pH, 0.6 of 1.1 pH units). The pH of all other solutions remained unaffected.

3.2.2.5. There is practically no effect of heat sterilization or storage time on the pH of those solutions with a pH value less than 6 after storage at 4°, 20° and 37° for a period of 350 days.

4 Discussion and conclusions of the results of colour and pH determination

4.1. The development of colour is dependent on pH of the solution and storage temperature. 71

4.2. Though nitrogen checks the oxidation of the phenolic OH group, it is not sufficient to stabilize the sympatol solutions, as we found that the sympatol solutions containing nitrogen become coloured after one year.

4.3. Of the two antioxidants used, sodium metabisulphite was more useful to check the development of colour than rongalit.

4.4. In order to obtain the optimum stability of the autoclaved sympatol solutions the following conditions must be adhered to :

— as low as (a) pH value should be possible (pH 3) .

(b) — air must be repalced by nitrogen.

— be (c) as antioxidant 0-1% of sodium metabisulphite should added.

(d) — storage temperature must be as low as possible.

On the grounds of the above findings, the following instructions solutions are proposed for the preparation and storage of the parental of sympatol.

5. Injection of sympatol

The following formula is recommended for the injection of sympatol:

Sympatol 6.0 g Sodium metabisulphite 0.1 g Acid tartaric 3.1 g Aqua bidistillata to make 100 ml

Dissolve sodium metabisulphite and tartaric acid in freshly boiled and cooled double distilled water in an atmosphere of nitrogen. Then add sympatol, filter and make up the required volume. Fill and seal the ampules in an atmosphere of nitrogen.

Sterilization. Sterilize by autoclaving at 120° for 15 minutes.

pH. Between 2.8 to 3.2.

Storage. The ampules should be stored at low temperatures and can be used till the development of colour is not more than the standard colour Y6, which does not take place till after at least 18 months at storage temperature of 37° and 24 months or more at a storage temperature of 20°. STABILITY STUDIES OF NORADRENALINE (NA) SOLUTIONS WITH RESPECT TO RACEMIZATION

CHAPTER VIII

INTRODUCTION AND PROBLEM Introduction. Recently NA has gained a great importance as a therapeutic agent, as would be clear from the fact that it has been recognized by several pharmacopoeias. The conditions for the stability of NA solu¬ tions were described by West (33). He recommended a pH of about 3.6 and the presence of 0*1% of sodium metabisulphite. March (110) has assayed solutions of NA prepared according to Ph. Dan. IX. Add. 1954 (111). However none of the papers deal with the problem of-racemiz¬ ation of NA though similar studies for adrenaline were available by Kisbye and Schou (40), Kisbye (41) and Rosenblum el al. (112).

As it is well known that the laevo form of NA is about 40 times more active than the dextro form, it is important that solutions of NA do not undergo racemization during sterilization and storage. No test for the specific rotation of NA solutions is given by any of the pharmaco¬ poeias, obviously due to the fact that it is not possible to determine the rotation of such dilute solutions. Hellberg (113) has described a method for concentrating the dilute solutions of adrenaline and NA and then determining the rotation.

Problem. In the following study

(a) the conditions for maintenance of optimum optical activity of the dilute solutions of 1-NA during sterilization and storage have been investigated.

(b) by carrying out the stability experiments at higher temperatures we have determined the velocity constant of the racemization of NA solutions and the temperature coefficient for a difference of 10° and then have predicted the stability with respect to racemization of NA solutions at storage temperature on the basis of these results. CHAPTER IX

NORADRENALINE : SYNONYMS, SYNTHESIS, PHYSICAL PRO- PERTIES, QUALITATIVE TESTS, METHODS OF QUANTITA¬ TIVE ANALYSIS, PHARMACOLOGICAL ACTIONS, CLINICAL USES AND DOSAGE.

1. Synonyms and proprietary names Levarterenol bitartrate U. S. P. XV (114) Nor-adrenaline acid tartrate Brit. Ph. 1958 (115) Nor-adrenaline bitartrate Ph. Int. I (116) Nor-adrenaline bitartrate Ph. Dan. IX. Add. 1954 (111) 1 -Norepinephrine Levophed bitartrate Winthrop-Stearns 1-Arterenol Hoechst Aktanin Schering (Germany) Noradrec Recordati Noradrenaline Boehringer, Byk

Wt. 337. 3 . Mol. C,Hu08N.C4Ht0,.Hi0 . OH 0

II — CHOH-C-O HO—6}—CH—CH3—NH3 1 A CHOH-C-OH H Ha0 II J 0

Chemical name. Noradrenaline bitartrate is (—)— o<—3:4—dihydroxy phenyl -/}- aminoethanol d—bitartrate monohydrate, or the monohydrate of (—)—2— amino —1—3' —4' dihydroxyphenyl ethanol acid tartrate. 2. Synthesis

NA was first synthesised by Stolz (117) in the year 1904. According to his method NA may be prepared by treating pyrocatechol with chloro- acetylchloride and phosphorous oxychloride. The reaction product is then treated with ammonia and, finally, moderate reduction of the ketone gives dl—NA.

OH OH H0*0 + G1COCH»C1 -» CICH/XX)-^

OH

POG1 HO-6 >COCHaCl a > -\J

OH OH

NHg HO-^>-COCHaNH., > HO-^>—CH-CH3-NHa OH 74

Several other methods are also available for the synthesis of NA, for which the following literature is cited : (118), (119), (120), (121), (122), (123).

However the resolution of di—NA was reported only recently by Tullar (124) and Tainter et al.' (125) in 1948. The separation was based on the fact that only 1—NA forms a hydrated salt with d-tartaric acid. This 1-noradrenaline d-bitartrate monohydrate possesses greater'solubility in aqueous methanol and considerably lower water solubility than does the nonhydrated d-noradrenaline d-bitartrate, permitting an easy separation of the diasteromers.

Noradrenaline acid tartrate is required to comply with the following tests as given in Brit. Ph. 1958 (115)

3. Physical properties 9 3. 1. Description

A white or nearly white, crystalline powder; odourless; tastes bitter; gradually darkens on exposure to air and light. 3. 2. Solubility

Freely soluble at 20° in 2.5 parts of water, slightly soluble in ethanol 95% and practically insoluble in ether and chloroform.

3. 3. Specific rotation

— _ 10° to 12° U.S.P. XV

"

— _ 9.4° to 12° ; Ph. Dan. IX. Add. 1954

— 9.8° to — 11.2° Ph. Int. I

3. 4. Melting range

102° Brit. Ph. 1958

100° to 106° U. S. P. XV, Ph. Dan.

IX. Add. 1954, Ph. Int.l

4. Qualitative tests

4. 1. Identification tests

» 4.1.1. Dissolve 10 mg of NA-acid tartrate in 1 ml of water, add 1 drop of ferric chloride solution (15% w/v), an intense green colour is produced which changes to blue and then to red on the gradual addi¬ tion of sodium bicarbonate solution (5°/0 w/v). 4. 1. 2. Acidity

pH of a l°/0 w/y solution : 3.5 to 5.0.

4. 1. 3. Extinction

Extinction of a 1-cmla.yer of a0.005o/o w/v solution in N/100 HG1 at 279 m^: about 0.40. i 75

4. 1. 4. Tartrates

Moisten a few milligramsNA—acid tartrate with a drop of water, add to 2 ml of sulphuric acid followed by a crystal of resorcinol : on carefully warming a red violent colour is formed. 4. 2. Distinction from similar substances (adrenaline & isoprenaline)

Mix 1 ml of 0.1 % NA-acid tartrate solution with 10 ml of a standard solution of pH 3.6, add 1 ml of N/10 iodine, allow to stand for 5 minutes, and add 2 ml of N/10 Na2Sa03 solution : not more than a very faint red colour is produced. Repeat the operation using solution of standard pH 6.6: a strong reddish violet colour is produced. 4. 3. Purity tests 4. 3. 1. Aminomethyl—(3,4—dihydroxyphenyl)—ketone, (Norad- renalone) Prepare a 0.1% solution of NA—acid tartrate in 0-01 N HC1, measure the extinction at 310 m/i in 1 cm cuvettes : the extinction

° must not be more than 0.2 (i. e. E, must not be more than 2). 1 cm

4. 3. 2. Water 5.0 to 6.0 % w/w when determined by Karl Fischer method.

4. 3. 3. Loss on ignition

On ignition NA-acid tartrate must not give a residue of more than 0.1 % w/w.

5. Methods of quantitative analysis described in the literature

For the quantitative estimation of NA many methods are available. They may be divided into two categories.

5.1. Biological 5.2. Chemical

5. 1. Biological methods

Biological methods are in some cases more specific and useful when it is to estimate required the undecomposed product in a solution. However the great disadvantage of the biological methods is that they are time very consuming, costly and there is always a great deviation in the results. Consequently we have based our results on chemical methods of analysis.

5. 2. Chemical methods

The chemical methods for the quantitative determination of NA may be classified into four categories. 5. 2. 1. Volumetric

5. 2. 2. Colorimetric

5. 2. 3. Fluorometric

i 5. 2. 4. Spectrophotometry 76

5. 2. 1. Volumetric methods

5. 2. 1. 1. By determination of nitrogen content The method is based on the determination of the nitrogen content of the cation (+) portion of the molecule. The method is official for the assay of NA-acid tartrate in U. S. P. XV (114).

5. 2. 1. 2. By titration in a non-aqueous medium In this method the NA content is determined by titration in a non¬ aqueous medium like glacial acetic acid with perchloric acid. The method is official for the assay of NA-acid tartrate in Brit. Ph. 1958 and in Ph. Dan. IX. Add. 1954.

5. 2. 2. Colorimetric methods

Most of the colorimetric methods are based on the formation of noradrenochrome or iodonoradrenochrome by such oxidizing agents as iodine, permanganate, ferricyanide etc. The reaction may be for¬ mulated as follows :

HO— ff^, CHOH 0=(^% CHOH 0= ^ CHOH HO-ll^ | -» 0=kJ I -> 0=1^ | /CHs /CH2 \/CHa N N N

H2 Ha H Further, some diazonium compounds have also been used for the formation of a coloured product with NA. It is to be noted that the results obtained by colorimetric methods do not differentiate between the racemic and the I-form which latter is physiologically more active.

5. 2. 2. 1. Iodine method

Euler aud Hamberg (126) have worked out a method for the deter¬ mination of adrenaline and NA in a mixture. When a solution of NA buffered at pH 6 is treated with iodine, the maximum formation of noradrenochrome takes place after 3 minutes iodine treatment. The colour is measured at 529 m/j, after neutralizing the excess of iodine with sodium thiosulphate solution. Oesterling (127) showed that the noradrenochrome is more stable if the concentration of the buffer is N/10 rather than N/2. The above method has been adopted in Ph. •Int. 1 (128) for the assay of the injection of NA-acid tartrate.

5. 2. 2. 2. Permanganate method In this method Suzuki and Ozaki (129) used a solution of potassium permanganate in lactic acid at pH 6.5 and 3 minutes oxidizing time. showed They that their method gives a uniform tint of colour in comparison to the iodine method of Euler and Hamberg. 5 2 2.3 Method of Folin, Cannon and Denis

The phosphomolybdic acid reagent (130) and the phenol reagent which colour (99) give reactions with polyphenols can be applied for the estimation of NA. But the colour formed is very unstable and further, metabisulphites interfere with the reaction as reported by Somer and West (131). 77

5.2.2.4 Ferrocitrate method

This method is based on the colour reaction of the phenolic OH * groups by Fe* ions. The intensity of the coloured product is measured in a suitable photometer and the NA content is found by means of a standard curve. Richter (132) has given the details of the method for the estimation of adrenaline and NA. The method is used in U.S.P. IV (133) for the estimation of adrenaline content.

5.2.25. Other coloritnerric methods The method of Auerbach and Angell (134), where the colour is deve¬ loped by the action of NA with sodium-napthoquinone-4-sulfonate and benzalammonium chloride, is very lengthy and too cumbersome.

Schuler and Hdnrich (135) have described a method in which they used the napthalene sulfonates of 2 or 3 halogen-4-nitro-l-aminobenzene as the reagent. The formation of the colour is assumed to be due to transformation or decomposition of the diazonium salt caused by phenolic compounds in an acid medium.

5 2.3. Fluorometric methods

Fluorometric methods are based on the formation of a fluorescent product, when NA is oxidized in an alkaline medium. Lund (136), (137) has established the constitution of the fluorescent product formed by the oxidation of adrenaline which is adrenolutine. It is assumed that NA also follows the same course with the formation of noradrenolutina sb follows : HO— r**S CHOH 0=^ CHOH HO—%J J -^O :Cr J,

-»HOl1

H H keto Noradrenolutine enol

Most of the fluorometric methods have been designed to estimate adrenaline and NA in mixture in body fluids ; however all the methods can be profitably employed to estimate NA in pure solutions.

5.2.3.1. Method of Lund

The method is based on the formation of 3:5:6-trihydroxyindole which fluoresces in ultraviolet light (138). NA is oxidized to noradreno- chrome with manganese dioxide, and the noradrenolutine is formed by the addition of a strong alkali. Ascorbic acid is added along with the alkali so that the oxidation does not proceed further than the norad¬ renolutine stage. The NA content is determined by measuring the fluorescence of the noradrenolutine and subsequent comparison with a standard curve. 78

5.2.3.2. By condensation with ethylene diamine

The observation of Natelson el al. (139) that the catechol amines form strongly fluorescent condensation products with ethylene-diamine in the presence of oxygen has been used by Weil-Malherbe and Bones (140) for the estimation of adrenaline and NA. The samples of adrenaline and NA are heated with ethylenediamine and the fluorescent product is quantitatively extracted with isobutanol. The fluorescence is then measured in a suitable spectrophotometer. The reaction probably takes the following course :

/^—CHOH HO- O = r^s CH.OH HO—^ !| §:Q- GHa+02 > \/CH2 I N NH, I H

CHaNHa N - + I r^r5^—CHOH

N \/CHa N A

The method has the advantage that the fluorescent products are with of very stable as compared the method Lund; however, the method cannot be applied in the presence of metabisulphites.

Erne and Cant ack (141) have modified the original method of Weil- Malherbe and Bones for the estimation of adrenaline and NA and have further proposed a combination of ion-exchange and subsequent fluorimetry to remove metabisulphites. 5.2.3.3 Fluorescent method of Euler and Floding (142), (143)

In this method potassium ferricyanide is used as the oxidizing agent for NA at pH 6. After treating the oxidation product with strong alkali and stabilization of the fluorescent product with ascorbic acid, the fluorescent intensities are measured.

5.2.4 Spectrophotometric method

NA gives an absorption maxima at 221 and 279 mp-. The absor- 1% bancy index, E at 279 mju for NA-acid tartrate monohydrate being 1 cm 80. The above property can be utilised to estimate the NA content in solu¬ tions. In U.S.P. XV (144) this spectrophotometric method is used to deter¬ mine the NA content only in the injections of NA-aicd tartrate, while in Ph. Int. I (116) the method is official for the assay of NA-acid tartrate. However, it is to be noted that for the etimation of NA in a solution, part of which might have been oxidized, it is essential that the oxidized product does not give any appreciable absorption at the wavelength at which the absorption readings for NA are to be taken. 79

6. Pharmacological actions

Comparative pharmacology of NA was first described by Barger and Dale (54). It is a more general systemic vasoconstrictor agent than adrenaline. The pharmacological actions of NA have been discussed in .a comprehensive review by' Euler (145), Lands (146) and Burn and Hutch'e'on (147). We shall describe them below.

6.1. Action on heart and coronary vessels

The action of NA on the heart is in many respects similar to that of stimulation of the sympathetic heart nerves.' When given intravenously NA causes bradycardi a and a reflex slowing of the heart. NA produces only a slight and transient increase in ventricular rate, while adrenaline shows a considerable rise in heart frequency.

NA dilates the coronary arteries and its action is about two and one- half times than that of adrenaline.

6.2. Systemic circulation

There is an almost parallel rise in systolic and diastolic blood pres¬ sures after intravenous infusion of NA, with practically no increase in cardiac output, showing general vasoconstriction where as, a similar dose of adrenaline, though raising the ventricular pressure lowers the diastolic pressure with little or no change in mean pressure. Since the cardiac output is simultaneously increased the action implies vasodilation. 6.3. Muscle and skin circulation Both adrenaline and NA cause constriction of the skin vessels though the action of adrenaline is more pronounced. The results of different authors show that whereas adrenaline causes dilation of the vessels of the muscle, NA causes vasoconstriction. 6.4. Kidneys

NA has a constrictor action on the vessels of the kidneys, the effect, however, being smaller than that of adrenaline. The effect is attri¬ buted to the indirect action of NA on systemic circulation. 6.5. Smooth muscles

Usually NA has a weaker effect on smooth muscles than adrenaline, and is not as potent a bronchodilator as adrenaline. 6.6. Glandular secretion In general NA increases the glandular secretion, for example of sali¬ the liberation of adrenaline and NA inhibits vary glands. Though gast¬ ric secretion. 6.7. Relative potency of Isomers Luduena et al. (148) found the 1-form of NA to be 12 to 60 times as inhibition or excitation active as the d-form, irrespective of the fact whether the 1-form to be 45 times was caused. Swanson and Chen (149) found of more active than the d-form when tested on the blood pressure response pithed cats and dogs. 6.8. Toxicity NA is considerably less toxic than adrenaline. In the following table given by Euler (145) the comparative lethal doses of adrenaline and NA is given as found by different workers. 80

Table 30.

Acute toxicity of intravenously injected adrenaline and noradrenaline in mice after 24 hours.

LD -. in LD Substance group cages ,- in single cages 50 50 mg/kg mg/kg

1—Adrenaline 2.4 ± 0.2 2.5 ± 0.2

1—Noradrenaline 6.1 =fc 2.0 20.5 d—Noradrenaline 70.7 ± 428 66.0

7. Clinical uses

Since the findings of Euler (150), (47), Holtz et al. (151) and others show that NA is naturally present in the animal body, it has gained impor¬ tance as a therapeutic agent.

Its NA is used as a general vasoconstrictor. usefulness as a pressor We shall describe below drug was first reported by Goldenberg et al. (152). its main uses taken from the review on NA by Euler (145) :—

7. 1. In acute hypotension due to loss of vasomotor tone and also after central vasomotor depression of various origins.

7. 2. In shocks of various kinds like haemorrhage, post operative It has been to be the best vaso¬ blood pressure depression etc. reported constrictor agent against the circulatory shock in acute myocardial infarct.

7.3. To prolong the duration of spinal anesthesia NA is more advantageous than adrenaline in conjugation with local anesthetics due and absence of to its greater resistance to oxidation the tachycardia- shortcomings which are comnlonly associated with adrenaline.

Churchill Davidson (153) has in detail discussed the uses of NA.

8. Dosage

By intravenous infusion 5 to 25 micro grams per minute, according to the blood pressure of the patient. CHAPTER X

TESTING OF THE MATERIAL 1. Noradrenaline hydrochloride

In our expriments we have made the racemization studies of dilute solutions of NA. It was necessary to obtain maximum optical rotation within the limited concentrations of NA. So we have used 1-NA hydrochloride in our experiments because the specific rotation of 1-NA hydrochloride is about 4 times that of 1-NA acid tartrate.

1-NA hydrochloride must confirm to all the tests as described for NA acid tartrate on page 74, as all of these tests are based on the cation (+) portion of the molecule, except tests of melting range, specific rotation and tartrates which are for NA hydrochloride as follows : Tullar Melting range. 145.2° to 146.4° (124)

Specific rotation. — 40° Throughout our experiments we have used the sample of (1-Noradrenaline hydrochloride (Hoechst) which complied with the tests described below.

1.1. Physical tests

1.1.1. Melting range 144°—147°. The melting range of NA hydrochloride was between 1.1.2. Specific rotation A 2 % w/v solution of NA hydrochloride was prepared in water, and the rotation was measured at 20°, using a 200 mm long polarimeter tube. The specific rotation was found to be —40°.

1.2. Chemical tests

1.2.1. Identification tests

1.2.1.1. Test withferric chloride. NA hydrochloride gave a positive test with ferric chloride solution as described on page 74.

1.2.1.2. Acidity. pH of a 1 % w/v solution of NA hydrochloride was 4.35.

1.2.1.3. Chloride. NA hydrochloride gave a positive test for chloride ions.

1.2.2. Distinction from similar substances. (Adrenaline and isopre- with the naline) NA hydrochloride complied test as given on page. 75. 1.2.3. Purity tests 1.2.3.1. Test for aminomethyU'3, 4-dihydroxyphenyl)-ketone On nor-adrenalone)). measuring the extinction of a 5 mg % solution of NA hydrochloride at 310 m^ using 1 cm cuvettes, the value of 1% E was 0.1., showing that the sample complied with the test described on 1 cm. page 75. 82

1.2.3.2- Test for copper ions. A 5 mg% solution of dithizone (diphenylthiocarbazone) was prepared in carbon tetrachloride. 5 ml of the 1% solution of NA hydrochloride were shaken with 0.1 ml of the dithizone reagent for 60 seconds. There was no change in the colour of the carbon tetrachloride layer showing the absence of Cu "*" ions.

1.2.3.3. Loss on ignition. NA hydrochloride complies with the test of loss on ignition as described on page 75.

1.3. Quantitative estimation of NA

The sample of NA hydrochloride was assayed according to the Method of Ph. Int. I (116).

1.3.1. Method

A 5 mg % solution of NA hydrochloride was prepared in 0.01 N HC1. Extinction readings at 279 m^ were taken in the Zeiss Spectropho¬ tometer, using 1 cm cuvettes to hold the solution and the solvent blank. 1.3.2. Results

The results are given in table 31. below.

Table 31

- Extinction values at 279 ir/l for a 5 mg % solution of NA Hcl.

Extinction value NA hydrochloride content

0.640 calculated from the value

0.645 eJ c% =80 for CsHnOgN.C^Os-t^O

0.650 98-5 % CHAPTER XI

DETERMINATION OF FRESH AND DETERIORATED NA IN SOLUTIONS

1. Introduction

1.1. Oxidation of NA

Solutions of NA are liable to undergo spontaneous oxidation in oxidation the presence of oxygen. The proceeds quicker in an alkaline medium than in an acid medium. The oxidation is accelerated by the Cu"*" presence of metallic ions, especially ions as reported by Schou (34). In recent years the chemistry of the oxidation of NA has been thoroughly investigated.

1.1.1. Noradrenochrome

On oxidation NA gives a red colour which has been attributed to the formation of noradrenochrome. Noradrenochrome has been identified by Beaudet (154). He prepared it by treatment of NA with silver oxide. Noradrenochrome gives absorption maxima in aqueous solutions at 490, 310 and 205 m/*. The same results were obtained by Marquardt and Carl (155). According to Green and Richter (156), when adrenaline is treated with iodine the first stage is the formation of adrenaline quinone, which is then transformed into adrenochrome. It is assumed that NA follows the same course. BulockandHarley-Mason (157) have discussed the mechanism- of oxidation of adrenaline and NA.

1.1.2. Iodonoradrenochrome

When NA is treated wit.li potassium iodate iodonoradrenochrome is formed. It was prepared by Barer et al. (158), and has been given the following structure :

0=,^ CHOH

\/CH.I N H

However, we did not find any absorption study of iodonoradreno¬ chrome in the literature.

All the colorimetric and fluorometric methods which we have described on page 76, are based on the formation of a colour product by oxidation with iodine, permanganate etc., or on the coupling reaction with a suitable diazo reagent, or on the formation of a fluorescent product.

However by looking into the literature it was not clear if these methods of analysis of NA could be used to determine the deteriorated NA in a so¬ lution. Therefore we have investigated the following methods to find out if they can differentiate quantitatively between NA and the deteriora¬ ted NA in a solution. 84

2. Spectrophotometry method (UV.) 3. Colorimetric methods 3.1. Iodine method 3.2. Persulphate method 3.3. Ferrocitrate method

2. UV spectrophotometric method This method is official in Ph. Int. I. (116) to assay the sample of NA-acid-tartrate while in U. S. P. XV (144) only for the injection of NA-acid-tartrate. it is However, not clear if the method could differentiate between NA and its oxidation products quantitatively So in this we have tried to investigation use the method for the estimation of deteriorated NA in solution.

2.1. Method

A 5 mg % solution of NA base (1) was prepared in 0.01 N HC1, and that of NA hydrochloride (II) and NA-acid-tartrate monohydrate (III) in water. The extinction readings between the wavelengths of 210 to 300 rmi were taken for all the 3 solutions (I, II, III) which are given in table 33.

2.2. Results

NA gives absorption maxima at 220-221 m/t and at 279 m/*. The ratio of the extinction values at the 2 maxima is 2.26:1. The graphical relationship between extinction and wavelength has been shown in fig. 7.

' In table 32 the extinction values at 279 m^ for the different salts of NA, together with their corresponding molecular weights are given.

Table 32

Extinction values for NA base, NA hydrochloride and NA acid tartrate monohydrate at 279 m/^along with their molecular weights

Data NA base NA hydro- NA acid tartrate chloride monohydrate

(I) . (II) (III)

Molecular weight 169-1 £05G 3373

Ratio of mol. weights 1:11 = 0.82 I:III a 050

Extinction values for a concen¬ 0-775 0-640 tration of 65 mg . 0-395

Ratio of extinction values II: I =s 0.82 III: I = 0'50

From the above table it is clear that the extinction value is dependent of only on the cation (+) portion of the molecule NA. 85

Table 33 Relation between extinction and wavelength for (I) Noradrenaline base (solution in 0.01 N HC1) (II) Noradrenaline hydrochloride (aqueous solution) (III) Noradrenaline acid tartrate monohydrate (aqueous solution) (IV) A 2 % w/v solution of NA base in 0.01 N HGl, which was heated at 210° for 2 hours in the presence of oxygen. The precipitate was filtered after heating.

Concentration of each of the solution was 5 mg %,

Extinction values at a cone, of 5 mg% (4%*) \ f I II III IV Wavelength NA base NA hydrochloride NAacid Heated solution in mC tart of NA base rate

210 1-90 1-80 1-35 1-90 215 1-65 1-375 0-88

217 1-675 1-40 0-895

218 1-725 1-425 0-8975 1-725 219 1-725 1-45 0-90 1-725

220 1-75 1-45 0-9025 1-75

221 1-75 1-45 0-9025 1-75 222 1-725 1-425 0-90 1-725 223 1-70 1-425 0-8875

225 1-60 1-38 0-88 1-65

230 1-40 115 0-72 1-40 235 0-83 0-69 0-42

240 0-31 0-26 016 0-36 245 012 008 005

250 009 005 0035 0105 255 0092 0078 0052

260 0175 015 0094 0-22

265 0-32 0-265 0165 0-34

270 0-51 0-4225 0-265 0-535

275 0-70 0-58 0-3575 0-705 276 0-73 0-60 0-375 0-7375 277 0-755 0-62 0-385

278 0-77 0-635 0-3925 0-7725 279 0-775 064 0-395 0-775 280 0-77 0-635 0-3925 0-77

281 0-765 0-63 0-3S0

285 0-65 0-5375 0-33 0-66

290 0-285 0-245 015 0-3125 016 295 0038 0-036 0024 0002 0-035 300 000 0002 C-029 310 320 0-026 86

2.3- Effect of heating on the U.V. absorption carve of NA For this investigation a 0.1 % solution of NA base in hydrochloric acid of pH 3 was filled in 5 ml ampules to half their capacity in the pre¬ sence of oxygen. The ampules were heated at 120° for 2 hours in an autoclave. The solution turned brown with a black precipitate after heat treatment showing some destruction of NA. The solution was fil¬ tered and the extinction readings between the wavelengths of 210 to 320 tnn were taken by using a concentration of 5 mg%.

Thejreadings of the extinction values are given in table 33 and a graph showing the relationship between extinction and wavelength is shown in fig. 7.

,.5mgtf 3 2 "1cm ABSORPTION CURVES OF NORADRENALINE 0,8

0.6-

0,2.

300 mu

1.NA BASE,solutionin 0-01 NHCl . solution 2.NA Hydrochloride,aqueous .

3.NA Acid tartrate .aqueous solution . 4 NABASE.heated solution (120°-2 h. in presence of02) Fig. 7 87

2.3.1. Discussion

The extinction wavelength curve of NA remains practically unchanged after heating the solution at 120° for 2 hours, except that the extinction values between the wavelengths of 280-320 m/* are slightly higher than that of the freshly prepared solution of NA, as is clear from fig. 7.

So we conclude that the method can not be used to estimate the deteriorated content of NA in a solution. 3. Colorimetric methods. 3.1. Iodine method. For the iodine oxidation of NA the method of Euler and Hamberg (126), (159) has been applied. They presumed that the development of colour was due to the formation of adrenochrome from adrenaline and noradrenochrome from NA. However, in Ehrlens (160) opinion, adrenaline on oxidation with iodine gives a mixture of adrenochrome and iodoadrenochrome. The formation of iodoadrenochrome is maximum at lower pH values by the method of Euler and Hamberg. It is assumed that NA follows the same course.

3.11. Method of oxidation

To 1 ml of the NA solution in a 20 ml volumetric flask 10 ml of the citrate buffer was added followed by 1 ml of iodine solution. Exactly after 3 minutes the excess of iodine ws neutralized by the addition of 1 ml of sodium thiosulphate solution. The volume was made to 20 ml by the addition of distilled water. Within a period of 5 minutes the extinction readings were taken between the wavelengths of 230-600 m, using a concentration of 2.5 mg % of NA base, in 1 cm cuvettes against a blank which was prepared in the same way but without NA in the Zeiss-spectrophotometer described on page 41.

3.1.2. Reagents 6 3.1.2.1. Citrate buffer of pH which was prepared as follows : Na2HPCv 2H,0 0.2 M (35.6 g/litre) 63.1 ml Citric acid 0.1 M (21.01 g/litre) 36.9 ml 3.1.2.2. 0.1 N iodine solution 3.1.2.3. 0.1 N sodium thiosulphate solution. 3.1.2.4. Solution of NA base. A 0.5 mg per ml solution in hydro¬ chloric acid 3 of pH .

3.1.3. Results

The extinction readings between the wavelengths of 230-600 m/* are given in table 34. The coloured product gave two maxima ; one in the in ultra violet range i. e. at 295-296 me and other the visible range

i . e. at 520-525 m/*. The extinction values at the two maxima were :

295.296 =1-12 m, ^ mg% l cm 520-525 m/x = 0.37 88

3.2. Persulphate method

The method for the persulphate oxidation is that of Barker et al. (161), which has been used by Stock and Hinson (162) for the oxidation of adrenaline and NA.

3.2.1. Method of oxidation

10 ml of the NA solution were mixed with 10 ml of the buffer solution in a 20 ml volumetric flask, so that the final concentration of NA base in the solution was 2.5 mg %. The flasks were kept at room temperature for 35 minutes and the extinction readings were taken between the wavelengths of 230-600 mfi in 1 cm cuvettes against a blank which was prepared in the same way but without NA in the Zeiss-spectrophoto¬ meter described on page 41.

3.2.2. Reagents 3.2.2.1. Buffer solution. A buffer solution of pH 5.5 containing the following quantities of the reagent was prepared. Na^HP04.12H20 0.329%

NaHiP04.2 H20 0.937%

NaCl 1.0%

Potassium persulphate 0.2%

3.2.2.2. A 5 mg % solution of NA base in the minimum amount of hydrochloric acid of pH 3.

35.2. Results

The extinction readings between the wavelengths of 230-600 m/* are given in table 35. The coloured product gave 2 maxima. One at at 490-495 The extinction values at the 288-290 m/j and another m/j. 2 maxima were :

' 288-290 mM ^JO1^ = °-98 l cm 490-495 mju = 0.34

3.3. Determination of deteriorated NA in solution by Iodine or Persulphate method

3 was heated For this investigation a 0.1% solution of NA base ofpH solution turned at 120° for 2 hours in presence of oxygen. After heating the filtered and light brown with a black precipitate. The precipitate was the solution oxidized with iodine and with persulphate respectively as described before. Then the extinction was measured at different maxima.

3.3.1. Results The results of the extinction measurements are given in table 34. -89

Table 34

Extinction values of NA solutions after iodine and persulpliate oxidation.

Extinction values at a cone, of 2.5 mg% of NA base

Wave length Iodine method Persulphate method in m/* NA solution NA solution

Frseh Deteriotrated % loss Fresh Deteriorated % loss

295-296 1.125 1.080 4.0 %

520-525 0.3675 0.350 4.8 %

288-290 0.9825 0.915 6.8 %

0.3185 5.6 490495 0.3375 %

The above readings are the mean of 3 sets of observations.

Table 35

Extinction reading) of NA after Jodine and after Persulphate oxidation.

Extinction values for a cone, of 2.5 mg% of NA

Wave length in m/i Iodine method Persulphate method

230 0.476 1.100

240 0.560 0.740

250 0.650 0.575

260 0.690 0.550

270 0.750 0.695

280 0.950 0.900

284 0.96

285 1.100 286 0.97

288 0.9825

290 1..100 0.9825

'

- 295 1.125 . 0.960

296 1.125

0.900 300 1.100 ...

' 310 • 0.933 0.655

0.3825 , 320 . 0.710 90

Extinction values for a conc.of 2-5 mg % ofNA

Wave length in m/»

Iodine method Persulphate method

330 0.430 0.180

340 0.290 0,092

0.062 360 0.170

0.084 380 0.147

400 0.120 0.130

420 0.123 0.195

440 0.173 0-260

49Q 0.240 Q.313

• 0.333 460 0,302 >

«90 0.3375

0.3375 495

"&0 0.343 0.326

520 0.3675 0295

625 0.3675

530 0.360

540 0.3425 0.220

560 0.300 0.145

580 0.225 0.086

600 0.160 0.050

3.4. Discussion of the results of iodine and persulphate methods

3.4.1. Iodine method

The absorption intensity of the curve after iodine oxidation is greatly increased and the maximum is shifted from 279 m/t to 295 m,ti in the ultra-violet region. Further, another maximum is obtained between that 520-525 m/i in the visible spectrum. By looking into fig. 8 one finds 91 the general characteristic of the absorption curve in the V. V. region re¬ mains practically unchanged. The differences are only quantitative.

ncrrt.

ABSORPTION-CURVES

OF OX i DIZED AND NONOXIDIZEO I-NORADRENALINE

Fig. 8

1. Noradrenaline-Base, nonoxidized Emax. at X- 279 m/t. 2. Noradrenaline-Base, oxidized with iodine 3 min. Emax. at A.=295—96 m^520—25 mjx 3. Noradrenaline—Base oxidized with persulphates 35 min. Emax. at A=288—90 m^ & 490—95 m/* 3.4.2. Persulphate method After persulphate oxidation the maximum in the ultra violet region shifts from 279 to 288-290 ni/i, with a general increase in the absorption 92

from 8." Further, another maximum in intensity, as may be seen fig. is obained between 490-495 the visible region of the spectrum mj*.

3.4.3. The maximum loss shown by the iodine method for a solution in the of of NA which was heated at 120° for 2 hours presence oxygen a loss of 6.8 If is 4.8 % while the persulphate method showed %. those West who found that solutions we compare the results with of (33) of NA in half filled ampules lose 25% of the activity after heat sterili¬ zation at 115° for 30 minutes when tested biologically, it would be clear does not results which show that the iodine or persulphate method give full deterioration of NA.

So neither of the methods can be used to estimate the deteriorated NA content in solution.

Ferro-citrate method 3.5. , Rickter (132) described the details for the estimation of adrenaline and NA by the well known reaction of phenols with Fe+ +ions. We have investigated the possibility of using this method for the estimation of deteriorated NA in solution.

3.5.1. Procedure

1 of Pipette a solution representing about mg NA hydrochloride in a 20 ml volumetric flask. Add 15 ml of sodium bisulphite solution. Then add 0.2 ml of ferro-citrate solution followed by 2 ml of the buffer solution. Make the volume to 20 ml by the addition of sodium bisulphite solution. After 25 minutes, when the development of the colour reached the maxi¬ mum, read the extinction at 350 mju, using 1 cm cuvettes against a blank without NA.

3.5.2. Reagents

3.5.2.1. Ferro-citrate solution. 1.5 g of ferrous sulphate (FeS04. to which 7 H^O) were dissolved in 200 ml of water 1 g of sodium N acid were added bisulphite and 1 ml of hydrochloric .

0.60 g of sodium citrate (Ph. Helv. V) were dissolved in the above solution. This solution was freshly prepared each day.

3.5.2.2. Buffer solution. 42 g of sodium bicarbonate and 50 g of potassium bicarbonate were dissolved in about 180 ml of water. To another 180 ml of water 37*5 g of amino-acetic acid and 17 ml ofammonia solution (30% ) were added. The two solutions were mixed and the to ml. volume was made up 500 '(pH 7.5 approximately) . 3.5.2.3. Sodium bisulphite solution. A 0.2% solution of NaHSOa in water.

3.5.3. Standard curve

NA hydrochloride solution representing 400, 600, 800, 1200 and 1600 Ug of NA hydrochloride was taken in a 20 ml volumetric flask and the colour was developed as described before and the extinction was measured at 530 m^ in 1 cm cuvettes. The readings are given in table 36. '93

Tabic 36

Relation between cone, of NA hydrochloride and extinction at 530 m/*

Final cone, of Extinction at 530 m/t using NA 4iydrochloride 1 cm cuvettes

20 M3/nil 01825

30 P£/ml 0.2725

40 /ig/ml 0 3650

60 Ms/ml 0.5425

80 /ig/ml 0.7275

(The above values are the mean of 3 different sets of readings). The relationship between the concentration of NA hydrochloride and extinction values is shown in fig. 9.

*

GS

0,0- - v

7" O

y a*- P X m

'STANDAiiD CURVE F0F! NORADRENALINE HCL AT530mp

Fig 9.

3.5.4. Application of the ferro-citrate method for the estimation of deteriorated NA in solution

For this investigation a 2% w/v solution of NA hydrochloride of different pH values was prepared by the addition of either sodium bicarbonate or IN hydrochloric acid. To half of the solutions of each pH value, 0.1% of NatS208 was added. The solutions were filled in 5 ml ampules to half of their capacity and sealed in the presence of air. The ampule's were then heated at 120° for 10 hours. 94

All the solutions turned brown with a black precipitate. The precipitate was filtered and the NA hydrochloride content was determined by ferro-citrate method as described on page 92.

3.5.5. Results The results are given in table 37. They are the mean of 3 different sets of observations.

Table 37

The effect of heating at 120° for 10 hours on the NA hydrochloride content, of solutions of NA hydrochloride of different pH values, with and without sodium metabisulphite

Initial pH of the solution %loss Initial pH of the solution % loss without NajSaOj with 0.1% NaaSa05

3.1 2.8 3.4 0.5

4.3 6.5 4.2 2.8

5.5 7.1 5.1 3.3

The maximum loss shown by this method is 7 % for a 2 % w/v solution of NA hydrochloride of pH 5.5, which seems to be much less than the values of West (33) who obtained a loss of 30% after 6 hours heating at 115°, the estimations being done biologically. The possible expla¬ nation for the above high values of intact NA may be that the oxidized product itself reacts with the ferro-citrate reagent and gives the colour.

So we conclude that this method is not good to estimate the oxidized NA in solution.

4. Summary

4.1. U.V. Spectrophotometric method

of the The extinction values of NA are independent anion (—) por¬ tion of the molecule. The extinction wavelength curve of a fresh solution and that of a heated solution (120°-2 hours, in presence of oxygen) remains unchanged.

4.2. Iodine method

After iodine oxidation NA gives maxima at 295-296 m/t and at 520- ratio of the extinction values at the 525 m/». The two maxima is 3:1.

The maximum loss shown by this method for a solution of NA which 120° for 2 hours in of is was heated at presence oxygen only 4.8 %.

4.3. Persulphate method

oxidation NA After persulphate gives maxima at 288-290 m^ and at 490-495 m/*. The ratio of the extinction values at the two maxima is 2.9:1. 95

The maximum loss shown by this method for a solution of NA which was heated at 120° for 2 hours in presence of oxygen is 6.8%.

4.4. Ferro-citrate method

NA gives maximum at 530 m/» and the colour obeys the Lambert- Beer's law within the range of concentration of NA studied. The maximum loss shown by this method for a solution of NA which was hea¬ ted at 120° for 10 hours is 7%. CHAPTER XII

RACEMIZATION STUDIES OF THE DILUTE SOLUTIONS OF NORADRENALINE

In the following study we have determined the effect of storage time at room temperature (18°-22°) on racemization of 0.2% solutions of NA hydrochloride under the following variables : (a) after 30, 60, 160 and 290 days. (b) at pH values of 2, 3, 4 and 5. (c) with and without 0.1% of sodium metabisulphite. (d) in presence of nitrogen and air. 1. Method for concentrating the solution and for determining the optical rotation The following method described by Hellberg (113) for concentrating the dilute solutions of adrenaline and NA has been used. 1.1. Column

About 0.9 g of the resion formed a column 3 X140 mm (0.99 Cm3) in chromatographic tube,

1.2. Absorption 20 ml of the dilute solution corresponding to 40 mg of NA hydro¬ chloride was passed through the column with a velocity of 1 ml per minute. Then the column was washed with 20 ml of distilledjvater.

1.3. Elution of noradrenaline

For elution, 2N hydrochloric acid was used, which was allowed to pass through the column with a velocity of 0.5 ml per minute. The first 0.8 ml was discarded because this portion of the elute is very poor in NA content and the follwoing 1.2 to 1*5 ml was collected.

1.4. Optical rotation of the elate The optical rotation of the elute was measured immediately after elution. At least 5 different readings were taken for each observation. The results of the optical rotation measurements given in the following tables are the mean values'of these readings.

1.5. Noradrenaline content of the elute

0.05 ml of the elute was diluted to 20 ml with distilled water, and the extinction was read at 279 rmi, using 1 cm cuvettes in the spectropho¬ tometer. Three sets of observations were taken for each solution. The amount of NA content was then calculated by means of a standard, prepared from the same batch samples of NA hydrochloride. The specific rotation was then calculated from the readings of the optical rotation and the concentration of the elute.

1.6. Material 1.6.1. 1-noradrenaline hydrochloride confirming with the tests as described on page 81 was used. 97

1.6.2. Double distilled water was prepared and tested as-described on page 39.

1.6.3. 25 ml ampules which confirmed to the powdered glass test XV were used. The results of the of U.S.P. (15) test are given on page 40. 1.6.4. Amberlite IRG 50, a cation exchange resin with carboxyl in groups as active centres, its neutralized, ammonium saturated form has a certain exchange capacity tpwards alkaloidal cations ; Winter and Knnin (163).- The ion exchanger amberlite IRG 50, IV mesh Ph. Helv. V (164) was transformed form its proton form to ammonium form by treating with an excess of 2 N ammonia. The resin was then washed with / distilled water and air dried.

1.7. Apparatus

1.7.1. A chromatographic column with an inner diameter of 3mm and 140 mm length, with a capillary fitted with a stop cock was used for concentrating the solution. 1.7.2. The optical rotation was measured with F. Schmid-Haensch- Polarimeter, using 10 and 20 cm long polarimeter tubes with an inner mm A diameter of 2 mm and 1.5 respectively. Zeiss sodium vapour lamp was employed as light source. 1.7.3. All the measurements were made with Methrom Poten¬ . pH tiometer with a glass electrode.

1.7.4. For spectrophotometric measurements the apparatus des¬ cribed on page 41 was used.

1.8. Discussion of the method

1.8.1 The interfering anions as sulphate or tartrate are removed during the exchange process. » 1.8.2. The maximum error of the method was 3 to 5 %. 1.8.3. There is no risk of racemization during the elutiori step as would be clear from the results given in table 38.

. In 1.8.4.. our experiments we obtained with this method a maxi¬ mum concentration of 8 times of the original which was sufficient for our work.

2. Preparation of the solution NA hydrochloride was accurately weighed and dissolved in freshly distilled water 0.8 % of prepared containing sodium chloride, so as t6 of in give a concentration 2 1000. The solution was then divided into and the four equal parts pH was adjusted to approximately 2,3,4 and 5 of either by the addition sodium bicarbonate or hydrochloric acid. Then to half of each of the above solutions of sodium 0.1% metabisulphite was added. v

2.1. Filling

• ml of the above solutions 25 portions were filled in 25 ml glass am¬ pules. Ampules containing the solutions with sodium metabisulphite 98 were sealed after replacing the air with nitrogen, while'the other ampules were sealed in presence of air.

2.2. Sterilization .,,-..

were sterilized at 120° for 30 minutes. - The above ampules ,

' 2.3. Storage , , „ . The sterilized ampules were stored at room temperature (I8°-22°), after protected from light, for a period of 30, 60, 160 and 290 days, to the method des¬ which they were analysed for racemization according cribed in previous page*. .

3. Results

Table 38

Effect of elation on racemization of NA hydrochloride solutions,

" ' pH - Specific rotation of

2% w/v solution Same solution 10 times diluted and the rotation measured after cone, by elution

2 0 40* 40°

30 40» 40 5'

4-2 40° 40°

51 40° 39-5°

The results of the optical rotation measurements of the sterilized solutions of NA hydrochloride after keeping at room temperature for 4, 30, 60, 160 and 290 days are given in the follwoing tables.

Table 39

Specific rotation- of the sterilized solutions of noradrenaline hydro¬ chloride after keeping for 4 days at room temp. (18°—22°).

Solutions without sodium mctabisulphite in air filled ampules

Initial pH of the Optical Extinction at NA hydrochlo¬ Specific solution rotation 279 mm in 1 ride content rotation 20 cm tube cm cuvettes

20 — 0-265° 0 35 1-125 % — 11-0"

30 — 0-915' 0-42 1-31 % — 34-9''

4-3 — 1-0C° 0-44 1-375 % — 39-3°

5-4 — l-06» 0-415 1-30 % — 40-8° Solutions with 0;1 % of sodium metabisulphite inNa filled ampules

Initial pH of Optical rotation Extinction at NA hydrochloride Specific the solution 20 cm tube 279 mp. in 1 eta content rotation cuvettes

2.1 — 0-41° 0-335 1-05 % — 19-5°

3.3 — 067° 0-265 0-83 % — 40-4"

4.1 — 0-97" 0-385 1-20 % — 40-4°

' — 5.0' — 1-14° 0-447 1-40 % 41-0"

Table 40 Specific rotation of the sterilized solutions of noradrenaline hydroch¬ loride after keeping for 30 days at room temp. {l8°-22°).

Solutions without sodium metabisulphite in air filled ampules

Initial pH of Optica] .Extinction at NA hydrochloride Specific the solution rotation 279 mp in 1 cm content rotation 20 cm tube cuvettes

2-1 — 0-225° 0-445 1-39 % — 81°

- 30 — 0-705° 0-37 1-17 % — 31-4°

4-3 — 1-06° 0-51 1-59 % — 33-3°

5-4 — 0-965° 0-428 1-34 % — 360°

Solutions with 0-1 % of sodium metabisulphite in N2 filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific the solution rotation 279 tnfi in 1 cm content rotation 20 cm tube cuvettes

JU —0-50° 0-4625 1-44% — 17-3°

3.3 —100° 0-4425 1-38 % — 36-2°

4.1 — 0-80° 0-345 1-08 % — 370°

5.0 ~ 0-93° 0-3825 1-19 % • — 390°

Table 41 Specific rotation of the sterilized solutions of noradrenaline hydroch¬ loride after keeping for 60 days at room temperature (18°-22t>). Solutions without sodium metebisulphite in air filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific the solution rotation 279 m(t in 1 cm content rotation 20 cm tube cuvettes

20 — 016° 0-430 1-34-% — 60°

30 — 0.43° 0-3175 1-0 % — 21-5° 4-3 N.D, N.D. N.D. N.D.

5-4 — 0-77° 0-360 1-12% — 34-2°

N.D. =Not determined. 100

Solutions with 0~1 % of sodium metabisulphite in N2 filled ampules^-

Initial pH of - Optical Extinction at NA hydrochloride Specific the solution • rotation 279 m/t in 1 cm content rotation 20 cm tube cuvettes

2-1 — 0-37" 0-3575 1-12 % — 16-5°

33;. — 0-76°' 0-34 1-06 % — 35-8°

< — 1-16 — 4-1 , 0-87° 0-3725 % 37-0*

50 — 0-89" 0-3725 M6 % — 33-2°

Table 42 Specific rotation of the sterilized solutions of noradrenaline hydro¬ chloride after keeping for 160 days at room temperature. (18°-22°) Solutions without sodium metabisulphite in air filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific ' the solution rotation 279 m/* in 1 cm content rotation 20 cm tube cuvettes

20 — 0055° 0-52 1-62 % — 3-4°

- 3-0 — 0-43° • 0-485 1-51% — 28-5°

4-3 — 0-39° 0-335 1-30 — % 300O .

5-4 — 0-42° 0-45 1-40 % — 300°

Solutions with 0-1% of sodium metabisulphite in N2 filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific the solution rotation 279 m/x in 1 cm content rotation 10 cm tube cuvettes

21 — 0-16" 0-4325 1-35 %' — 11'8°

3-3 - — 0-50", 0-52 1-62 %c — 30'8°

41 — 0-59° 0-515 1-61 % — 36-6°

— — 0-57° - 0-51 1-59 . 35-8° , 5-0 %

• Table - '> , 43 Specific rotation of the sterilized solutions of noradrenaline hydro¬ chloride after keeping for 290 days at room temp. (18°-22°). Solutions without sodium metabisulphite in air filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific the solution "rotation 279 m/i in 1 cm content rotation 10 cm tube cuvettes

20' 0-00 — 00 000

• 3-0 — 0-41° 0-413 1-50 % — 27-3°I

4-3 — 0-46" 0-42 1-53 % — 30'0°

— 0-345 1-26 — 5-6 0-37° % . , .., 300° 101

Solutions with 01% of sodium metabisulphite in Na filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specific rotation in 1 cm content the solution rotation .. 279 in/* 10 cm tube -cuvettes

1-45 — 80" 21 — 0-23° 0-40 % .f

1-38 — 280° 3-3 — 0-3?" 0-38 %

— 340° 41 — 0-47° 0-385 •1-38 %

_ 1-53 , 28-7° 50 — 0-44" 0-42 %

Table 44

Specific rotation of the sterilized solutions of noradrenaline hydro¬ chloride without sodium metabisulphite in air filled ampules.

Initial pH, of Specific rotation " the solution '

Time in days after sterilization at which the optical rotation was measured

4 30 60 160 290

' ' ; I t

— — — 2.0 11-8° 81° 6-C — 3-4° - 0.0 -

3.0 — 349° — 31-4° — 21-5° — 28-5° — 27-3°

4.3 — 39-3° — 33-3"» - — 30-0" — 300°

— — — 5.4 40-8° 36-0"" 34£° — 300° — 300°

Specific rotation of the sterilized solution of noradrenaline hydro- ide containing 0.1 % of sodium metabisulphite in Nj filled ampules.

Initial pH of Specific rotation the solution

Time in at which days after sterilization the optical rotation was measured

4 30 60 < 160 2C0

' " —19-5° — - 21 17-3° —16-5° —11-8° — 80°

'

— — — 3-3 40-4°' 36-2° 35-8° — 30-8° —280°

- — — — 4-1 40-4° 37-0° 37-3° —36-6° — 340"

_ — — . 6>0 41-0- 390° 3-82° — 358 — 28-7" 102

Tabic 45

Table showing the relationship between storage time and the percentage of noradrenaline hydrochloride racemized at different pH values, for solutions of NA hydrochloride without j^faQS:i0B in air filled ampules.

Initial pH of Time in days

the solution 4 30 60 160 290

'- Percentage of noradrenaline hydrochloride racemized

2-1 70-5 80-0 850 .91-5 100

30 12-5 21-5 460 290 320

4-3 20 170 250 250

-5-4 00 10-0 14-5 25-0 . 250 • - ^

Table 46

Table showing the relationship between storage time and the Percentage ofnoradrenaline hydrochloride racemized at different pH values, for solutions of NA .hydrochloride with 0.1% of Na^O,, in N, filled ampules.

Initial pH of Time in days

the solution 4 30 60 160 290

Percentage of noradrenaline hydrochloride racemized

2-1 510 570 690 70-5 800

3-3 00 9-5 11-0 230 300

41 00 70 70 86 150

50 00 2.5 4-5 10-5 280

4. Summary of the results of racemization studies

4.1. Effect of pH on racemizationof NA solutions

4.1.1. The solution of pH 2 got more or less completely racemized after about 10 months, irrespective of the fact whether they contained sodium metabisulphite or not. While the solutions of pH 3 showed about 30% racemization after this time.

- 4.1.2. The minimum racemization was with'the-solutions of pH 4.1 containing 0.1% of sodium metabisulphite in ampules filled with nit¬ of which showed a racemization of rogen instead air, 15% after 290 days, while the solutions without sodium metabisulphite showed a stabilization of the racemization at 25% after about 5 months. 103

" ; The effect of storage time x>n racemization is graphically shown in fig. 10.

-«—^0.2%solution of NA Hydrochloride with 0.l7oNa2S205 Fn N2 filled ampules.

without — j0.2°/o solution of NA Hydrochloride Na2S20gin air filled ampules. Fig. 10

4.1.3. For the solutions of pH 5 containing 0.1% of sodium meta- bisulphite the racemization is only 5% after 60 days, but the racemization curve goes still constantly upwards after 290 days when the racemization is 28%. While the solutions of pH 4 and 5 containing no sodium meta- bisulphite show nearly the same loss of optical activity, namely 25% after 160 and 290 days.

' 4.2. Effect of sodium metabisulphite on racemization Sodium metabisulphite helps "at least to some extent in' checking the racemization, which is most prominent at pH value of 4. The effect 104

has been graphically shown in fig. 11 for solutions of different pH values

after a period of 290 days. .

^SJ sterilized solution of NA-hydrochloride containing 0.1 e)P of N^S2Os> in Ngfilled ampules.

sterilized solutions of NA hydrochloride, i n air filled ampules.

Fig. 11

The racemization curves in fig. 10 for the solutions without metabi¬ sulphite are flat after about 60 days, showing that the rate of racemi¬ zation is very slow afterwards, while for the solutions with metabisulphite' the curves show a slight slope indicating that the racemization equilibrium is not reached even after 290 days. . . yet ., 105

5. Discussion and conclusions

5.1. time at room Storage temperature (18°-22°) has very little influence on the racemization. During the first 160 days the influence of time is after pronounced, which there is very little change.

5.2. Racemization is highly dependent on pH of the solution. The best value was in the pH neighbourhood of 4 .

5.3. Sodium metabisulphite has some influence on checking the racemization.

On the basis of these the findings following instructions are proposed for the injection of NA, with respect to optical stability.

5.4. Injection of noradrenaline

Noradrenaline 0.1%

Sodium chloride 0,9% Sodium metabisulphite 0.1% Aqua bidistillata to make 100 ml.

Dissolve sodium chloride and sodium metabisulphite in freshly prepared double distilled water. Add noradrenaline, filter and make the required volume. The above operations should be carried out in an atmosphere of nitrogen.

Fill and seal the ampules in an atmosphere of nitrogen. Sterilization. Sterilize by autoclaving at 120° for 15 minutes. pH between 3.7 to 4.5

It is to be noted that the pH values recommended by different phar¬ macopoeias vary to a great extent for the injections of noradrenaline acid tartrate, as may be seen from table 47.

Table 47

Recommeded pH values for the injections of NA acid tartrate

Pharmacopoeia Recommended pH value

Ph. Int. I (128) 2-5 — 4-5

Ph. Dan. K Add. 1954 (164) 2-5

U. S. P. XV (144) 3 —4-5

On the basis of our findings we recommend that in order to maintain the maximum optical activity the pH value of the dilute solutions ofNA must be between 3.7 to 4.5j which also agrees very closely with the findings of West (33) who recommended a pH value of 3,5 to 3.9 for the injecions of NA, with respect to biological activity. CHAPTER XII

PREDICTION OF THE STABILITY OF THE SOLUTIONS OF NORADRENALINE WITH RESPECT TO RACEMIZATION.

1. Introduction

stream of In recent years one finds a continuous new pharmaceutical preparations being introduced to the market. For the pharmacist as well as for the doctor it is essential to know for how long the preparation will keep its labelled amount of the drug on storage at room temperature.

One way of knowing the stability of the new preparation is by storing it at the storage temperature, say for a period of 1 to 2 years, and then finding out the activity by suitable chemical or biological methods from time to time, and in case the preparation is not stable, then carrying out the lengthy time consuming process once again. But in these days of competition pharmaceutical firms can ill-afford to wait for such a long time before they can put their preparation on the market. However, if the reaction, which inactivates the product, is of the first order and the temperature coefficient known, it should be possible to predict the stability of the preparation at the storage temperature by carrying out the experiments at elevated temperatures within a compera- tively short period. In the following pages we shall discuss the basis of the above method (40), (165).

2. O'der of reaction

The rate of a chemical reaction is proportional to the concentrations of the reacting substances according to the law of mass action. As the reaction proceeds the concentration of the reacting substances goes on decreasing and so does the reaction rate.

The reactions can be classified according to their molecularity, i; ei the number of molecules or atoms taking part in the chemical reaction. If only one molecule is involved the reaction is said to be unimolecular, if two molecules are involved bimolecular, and so on. The example of a unimolecular reaction is the decomposition of nitrogen pentoxide as shown below :

N308 > N.,04 + 1/2 O,.

For a bimolecular reaction hydrolysis of an ester may be cited as an example :

' • CH3.COO.CaHs + Hp -» CH3.COOH + G2H8OH

From a quantitative standpoint reactions may be classified according to the order of the reaction 1. e., the number of atoms or molecules whose concentrations (or for gases pressure) determine the rate of the reaction. The molecularity and the order of the reaction are often identical, but not always ; for example, though the hydrolysis of an ester is a bimolecular., first order reaction, it is a reaction because the concentration of water may. be assumed to be constant since it is present' in large excess. The rate' of the reaction is therefore solely determined by the concentration of the ester. ' • "107

2.1. First ordet reaction

In a first order reaction the rate is directly proportional to the concentration of the reacting substances. If c is the concentration of the reacting substance, t the time and k the velocity constant the above con¬ dition may be expressed as : dc -— =.kc (1) dt

According to the equation (1), the rate of decrease of the concehtra- dc

z. e. at instant the tion with time, , any represented by time t, is pro- dt

tional to the concentration c at that instant.

The equation (1) can be put in another form. If a is the initial concentration of the reacting substance and x the decrease during the time t, then (a-x) would be the amount remaining which is equal to concen¬ tration c at any instant. Substituting this in equation (1), we get. d (a-x) = k. (a-x) (2) dt

and since the initial concentration a is constant d (a-x) dx

dt dt

and hence equation (2) may be written as dx = k. (a-x) (3) dt

equation (3) can be rearranged so as to carry out the integration dx

= k . dt (4) a-x

now when t is zero, the value of x is also zero, while after time t the change is x. By integrating equation (4) we get

1 a 2.303 a

= k = In . log (5) t a-x t a-x

This equation (5) is known as the kinetic equation for a reaction of the first order.- By studying this equation, the following conclusions can be drawn.

a (a) The quantity is the ratio of the concentrations and a-x

that the same units are so it is independent of the units used, provided used for both a and x. 108 •

' (b) The time to complete any definite fraction tif the reaction is independent of the initial concentration.

2.1.1. Velocity constant

For a first order reaction the value of k should be a constant one which can be easily determined by carrying out the stability experiments and then at a definite temperature for different values oft, by calculating the value of k by introducing the experimental values of a, x and t in equ¬ ation (5), or a graphical method may be used. By rearrangement equa¬ tion (5) becomes 2.303 2.303

= t .log a . log {a-x) (6) k k

Now if t is plotted as ordinate and log (a-x) as abscissa, the plot should be a straight line if the reaction is of first order.

2.1.2. Examples of first order reaction

Hydrolysis of an ester, inversion of sucrose, decomposition of nitrogen pentoxide in liquid or gaseous state are a few examples of the first order reaction. 3. Temperature coefficient

The velocity of a chemical reaction is invariably increased to a mar¬ ked extent by raising the temperature. In general, by raising the tem¬ perature by 10°, the reaction velocity is doubled, though it is not always the case.

The relation between time and temperature was first given by Arrhenius, as the relation between reaction velocity and temperature with the equation Ea 1

k = log . + q where (7)

T = absolute temperature

R = gas constant

Ea = critical increment

q = a constant

Now the temperature coefficient is the coefficient by which to multi¬ ply the velocity constant in order to obtain the velocity constant at any temperature. The above statement will be clear from the following example :

We shall consider the hydrolysis of procaine where we know the relation between time and destruction at 20° and wish to know the stability at 10°. For the hydrolysis at the two temperatures we have :

2.303 a

= kI0° . log (8) ° t,tio° a-x 109

2.303 a

= — kaoo . log (9) tj0 a-x

If we now specify the time t to olrtain the same amount of hydro-

a lysis, using the same initial -concentration log would have the same a-x value in the two equations. Now by division we have

» <10) ° ° Tc t

If the temperature coefficient for this process is 0.2 we get

Klo Vjo fll) 0.2 t k10°. (20-10). 10 Experimentally the temperature coefficient is determined by carrying out the stability experiments at definite temperatures at intervals of 10°, and if the time t is plotted as abscissa and the destruction as ordinate' of one will obtain at each specific value the ordinate the relation between the two times tx and t2, thus giving the temperature coefficient of the be clear from 15. process for 10° as would fig. '4. Experimental

In the following experiments we have made the racemization studies of 2 % w/v solutions of NA hydrochloride of (a)

4.1.2. The polarimeter which has l>een described on page 97 inner using a lOcmlongpolarimeteriubewithan diameter of2 iron was used.

4.1.3. pH measurements were made with Methrom-potentiometer The of the with a glass electrode. accuracy apparatus was 0.05 pH units.

4.1.4. Heating at 60" was done in an electric oven, -while for heating at 100° and 120°, the Egro autoclave was used. The ampules were when a placed in the autoclave only temperature of about 100° was attained. After the duration of heating the ampules were taken out of the autoclave as soon as possible and stored in a refrigerator at a temperature of 4° till the optical rotation was determined. The heating ti me was noted from the moment the autoclave registered a temperature of 100° or 120° 110

as the volume of the solution in each ampule was only 2.5 ml and not heated at one time. more than 20 ampules were

4.2. Determination of the optical rotation

All the measurements were made between the temperatures of 20° and 22°, except for the solutions of pH 5.5/100° and of pH 5.5/120° whose rotation was measured at 23°-24°. The deviation in the optical rotation for 15° is about 0.01° therefore the error of the measurements between the temperatures of 20° and 24° is negligible.

Solutions which were turbid due to the formation of precipitate filter were filtered through a small paper before the determination of the optical rotation. Further those solutions which were dark coloured were diluted so that readings could be taken.

The following results are the mean of 5 to 10 readings fQr each solu¬ tion, the mximum deviation .from the .mean. being± 2.5 %.

4.3. Working procedure 4.3.1. Preparation of the solutions NA hydrochloride was accurately weighed and dissolved in freshly prepared double distilled water in a volumetric flask. The solution 4 and the was divided into equal parts pH value of different portions of the solution was adjusted to approximately 2, 3, 4 and 5 by the addition of either sodium bicarbonate or 1 N hydrochloric acid. The solutions then of different pH values were divided into two equal parts, and to solution of each value one portion of the pH 0.1% of sodium metabis- added. The final concentration of the ulphite was different solutions was adjusted to 2 g of NA hydrochloride per 100 ml of the solution. The the NA content was checked by measuring extinction at 279 mju.

and 4.3.2. Filling sealing ,

2.5 ml quantities of the above solutions were filled in freshly cleaned and dried 5 ml ampules. The ampules containing the solutions without metabisulphite were sealed in the usual way in the presence of air, while those ampules which contained the solution with metabisulphite were sealed after replacing the air by nitrogen as described on pages 62 and 63 The ampules were then stored in a refrigerator at a temperature of 4° for a maximum period of fifteen weeks.

Throughout the operation, care was taken that the solutions did not get in contact with any metal.

4.3.3. Heating of the ampules • The above ampules were then heated for 1, 3, 6 and 10 hours at 60°, 100° and 120°. 4.4. Results

4.4.1. of . pH 1-noradrenaline hydrochloride solutions f' 2%w/v( solutions of NA hydrochloride with and without sodium metabisulphite were prepared, the pH values of which were measured immediately after preparation. The results are shown in table 48. Ill

Table 48 pH values of I-noradrenalinc hydrochloride solutions

pH Values

NA hydrochloride solutions without NA hydrochloride solutions Na2SaOc, in air filled ampules •with 0.1% of Na2S3Os, in nitrogen filled ampules

=. 2-2 2-4

" 3-1 34 4-3 4-2 5-5 5-1

4.4.2. Results of the optical rotation determinations

* The following observations have beem made between '20°, and 24° and are without temperature correction.

In tables 49 and 50 the stand for : ^ following signs

Solution = A freshly prepared solution, whose rotation was measured immediately after preparation. * solution .for the determination two times diluted. + solution for the determination three times diluted. x solution for the determination four times diluted. Table 49 Specific rotation of l-noradrcnalinc hydrochloride solutions without sodium metabisulphitc in air filled ampules

Specific rotation of 2% w/v NA hydrochloride solutions without Na3S20s, heated to a temp, of

treating time in hours pH 60» IOC 120°

Solution A 39-5* — 40 0° — 400°

1 390° — 270° • — JD-B*

3 2.2 380° — 17-2° 00°

6 370° — 50° 0 0°

10 34-5° — 1-0* 0 0°

Solution A 395° — 40 0° — 400°

1 390° — 38-5° — 26-5°

3 3.1 380# — 32-5° — 16-5' +

6 370° — 24-0' + — 40° X 10 35 0° — 12-0'X 0 0°X

Solution A 39-5° — 400° — 40 0'

1 390° — 37-5° — 27-5"

3 4.3 38 0' — 32-5° — 150°*

6 370' — 310°* — 80° X 10 31-5" — 270** — 40° X

Solution A 39-5" — 40 0" — 40 0°

1 390" — 360°* — 26-0«*

• 3 5.5 38-0° — 330°* — 140° X

6 37-0° — 26 0°* — 00° X

' 10 350° — 24-0° + 00° X 112

Table SO

Specific rotation of I-noradrenaline hydrochloride solutions containing 0.1 % of sodium tnetabisuIpMte in N2 filled ampules

Specific rotation of 2% w/r NA hydro¬ chloride solutions with 0.1 % Na3S20e, heated to a temp, of

Heating time in hours pH 60° 100" 120°

Solution A. 39S* — 40-0* — 40-0'

1 39-5" — 320tt — 260'

a 2.4 380° — 255" — 42"

6 380" — 110" 00

10 36-5* — 30" 00

Solution A 39-5° — 400° — 40-0°

I 39-5* — 40-0° — 24-0"

3 3.4 390" — 33-5* — 10.0"

6 380" — 25-5° — 2-7"

• 10 370" — 13-5° — 1-Q"

' Solution A 39-5" — 400" — 400"

1 39-5° — 37-5" — 32-5°

3 4.2 390° — 330" — 18-5"

* 6 380" — 30-5° — 100"

10 37-5* — 26-5° — 60° +

Solution A 39-5° — 400° — 40 0°

1 39-5° — 36-5" — 28-5"

* 3 5.1 390° — 32-2" — 160?

C 380" — 260° — 4-0° X

10 37.0° — 25.0° o.o x 113

Table 51

Racemization of 1-noradrenaline hydrochloride solutions

% of 1-NA hydrochloride % of 1-NA hydrochloride racemized without pH racemized with 0.1 % of Na2S2Os, in air filled ampules Na2S2Os, in N2 filled ampules

Heating time pH in hours 60° 100° 120° 60° 100° 120°

1 1-27 32-5 73-7 • Nil - 200 350

3 22 3-8 570 1000 2-4 1-27 38-0 ' ' 900

6 6-35 87-5 1000 3-8 72-5 1000

10 127 1000 6-3 92-5 97-5 1000 .

1 1-27 375 33-75 Nil Nil 40 0

3 31 3-8 1875 53-75. 34 .1-27 16-25 750

6 6-35 400 900 3-8 36-25 93-25

10 11-4 700 1000 6-3 66-25 97-5

•1 1-27 6-25 31-2 Nil 6-25 190

4.3 3-8 18-5 62-5 4.2 1-27 17-5 53-7

6 6'35 22-5 800 3-8 23-7 750

10 12-7 32-5 900 51 33-75 .850

1 1-27 100 350 Nil 8-8 23-7

3 55 3-8 17-5 650 51 1-27 19-5 600

6 6-35 350 1000 3-8 350 90 0

10 11-4 400 1000 6-35 37-5 1000

4.5. Discussion of the results

4.5.1. Influence of sodium metabisulphite

In the racemization for solutions sodium metabi¬ ( general containing sulphite in nitrogen filled ampules is less when compared with the solutions containing no metabisulphite in air filled ampules.

4.5.1.1. At 60° the racemization for the solutions containing no metabisulphite in air filled ampules was practically double than that of the solutions with metabisulphite in nitrogen filled ampules.

4.5.1.2. At 100° the difference is much smaller and at pH values of 4 and 5 there is no difference. 114

4.5.1.3. At 120° there is some difference only after 1 hour heating time for the solutions of all pH values, while after heating for more than for solutions conta¬ 1 hour there is very little difference in racemization the ining metabisulphite in nitrogen filled ampules and those without metabi¬ sulphite in air filled ampules, as may be seen from the results given in table 51.

4.5.2. Influence of pH The results of table 51 show that the pH plays an important role on the rate of racemization.

4.5.2.1. At 60° there was an equal amount of racemization between pH values of 2 and 5.

4.5.2.2. At 100° the effect of pH is very clear. On comparing the racemization results of pH 2, 3, 4 and 5 after 10 hours heating at 4 time we find that the racemization at pH 3 is 25% less while pH and 5 about 60% less than the racemization at pH 2. i

4.5.2.3. At 120° the pH has the same influence as at 100°, except that the amount of racemization is greater. The minimum racemiza- tion is in the case of solutions of pH 4, 2.

4.5.3. Influence of time and temperature The influence of heating time and temperature for solutions of 1- noradrenaline hydrochloride, with and without sodium metabisulphite, at different pH values is graphically shown in figures 12 and 13.

' 3 6 TIME IN HOURS

RELATION BETWEEN THE PERCENTAGE OF NORADRENALINE HCL RACEMISED AND RELATION BETWEEN THE PERCENTAGE OF TIME AT DIFFERNT TEMPERATURESAND NORADRENALINE HCL RACEMISED AND TIME pH-VALUES AT DIFFERENT TEMPERATURES ANDpH-VAlUES

(2%wfv totutlonof Noradrenalint HCL with (2°/oWv solution of Noradranaltna HCl without 0,1% Sodium mttabtaulfite.1 Sodium matabtfiAfite)

Fig. 12. Fig. 13. The results show that by heating the solutions for longer periods and by increasing the temperature, the rate of racemization goes on increasing. 115

4.5.4. Calculation of the stability of noradrenaline solutions

In order to determine the order of the reaction for the racemization of NA hydrochloride solutions, the velocity constant k has been calculated from the formula :

2.303 a

= k . log where t a-x

a = initial cone, of the unracemized NA = 100 x «=%of racemized NA hydrochloride during t hours.

The k values for different tempratures are given in table 52. Table 52

Velocity constants for the racemization of 1-noradrenaline hydrochloride solutions at different temperatures.

Velocity constant k

2 % w/v solution of NA pH 2% w v solution ofNA hydrochloride without hydrochloride with 0.1% Na 2S2O5, in air filled NaaS205, in nitrogen ampules. filled ampules.

Heating time PH in hours. 60° 100° 120° 60° 100° 120°

1 00129 0-3915 0-3357 000 0-2232 0-4309

3 22 00127 0-2809 24 00043 01488 0-7676

6 00109 0-3456 00064 0-2152

10 00136 0-3689 00072 0-2590

1 00129 0-0283 0-4118 000 000 0-5108

3 31 00127 00691 0-2852 34 00043 00591 0-4621

6 00109 00851 0-3838 00064 00751 0-4487

10 00121 0-1204 00051 01090 0-3689

1 00129 0-0645 0-3740 000 00645 0-2107

3 43 00127 00683 0-3270 42 0-0043 00681 0-2567

6 00109 00425 0-2683 00064 0-0451 0-2311

10 00139 00393 0-2303 00065 00412 0-1897

1 00129 01055 0-4320 000 0-0921 0-3385

3 5-5 00127 00641 0-3499 5-1 00043 00722 0-3054

6 0-0109 00718 00064 00718 0-3838

10 00139 0-0511 00065 00470 116

The figures of the above table show that at 120° and at 100° there is an appreciable difference in the k values for different time intervals at all the pH values, showing that the reaction is not of the first order at 120° and at 100°. While at 60° the k values are more or less constant between the pH values of 2 to 5, proving that the reaction is of the first order at lower temperatures.

The above is also clear by the graphical relationship of the log of is shown the % of unracemized NA hydrochloride and time, which in fig. 14 for solutions of NA hydrochloride containing 0.1% of metabisulpliite in nitrogen filled ampules.

RELATIONBETWEENTHELOeoF THE%0F THE UNRACEMISEO NORADRENALINE HCIANO THE TIME AT DIFFERENT TEMPERATURES AND pH VALUES.

Fig 14

So we conclude that it is not possible to predict the stability of NA solutions with respect to racemization at storage temperature from the racemization studies at 100° or,120°. Further, in order to predict the stability at storage temperature, the stability experiments must be carried out in the neighbourhood of 60°.

It is to be noted that the rate of racemization at 60° is very slow and an error of 0-10° in the polarimeter reading will give a difference of racemization of 1*25% when the optical rotation readings are taken for a 2% w/v solution of NA hydrochloride, using 10 cm long polarimeter tube, i. e. there will be an error of as much as 20 % for a solution which has been heated for 10 hours. So it is necessary that the racemization studies at lower temperatures should be carried out over a much longer period so as to obtain a racemization ofabout 50%, and thus minimize the error. 117

5. Racemization studies at 5(P, 60°, 70°

we found that the best for the solu¬ In our previous experiments pH the reaction tions of NA hydrochloride was about 4, and that velocity of 60° with to racemization. was constant in the neighbourhood respect

So in the following studies we have further carried out the stability experiments at 50°, 60° and 70°, for a much longer period (about 200-400 of NA of sodium h.) for a 2% w'v solution hydrochloride containing 0.1% metabisulphite in nitrogen filled ampules of approximately pH 4.

5.1. Experimental

The method for preparation of the solution and determination of the on 110 optical rotation was exactly the same as described page except for the following differences :

(a) The ampules were heated in a liquid paraffin bath of which the maximum variation of temperature was ± 1°.

(b) All the optical rotation readings were taken between 20° and 22°

5.2. Results

In tables 53, 54 and 55 results of the optical rotation determinations at 50°, 60°, and 70°, are given together with the calculated k values.

Table S3

Specific rotation and the velocity constantkof I-noradrenaline hydrochloride solutions containing 0-1 % of Na2S205 in nitrogen filled ampules after heating at 50°

time rotation % of unracemized constant k Heating pH t Specific Velocity in hours NA hydrochloride

Solution A — 41-0° 100

48 — 37-5° 91-46 000186

96 — 35-0° 85-37 000164

144 — 330° 80-5 • 000150 4.25

192 — 30-5° 74-4 000154

240 — 27-25° 66-46 000170

•

290 — 26-0° 63-4 000157

400 — 23-5° 50-73 000170

Mean k value 0.00164 118

Table 54

Specific rotation and the velocity constant fc of l-noradrenaline hydrochloride solutions 01 containing % of Na2S2Os in nitrogen filled ampules after heating at 60°

time Heating PH Specific rotation % of unracemizcd Velocity constant k in hours NA hydrochloride

Solution A — 41-0° 100

^ 8 — 39-5° 96-34' 000460

16 — 38-5° 93-9 000390

48 — 34-5° 84-15 000360

96 — 27-5° 67-07 000416 4.25

144 — 24-0° 58-54 000370

192 — 190° 46.34 000400

240* — 18-0° 40-4 0-00380

Mean k value ...... 000397

Table 55

Specific rotation and the velocity constant k of l-noradrenaline hydrochloride solution containing 0-1 % of Na2S2Os in nitrogen filled ampules after heating at 70°

Heating time pH Specific rotation % of unracemized Velocity constant k in hours NA hydrochloride

Solution A — 41-0° 10000 000

8 — 38-25° 93-30 000866

16 — 35-7° 87-10 000860

24 — 33-5° 81-70 0 00840

4.25

48 — 27-0° 65-85 000870

72 — 22-25° 54-30 000847

96 — 18-0° 4390 000856

144* — 6-5° 15-9 001270

200* — 4-5° 110 001120

Velocity constant k, mean value .. •• 000856

Solution A = freshly prepared solution, the rotation of which was measured immediately after preparation. * = solution which turned slightly yellowish after heating. 5.3. Discussion of the results 5.3.1. Velocity constant The reaction velocity is practically constant for all the 3 temperatures has been calculated from the formula i. e. 50°, 60° and 70°, which 119

2.303 a

k = . log t a-x for example k value at 60° after 16 hours, when the value of a-x = 93.9 is 2.303 100

= = k . log 0.00390 16 93.9

The relation between the log of the percentage of the unracemized NA hydrochloride and time is graphically shown in fig. 15, which is linear showing that the reaction is of the first order.

/o racemized 0.0

20

40

60

80

100

300 hours

RACEMISATION OF NORADRENALINE AT 50°,60°&70o fora2%w/v solution of NA Hydrochloride of phU.2

Fig. 15 120

The solutions, when heated at 50° for 400 hours, remain colourless, but at 60° start developing a yellowish colour after about 240 hours at which the racemization is 60%, further, at 70° solutions start turning yellowish after 96 hours ofheating time, with a slight brownish precipitate and increase in reaction velocity after 144 hours when the racemization is about 85%. 5.3.2. Temperature coefficient The temperature coefficient for a difference of 10° is found from fig. 16 and from the reaction velocity constants given in table 56.

The temperature coefficient for the above process for a difference of 10° is 2-3.

togot 2.0- V\^ ^^^^**^_

1.8 - --^5{fc

1.6 _

x^ fc

U- A^S-

- Hours 100 200 300 too

RELATION'BETWEENTHELOG 0FTHE%OF THE UNRACEMJSED NORADRENALINE HCl &

THETIME AT DIFFERENT TEMPERATURES . Fig. 16.

Table 56 Velocity constants for the racemization of NA hydrochloride at 50°, 60°, and 70°; and the temperature coefficient for 10°

PH Temperature Velocity constant Temperature cofficient k

calculated from fig. 16

50° 0.00164

4.25 6G° 0.00397 2.4 2.3

70° 0.00856 2.15 2.3 121

5.3.3. Calculation of the stability at room temperature (20d)

Now if we know the time after which a certain % of NA is racemized the solutions 2.3 at a fixed temperature, it should be possible to keep times longer at 10° lower temperature. In the following table the racemization of has been cal¬ storage time for different percentage of NA culated.

Table 57

Calculated storage time for solutions of NA hydrochloride of pH 425; containing 0-1 % of NajSaOg, in nitrogen filled ampules

% racemization Temp. Time in h. Calculated storage time at 20°

10 50° 60 60 X 233 h. = 30 days

20 50° 136 136 X 2-33 h. = 78 days

30 60° 96 96 X 2-34 h. sa 112 days

40 60° 137 137 X 2-34: h. = 160 days

50 70° 82 82 X 235 h. a 220 days

60 70° 105 105 X 2-35 h. s 282 days

5.3.4. Conclusions 5.3.4.1. It is not possible to predict the stability of NA solutions with respect to racemization by carrying out the stability experiments at higher temperatures because the results of the experiments of the dilute solutions of NA hydrochloride at room temperature (18°-22°) show that for solutions of pH 2,3, 4 and 5 an equilibrium is attained with respect to racemization after about 60 days, as may be seen from fig. 10, page 103, and so there is a great difference in the predicted storage time and that found experimentally for the dilute solutions of NA hydrochloride. 5.3.4..2. In order to maintain maximum activity, NA injections must first be stored for about 2 months after manufacture, so that an equilibrium is attained with respect to racemization and only then they should be dispensed after determining their potency. 6. Summary

6.1. For the racemization of NA solutions the reaction is not of the first order at 120° and 100°, however at 70°, 60° and 50° the reaction is of the first order.

6.2. The temperature coefficient for a difference of 10° for a "2% w/v solution of NA hydrochloride of pH 4.25 containing 0.1% of sodium metabisulphite in nitrogen filled ampules is 2.3.

6.3. The minimum racemization was found for solutions with a pH value of about 4. 6.4. Solutions containing 0.1% of sodium metabisulphite in nit¬ rogen filled ampules showed less racemization when compared with the solutions containing no sodium metabisulphite in air filled ampules. CHAPTER XIV

SUMMARY 1. Sympatol 1.1. Methods of analysis

The following methods of analysis for the quantitative estimation of sympatol in solutions have been established : 1.1.1. Colorimetric method

Sympatol gives a colour reaction with sodium nitrite in the presence of mercuric sulphate. The coloured product gives a peak at 500 m/i and is stable at least for 50 minutes. Thus the method can be applied for the quantitative estimation ofsympatol content.

1.1 2. UV-Spectrophotometric method

.Sympatol gives absorption maxima at 223 and 273 mp. The ratio of the extinction values at the two maxima is 5.86 :1. Thus of any the maxima can be used to assay the sympatol solutions.

1.1.3. Paper chromatographic method Solutions of sympatol gave a linear reltionship between the spot area and the of log the amount of sympatol between 25 to 150 p.g. Thus the method may be used for the quantitative estimation of sympatol in solutions. On heating the sympatol solutions for a long time an orange product is formed, which gives absorption maximum at 290-295 mp.

However none of the above 3 methods can be used to estimate quantitatively the undeteriorated and deteriorated sympatol content in a mixture.

1.2. Stability of sympatol solutions

Since none of the previously examined methods can be used to determine the stability of" sympatol solutions, but as the decomposed product of sympatol is coloured, the stability of sympatol solutions has been determined by means of colour standards. Through the systematic variation of different influencing factors the following results have been obtained :

(a) The development of colour and thus the destruction of sympatol solutions is proportional to the increasing pH and storage temperature.

(b) Though nitrogen checks the oxidation in the phenolic OH group it is not sufficient to stabilize the sympatol solutions.

(c) Sodium metabisulphite helps to check the development of the colour.

In order to obtain the optimum stability of sympatol solutions the following conditions must be adhered to ' 123

— pH value should be 3 or lower,

— the ampules must be filled in an atmosphere of nitrogen, — as antioxidant 0.1% of sodium metabisulphite must be added,

— storage temperature must be as low as possible..

On the basis of the above, suggestions for the preparation of a stable solution of sympatol have been given, (see page 71)

2. Noradrenaline

2.1. Methods of analysis The following methods of analysis for the estimation of undeteriorated and deteriorated noradrenaline content in solutions have been investigated. 2.1.1. UV Spectrophotometric method. of The extinction wavelength curve of the deteriorated solution NA does* not show any change when compared with that of the fresh solution.* 2 1.2 Iodine method After iodine oxidation NA gives absorption maxima at 295-296 mf and at 520-525 m/*. The maximum loss shown by this method for a solution of NA which was heated at 120° for 2 hours in the presence of oxygen is 4.8%. 2.13. Persulphate method After persulphate oxidation NA gives absorption maxima at 288-290 mjx and at 490-495 rmt. The maximum loss shown by this method for a solution of NA which was heated at 120° for 2 hours in the presence of oxygen is 6.8 %. 2.1.4. Ferro-citrate method

NA gives an absorption maximum at 530 imt after reaction with ferro-citrate solution. The maximum loss shown by this method for a solution of NA which was heated at 120° for 10 hours in the pre¬ sence" of air is 7%.

None of these methods can be used for the estimation of the deteri¬ results and the orated NA content, as there is a great deviation in the above results of the biological assays carried out by other workers.

2.2. Racemization studies of the dilate solutions of NA (0.2%)

As the 1-form of NA is much more potent than the racemic form, the best conditions for the stability of the optical activity of NA solutions have been evaluated.

2.2.1. The racemization is highly dependent on pH. Solutions with a pH value in the neighbourhood of 4, show the maximum optical activity. 2.2.2. Storage time at room temperature (18°-22°) has very little influence on racemization. During the first 160 days, the influence of time is pronounced after which there is very little change.

2.2.3. Sodium metabisulphite has some influence on checking the racemization. J 24

2.3. Prediction of the stability of NA solutions with respect to i acemization.

2.3.1. At 120° and 100°, the reaction is not of the first order with respect to racemization.

2.3.2. At 50°, 60° and 70° the reaction is of the first order with coefficient of the for a respect to racemization. The temperature process 0.1% of difference of 10° for a solution of NA of pH 4.25 containing sodium metabisulphite is 2.3

2.3.3. On the basis of the racemization experiments of dilute solutions of NA at 20°, the different storage timings obtained for varying degree of racemization of NA, do not agree with the calculated storage time. For calculation of the stability, the racemization experiments have 60° and been carried out for a concentrated solution of NA (2%) at 50°, 70°. The dilute solutions ofNA between the pH values of 3 to 5, show an after for 2 months at 20 equilibrium of racemization storage , against the calculated storage timings under the same conditions.

ZUSAMMEMFASSUNG

1. Sympatol 1.1. Bestimmungsmethoden

Die folgenden Methoden fiir die quantitative Gehaltsbestimmung wurden : von Sympatollosungen ausgearbeitet

1.11. Kolorimetrische Methode. Natrium- Sympatol gibt bei Anwesenheit von Mercurisulfat mit nitrit eine Farbreaktion. Die Absorptionskurve des gefarbten Reaktionsproduktes, welches mindestens 50 Minuten lang stabil ist, fur die zeigt ein Maximum bei 500 m/i. Diese Reaktion kann daher Gehaltsbestimmung von Sympatol in Lbsungen benutzt werden. 11.2. UV-Spektrophotomelrische Methode. Sympatol selbst zeigt im UV-Gebiet zwei Maxima : eines bei Extinktionswerte 223 m/t, ein zweites bei 273 imi. Das Verhaltnis der bei diesen beiden Wellenlangen betragt 5.86:1. Beide Maxima sind daher fiir die Gehaltsbestimmung von Sympatollosungen brauchbar. 11.3. Papierchromatographiche Methode. Bei der Papierchromatographie von Sympatollosungen nimmt die Grosse der Sympatolflecken linearmitdem Logarithmus der Sympatol- menge zwischen 25 und 150 ^g zu. Diese Methode scheint daher auch geeignet zu sein, um den Sympatolgehalt einer L'dsung zu bestimmen.

Wenn Sympatoll'dsungen langere Zeit erhitzt werden, bildet sich ein orange gefa*rbtes Produkt, welches ein Absorptionsmaximum bei 290- 295 m/t aufweist.

Es zeigte sich jedoch, dass keine dieser 3 Methoden im Stande ist,,zersetztes und nicht zersetztes Sympatol quantitative nebeneinander zu erfassen. 125

1.2. Haltbarkeit von SympatoUosungen

Da keine der vorher geprliften Methoden zur Haltbarkeitsbestimmung und von SympatoUosungen tauglich ist, da ferner festgestellt wurde, dass die Zersetzungsprodukte von Sympatol gefarbt vvaren, wurde die Haltbarkeit der SympatoUosungen auf Grund der Farbveranderungen beurteUt.

Durch systematische Variation der einzelnen Einflussfaktoren ergab sich, dass

(a) die Farbentwicklung und damit der Abbau des Sympatols mit steigendem pH und steigender Lagertemperatur zunimmt.

(b) die Begasung mit Stickstoff die Oxydation der phenolischen OH-Gruppe wohl hemmt, aber fur eine normale Lagerzeit nicht geniigend verhindert,

(c) Natriummetabisulfit die Verfa"rbung von SympatoUosungen wcitgehend hintanhUlt.

Um moglichst stabile SympatoUosungen zu erhalten, sind folgende Bedingungen einzuhalten:

— der pH-Wert soUte 3 oder weniger sein,

— die Ampullen miissen mit Stickstoff begast werden,

— als Antioxydans ist 0.1% Natriummetabisulfit zuzufiigen,

— die SympatoUosungen miissen bei moglichst niedriger Tempera¬ ture gelagert werden.

Auf Grund der erhaltenen Resultate wurde eine Vorschrift fur eine haltbare SympatoUosungen vorgeschlagen (siehe page 71).

2. Noradrenalin

2.1. Bestimmnngsmcthodcn

Es wurde versucht, mit ciner der nachfolgcnden Methoden den und Gehalt von oxydiertem nichtoxydiertem Noradrenalin zu bestimmen.

2.1.1. UV-Spcktrophotometrische Methode. Die Extinktions- Wellenlangenkurve von weitgehend oxydiertem Noradrenalin zeigt keine wesentlichen Unterschiede gegeniiber frischzubereiteten Noradrc- nalinlosungen.

2.1.2. Jod-Methode. Mit Jod oxydiertes Noradrenalin zeigt Absorptionsmaxima bei 295-296 m/i und bei 520-525 m/*. Der mit dieser Methode nachweisbare abbau des Noradrenalins nach 2 Stunden Erhitzen auf 120° in sauerstoffgefiillten Ampullen betrug hochstens 4.8%.

2.1.3. Persulfat-Methode. Mit Natriumpersulfat oxydiertes Noradrenalin zeigt Absorptionsmaxima bei 288-290 tnp. und bei 490- 126

495 m/i. Der mit dieser Methode nachweisbare Abbau betrug nach 2 Stunden Erhitzen auf 120° in sauerstoffgefiillten Ampullen hochstens 6.8%.

2.1.4. Ferrozitrat-Methode. Das Reaktions produkt von Nora- ein drenalin mit Firrozitrat zeigt Absorptionsmaximum bei 530 tn/i. Der mit dieser Methode nachweisbare Abbau von Noradrenalin nach 10 Stunden Erhitzen auf 120° in luftgefullten Ampullen betrug hochas- Jtens 7%

Da die mit diesen Methoden erhaltenen Resultate unter sich nicht ubereinstimmen und vor allem grosse Abweichungen gegeniiber den im biologischen Versuch erhaltenen Resultaten anderer Autoren aufweisen, eignen sie sich fiir die Bestimmung des zersetzten Norad- renalins nicht.

2.2. Untersuchung iiber die Razemisicrung von Noradrenalin in verdiinnten Losungen

Da die Wirkung des 1-Noradrenalins viel starker ist als diejenige des Razemates warden die Einfliisse untersucht, die die Erhaltung der optischen Aktivitat begiinstigen.

2.2.1. Die Razemisierung, von 1-Noradrenalin ist sehr pH abh'angig Die optische Aktivitat wird bei ca. pH 4 am langsten erhalten.

2.2.2. Bei Zimmertemperatur (18°-22°) geht die Razemisicrung im Durchschnitt langsam vor sich. In den ersten 160 Tagen strebt sie ziemlich rasch einem Gleichgewichtszustand entgegen, welcher sich nach 160 Tagen kaum weiter verandert.

2.2.3. Natriummetabisulfit besitzt die Fahigkeit, den Razemisier-

zu . ungsvorgang hemmen. ,

2.3. Berechnung der Haltbarkcit von 1-Noradrenalinlosungen in Bezug auf die Razemisicrung

2.3.1. Im Bereich von 100° bis 120° folgt die Razemisierung nicht dem Gesetze der Reaktionen erster Ordnung.

2.3.2. Bei 50°, 60° und 70° folgt die Razemisierung dem Gesetze fiir Reaktionen erster Ordnung. Der Tempcraturkoeffizient fiir eine Temperaturdifferenz von 10° betragt fiir eine Noradrenalinlosung vom pH 4.25 mit einem Zusatz von 0.1% Natriummetabisulfit 2.3.

2.3.3. Bei der experimentellen Bestimmung der Razemisierung von verdiinnten Noradrenalinlosungen bei 20° resultierten fiir die verschiedenen Razemisierungsgrade Lagerfristen, welche gar nicht mit den berechneten iibereinstimmten. Als Grundlage fiir diese Berech- nungen dienten Razemisierungsversuche mit konzentrierten Noradrenalin¬ losungen (2%) bei 50°, 60°, und 70°. Die Razemisierung erreicht in den verdiinnten Noradrenalinlosungen vom pH 3-5 bei 20° nach 2 Monaten im Gegensatz zu den Berechnungen einen Gleichgewichtszustand. References

1. S. A. Schou, 1st Inte rnational Conference of Hospital Pharmacists, Basel, p. 58, 1952. 2. E. F. Cook and E. W. Martin, Remington's Practice of Pharmacy, 9th edition, p. 249, 1948 (The Mack Publishing Company, Easton) 3. E. Butikofer, Thesis E. T. H. Zurich, 1954. 4. S. A. Schou and A. Gredsted, Dansk T. Farm., 25, 164, 1951. 5. H. Spiegelberg and R. Schlapfer, Arzneim.-Forsch., 6, 75, 1956. 6. C. G. Lund, E. P. Nielsen and K. Pedersen-Bjergaard, The pre¬ paration of solutions isoosmotic with blood, tears and tissue, London/Copenhagen, 1947.

7. amer. I. J. Szekely and F. M. Goyan, J. pharm . Ass. (sci Ed.), 41, 30, 1952.

8. J. M. Wells, J. amer, pharm. Ass. (pract. Ed.), 33, 99, 1944. 9. F. M. Govan, J. M. Enright and J. M. Wells, J. amer. pharm. Ass. (sci'. Ed.), 33, 74, 1944. 10. Ph. Helv. V. Suppl. II, p. 189. 11. A. V. Hill, Proc. Roy Soc. London. Ser. A., 127, 9, 1930.

12. E. BUtikofer, Thesis E. T. H. Zurich, p. 126, 1954. 13. V. Dimbleby, J. Pharm. Pharmacol., 5, 969, 1953. 14. W. Fahrig, Pharm. Ind., 17, 494, 1955.

15. U. S. P. XV, p. 923.

16. Brit. Ph. 1958, p. 963. 17. Ph. Helv. V, p. 36. 18. F. Mylius, Angew. Chem., 34, 281, 1921. 19. H. Berry, J. Pharm. Pharmacol., 5, 1008, 1953. 20. F.H. Christensen, Farm. Tidende., 21 and 24, 1952. 21. K. MUnzel, Schweiz. Apoth. Ztg., 92, 33., 1954. 22. R. Dreweny, Scientia pharm., 25, 231, 1957. 23. U. S. P.« XIII, p. 628. 24. P.—D.E. Baumann, Sterilisation and Sterile Aufbewahrung von Sprtzein und Hohlandeln, 2nd edition, 1948, Basel (Benno Schwabe Co.). 25. C. Stich, Bakteriologie Serologic und Sterilisation im Apothekcn betriebe, 1950, Berlin. 26. V. Cazzani, Ipodermoterapia, 2nd edition, Milano (Industrie Grafiche Stucchi). 27. A. Lesure and J. Lavagne, Les Medicaments Injectablcs, 5th edition, 1942, Paris (Librairie E. Le Francois). 28. T. D. Whittet, Pharm. J., 165, 309, 1950. 29. G. E. Foster and G. A. Stewart, Quart. J. Pharm. Pharmacol., 21, 211, 1948. 128

30. H. Brunnhofer and K. Steiger, Thesis, University of Bern, p. 88, 1955. 31. H. Berry and G. B. West, Quart. J. Pharm. Pharmacol., 17, 242, 1944. 32. G. B. West, Quart. J. Pharm. Pharmacol., 18, 267, 1945. 33. G. B. West, J. Pharm. Pharmacol., 4, 560, 1952. 34. S. A. Schou, Pharm. Wbl. 4, 129, 1958. 35. M. P. Girard and M. G. Kerny, Ann. pharm. franc, 8,463, 1950. 36. P. Pfeiffer and W. Offermann, Ber. dtsch. chem. Ges., 75,1,1942. 37. H. Jager, Pharmazie., 3, 536, 1948. 38. Coleman, Amer. Chem. Soc. Abstr., 119th meeting, p. 17. 39. G. L. Jenkins and W. H. Hartung, The chemistry of organic medicinal products, 3rd edition, p. 645, 1949, (John Wiley and Sons, Inc., New York). 40. J. Kisbye and S. A. Schou, Dansk T. Farm., 25, 185, 1951. 41. J. Kisbye, Dansk T. Farm., 30, Suppl. II, 156, 1956. 42. J. Kisbye and T. Bols, Dansk T. Farm., 31 205, 1957. 43. Wilson and Schild, Clark's applied Pharmacology, 1952 (J. and A. Churchill). 44. J. C. Krantz and C. J. Carr, The pharmacological principles of medical practice, p. 562, 1949, Baltimore (The Williams and Wilkins Company). 45. O. Loewi, Pfliiegers Arch., 189, 239, 1921. 46. W. B. Cannon and A. Rosenblueth, Amer. J. Physiol., 104, 557, 1933. 47. U. S. von Euler, Acta physiol. Scand., 12, 73, 1947. 48. U. S. von Euler, Pharmacol. Rev., 3, 247, 1951. 49. E. Bulbring and J. H. Burn, Brit. J. Pharmacol., 4, 202, 1949. 50. J. H. Gaddum and L. G. Goodwin, J. Physiol., 105, 357,. 1947. 51. G. B. West, J. Physiol., 106, 418, 1947. 52. L. Goodman and A. Gilman, The pharmacological basis of therapeutics, 1946, New York (The Macmillan Co.).

53. H. Gold, Ind. engng. Chem., 37, 117, 1945. 54. G. Barger and H. H. Dale, J. Physiol., 41, 19, 1910. 55. W. H. Hartung, Chem. Rev., 9, 389, 1931. 56. W^ H. Hartung, Ind. engeng. Chem., 37, 126, 1945.. 57. D. Bovet and F. Bovet-Nitti, Medicaments du Systeme Nerveux Vegetatif, Editions S. Karger S. A., Bale,. 1948. 58. K. K. Chen, C. K. Wu and E. Henriksen, J. Pharmacol, exper. Therap., 36, 363, 1929.

59. J. Buchi, S. Ph. I., p. 5. 57 13, 1957. 60. A. B. Stockton, P. T. Pace and M. L. Tainter, J. Pharmacol. exper. Therap., 41, 11, 1931.

61. Nachtr. DAB 6., p. 55.

62. Ph. Int. I. Vol. II., English edition, p. 21. 129

63. Ph. Dan. IX. Vol. II., p. 568.

64. NNR., 1934, p. 423. 65. Schweizerisches Nachschlagebuch fur Pharmazeutische Speziali- 1956. taten p. 151, 15th edition, 66. F. StolzandJ. Hallensleben, Chem. Zbl., 102, II, 1056, 1931: D. R. P., 526, 087. 67. C. H. Boehringer Sohn, D. R. P., 566, 578. 68. J. Kamlet, Chem. Zbl., 110 II, 3451, 1939 : A. P., 2,151, 517. 69. H. Legerlotz, Chem. Zbl., 105 I, 1885, 1934: D. R. P., 522, 790.

70. H. Legerlotz, Chem. Zbl., 102 I, 1518, 1931; French. P., 695, 675. 71. H. Legerlotz, Swiss. P., 163, 397. 72. Ernst D. Bergmann and M. Sulzbacher, J. org. Chem., 16, 84, 1951.

73. G. Fodor and O. Kovacs, J. amer, chem., Soc, 71, 1045, 1949. 74. M. Asscher, (N. V. Philips-Roxane, Weesp., Netherlands), Rec. trav. chim., 68, 960, 1949: Chem. Abstr., 44, 3939 i. 1950. 75. E. Kallstrom, Pharm. Zhalle., 74, 269, 1933. 76. W. Awe and H. Stohlmann, Pharm. Zhalle., 96, 371, 1957. 77. H. Flaschka, A. Holasek and A. M. Amin, Arzneim—Forsch., 4, 38, 1954. 78. Lee Worell and W. R. Ebert, Drug Stand., 24, 153, 1956. 79. O. Aklin, Thesis, University of Strasbourg, 1957. 80. E. Sinidinos and R. VuiIIaume, Bull. Soc. Chim. biol., 32, 409, 1950. 81. A.Jindra,V. HamplandJ. Zyka, Ceskoslov. farm.,1, 76, 1952: Chem. Abstr., 46, 10538 i, 1952. 82. O. Ehrismann, Dtsch. med. Wschr., 53, 1263, 1927. 83. F. Lasch, Arch, exper. Pathol. Pharmakol., 124, 231, 1927. 84. O. Ehrismann and G. Maloff, Arch, exper. Pathol. Pharmakol., 136, 172, 1928. 85. G. Kuschinsky, Arch, exper. Pathol. Pharmakol., 156, 290, 1930. 86. C. E. Schuntermann, Klin, Wschr., 10, 1764, 1931.

87. K. Oberdisse, Verhandl. deut, Ges. inn. Med., 46th congress, 262, 1934; Chem. Abstr., 29, 8129, 1935. 88. M. L. Tainter and M. A. Seidenfeld. J. Pharmacol, exper. Therap., 40, 23, 130. 89. A. Schretzenmayr, Arch, exper. Pathol. Pharmakol., 159, 488, 1931.

90. G. Barkan and R. Matiisen-Dreybladt, Arch, exper. Pathol. Phar¬ makol., 172, 486, 1933.

91. B. Behrens and H. Taeger, Arch, exper. Pathol. Pharmakol., 178, 64, 1935. 92. M. R. Gurd, Quart. J. Pharm. Pharmacol., 10, 188., 1937. 130

93. G. L. Jenkins and W. H. Hartung, The chemistry of organic me¬ dicinal products, 3rd edition, p. 365, (John Wiley & Sons, Inc., New York). 94. R. Shuppli, Dermatologica, 96, 73, 1948. 95. W. Stepp and L. L. Kirchmann, Munch, med. Wschr., 86, 433, 1939. 96. H. Zischinsky, Munch.med. Wschr., 83, 1823, 1936. 97. C. H. Boehringer Sohn, Sympatol pamphlet, Winter 1952/53. 98. Analysmetoder., 8, 30, 1932. 99. O. Folin and V. Ciocalteau, J. biol. Chem., 73, 627, 1927.

100. J. W. H. Lugg, Biochem, J., 32, 775, 1938. 101. E. E. Arnow., J. biol. Chem., 118, 531, 137. 102. R. I. Ellin and A. A. Kondritzer. J. amer. pharm. Ass. (sci. Ed.), 41, 71, 1952. 103. S. A. Schou and J. M. Rhodes, Dansk T. Farm., 25, 350, 1951. 104. R. B. Fisher and R. Holmes, Biochem. J., 44, liv., 1949. 105. R. B. Fisher, D. S. Parsons and G. A. Morrison, Nature, London, 161, 764, 1948. 106. R. L. Reid and M. Lederer, Biochem. J., 50, 60, 1951. 107. A. Wickstrom and B. Salvesen, J. Pharm. Pharmacol., 4, 631, 1952.

108. R. Consden, A. H. Gordon and A. J. P. Martin, Biochem. J., 38, 224, 1944. 109. J. Biichi, Vorschlag fur Allgemeine Bestimmiingen, Ph. JJelv. VI 110. J. Morch, Pharm. Wbl., 4, 141, 1958. 111. Ph. Dan. IX. Add. 1954., p. 214-5. 112. H. Rosenblum, R. Goldman and H. Feldman, J. amer. pharm. Ass. (sci. Ed.), 38, 255, 1949. 113. H. Hellberg., J. Pharm. Pharmacol., 7, 191, 1955. 114. U. S. P. XV., p. 376.

115. Brith. Ph. 1958., p. 434. 116. Ph. Int. I. Vol. II., English edition, p. 164. 117. F. Stolz, Ber. dtsch. chem., Ges., 37, 4149, 1904. 118. K. W. Rosenmund, Ber. dtsch. chem. Ges., 46, 1049, 1913. 119. O. Hinsberg, Ber. dtsch, chem. Ges., 56, 854, 1923. 120. D. R. P., 157 300, Chem., Zbl., 76, I, 315, 1905. 121. D. R. P., 193 634, Chem., Zbl.., 79, I, 430, 1908. 122. W. Langenbeck and F. Fischer, Pharmazie., 5., 56, 1950. 123. G. Fodor, O. Kovacs and T. Mecher, Acta. chim. acad. Sci. Hung., 1, 395, 1951: Chem. Abstr., 49, 897, i., 1955. 124. B. F. Tuliar, J. amer. chem. Soc, 70, 2067, 1948. 125. M. L. Tainter, B. F. Tuliar and F. P. Luduena, Science, N. Y., 107, 39, 1948. 126. U. S. von Euler and U. Hamberg, Science, N. Y., 110, 561,1949, 131 e

127. M.J. Oesterling, Biochim. bioph'ys. Acta., 24, 178, 1957.

128. Ph. Int. I. Vol. II., English edition., p. 133. 129. T. Suzuki and T. Ozaki, Tohoku. J. Exptl. Med., 54, 322, 1951: Chem. Abstr., 46, 6693 h, 1952.

130. O. Folin, W. B. Cannon and W. Denis, J. biol. Chem., 13, 477, 1912. 131. G. F. Somers and G. B. West, Quart. J. Pharm. Pharmacol., 17, 308, 1944. 132. J. Richter, Z. anal. Chem., 145, 334, 1955. 133. U. S. P. XIV., p. 216. 134. M. E. Auerbach and E. Angell, Science, N. Y., 109,1537, 1949. 135. W. Schuler and P. Heinrich, Helv. chim. Acta., 31, 320, 1948. 136. A. Lund., Acta, pharmacol. et toxicol., 5, 75, 1949. 137. A. Lund, Acta, pharmacol. et toxicol., 5, 121, 1949. 138. A. Lund, Acta, pharmacol. et toxicol., 5, 231, 1949. 139. S. Natelson, J. K. Lugovoy and J. B. Pincus, Arch, Biochem., 23, 157, 1949. 140. H. Weil-Malherbe andA. D. Bones, Biochem, J., 51, 311, 1952. 141. K. Erne and T. Ca'nback, J. Pharm. Pharmacol., 7, 248, 1955.

142. U. S. von Euler and I Floding, Act physiol. scand., 33, Suppl., 118, 45, 1955.

143. U. S. von Euler and I. Floding, Acta physiol. scand., 33, Suppl., 118,57,1955. 144. U. S. P. XV., P. 377.

145. U. S. von Euler, Noradrenaline: Chemistry, Physiology, Spring¬ field, 111., Chas. C. Thomas, 1956. 146. A. M. Lands, Pharmacol. Rev., 1, 279, 1949. 147. J. H. Burn and D. E. Hutcheon, Brit. J. Pharmacol., 4, 373, 1949. 148. F. P. Luduena, E. Ananenko, O. H. Siegmund and L. C. Miller, J. Pharmacol, exper. Therap., 95, 155, 1949. 149. E. E. Swanson and K. K. Chen, J. amer. pharm Ass. (sci. Ed.), 41, 187, 1952. 150. U. S. v on Euler, Acta physiol. scand,. 11, 168, 1946.. 151. P. Holtz, K. Credner and G. Kroneberg. Arch, exper. Pathol. Pharmakol., 204, 228, 1947. 152. M. Goldenberg., V. Apgar, R. Deterling and K. L. Pines, J.- amer. med. Ass. 140, 776, 1949. 153. H. C. Churchill-Davidson, Brit. med. J., 29/12, 1551, 1951. 154. C. Beaudet, Experientia, 7, 291, 1951. 155. P. Marquardt and E. Carl, Naturwissenschaften., 39, 210, 1952 156. D. E. Green and D. Richter, Biochem. J., 31, 596, 1937. 157. J. D. Bulock and J. Harley-Mason, J. chem. Soc, 712, 1951. 158. R. Barer, H. Blaschko and H. Langemann, J. Physiol., 112,21 P. 1951. • 132

159. U. S. von Euler and U. Hamberg, Acta, physiol. scand., 19, 74, 1949. 160. Ehrlen, Farm. Revy., 47, 321, 1948. 161. J. H. Barker, J. C. Eastland and N. Evers, Biochem. J., 26, 2129, 1932. 162. F. G. Stock and W. Hinson, J. Pharm. Pharmacol., 7, 512, 1955. 163. J. C. Winter and R. Kunin, Ind. engng, Chem., 41, 460, 1949.

164. Ph. Helv. V., p. 5. 165. S. Glasstone, Text book in physical Chemistry, 2nd edition, p. 1044, 1953, New York London (Macmillan and Co. Ltd.). CURICULUM VITAE

of Devendra Kumar Agrawal I I was born at Bilgram (Uttar Pradesh), India, on the 26th of January 1932.

After attending School at Hardoi, I passed the High School exami¬ nation of the U. P. Board of High School and Intermediate Education in the year 1947. After" further studying for 2 years I passed the Inter¬ mediate (science) examination from the Government College, Allahabad, in the year 1949.

I studied at the Pharmaceutical department of the College of Tech¬ nology of Banaras Hindu University, and passed the final degree examination of the Bachelor of Pharmacy in June 1954.

After taking apprenticeship for 1 year at various manufacturing laboratories I joined the School of Pharmacy of the Swiss Federal Insti¬ tute of Technology, Zurich, in October 1955. After passing the admission examination in August, 1956, I started work on research, the results of which are herein submitted,

\