THE DISTRIBUTION OF IPRONIAZID IN BODY FLUIDS AND
TISSUES AFTER ORAL ADMINISTRATION AND
A METHOD FOR ITS DETERMINATION
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
CARL EDWARD MOYER, B. S., M. S.
<1 The Ohio State University
1959
Approved by
Department of Physiological Chemistry ACISOWLEDGSKEHT
The author wishes to express his sincere appreciation to Dr. Helen L. Wikoff for her counsel, interest and patience in preparing this work.
ii TABLE OP CONTENTS
Page
Introduction...... 1
Historical...... 4
Preparation and Chemical Properties of Iproniazid.... 8
General Methods for the Determination of Carboxylic Acid Derivatives...... 11 Experimental Procedures and Results...... 14
A. Attempts to Develop Colorimetric Procedures for the Determination of Iproniazid...... 14
B. Spectrophotometric Procedures...... 16
C. Attempts to Separate Iproniazid from Pyridine Carboxylic Acids and Their Derivatives...... 19
D. The Determination of Iproniazid in Aqueous Solutions...... 2 4
E. Quantitative Estimation of Iproniazid Added to Pooled Serum...... 26
P. Quantitative Estimation of Iproniazid Added to Urine...... 32
G. Procedure Finally Adopted for the Quanti tative Determination of Iproniazid In Blood and Urine...... 39
1. Reagents...... 39
2. Procedure...... 40
iii iv
Page
H. The teOiantitative Estimation of Iproniazid In Rabbit Serum Following Oral Admini stration...... lj.2
I. Determination cff the Amount of Iproniazid Excreted in the Urine...... i+9
J. Determination of Iproniazid In Serum And 21; Hour Urine Specimens From Psychotic Patients...... 53
K. Absorption of Iproniazid From the Gastro- intestional Tract of a Rat...... 55
L. Determination of Iproniazid In Body Tissues...... 57
Discussion...... 59
Summary...... 61;
Bibliography...... 67
Autobiography...... 70 LIST OP TABLES
Table Page
1 The Amounts of Iproniazid, Isonicotinic Acid anc: Isonicotinic Acid Amide Extracted by Organic Solvents from 1 ml. Portions of Aqueous Solutions Containing 25 Gamma of the Respective Compounds...... 20
2 The Amount of Iproniazid Extracted Prom 1 ml. of An Aqueous Solution Containing 25 Gamma Iproniazid by 50 ml. Chloroform After the Addition of 1 ml. of Various Bases in the Concentrations Listed...... 21
3 Effect of Time of Extraction With Chloroform on Iproniazid Recovery Prom 1 ml, Basic Aqueous Solution Containing 25 Gamma Iproniazid Per ml.. 22
Lj. Amounts of Iproniazid Recovered Prom Basic Aqueous Solutions by Various Quantities of Chloroform...... 22
5 Effect of Concentration of Hydrochloric Acid on the Amount of Iproniazid Recovered Prom 25 ml. Chloroform Solution Containing 22 Gamma Iproniazid Per ml ...... 23
6 Time Necessary to Extract Iproniazid Prom 25 ml. Chloroform'With 5 ml. of IN Hydrochloric Acid...... 2l|
7 The Determination of Iproniazid in Aqueous Solutions...... 26
8 The Amount of Added Iproniazid Pound in 1 ml. Solution When the Iproniazid Standard Was Extracted in the Same Manner...... 26
9 Absorbance at 268 nyt Shown by 1 ml. Samples of Sera Obtained Prom Ten Normal Persons...... 27
v vi
Table Pag©
10 Amount of Iproniazid Recovered From 1 ml. Serum Samples Containing 25 Gramma Iproniazid... 28
11 Effect of Time of Extraction With Chloroform On Iproniazid Recovery From 1 ml. Serum Containing 25 Gamma Per ml ...... 28
12 Stabilization of the Recovery of Iproniazid From Serum by the Addition of Ammonium Salts... 29
13 Effect of the Addition of Various Quantities of Ammonium Chloride on the Chloroform Extraction of luroniazid From 1 ml. Serum Containing 25 Gamma per ml ...... 30
lit Determination of Iproniazid In 1 ml. Pooled Serum...... 30
15. The Amount of Added Iproniazid Found in 1 ml. Serum When Compared With a Standard Dissolved in Serum...... 32
16 Minimum Amount Iproniazid That Can Be Determined Accurately In 1 ml. Serum...... 32
17 Absorbance at 268 my Produced by Extracts From Normal Urine Treated According to the Procedure for the Isolation of Iproniazid 33
18 Effects of Various Concentrations of Ammonium Hydroxide Added to Urine Before Extraction With Chloroform...... 33
19 Effect of Various Amounts of Ammonium Chloride Added to Urine Before Extraction With Chloroform on the Absorbance at 268 irjy.... 3^
20 Absorbance at 268 nyu Produced by 5 ml. Aliquots of 2l± Hour Urine From 57 Individuals...... 35>
21 Comparison of the Amounts of Iproniazid Found in 5 ml. Urine With That Added...... 37
22 The Amount of Added Iproniazid Found in 5 ml. Urine...... 37 vli
Table Page
23 The Amount of Added Iproniazid Pound in 5 ml. Urine Specimens When the Standard Consisted of Iproniazid Dissolved in Urine...... 38
2[|_ Absorbance at 268 mjf Produced by Extracts Prepared Prom the Serum of a Rabbit According to the Procedure For the Isolation of Iproniazid...... J4J4-
25 Absorbance and Amount of Iproniazid in the Serum of a Rabbit 2 Hours After Receiving 1.6 mg. Iproniazid......
26 Iproniazid Content of Rabbit Serum Drawn at Various Intervals After Oral Administration of Iproniazid...... lj.6
27 Concentration of Iproniazid in Rabbit Serum Following the Daily Administration of 2.0 mg. Iproniazid Phosphate Per kg. Body Weight..... 1+8
28 Concentration of Iproniazid in Rabbit Serum Following the Daily Administration of 1|,.0 mg. Iproniazid Phosphate Per kg. Body Weight 1+9
29 Amounts of Iproniazid Found in 21+ Hour Urine Specimens Obtained From Two Rabbits That Had Received Daily Oral Doses of 2.0 mg. Iproniazid - Phosphate Per kg. Body Weight...... 5>1
30 Amounts of Iproniazid Found in 21+ Hour Urine Specimens Obtained From Two Rabbits That Had Received Daily Oral Doses of 2.6 mg. Iproniazid Phosrhate Per kg. Body Weight...... £2
31 Concentration of Iproniazid in Serum and Urine of 6 Male Mental Patiert s Receiving 5o nig. Marsilid Phosphate (32.ip mg. iproniazid) Daily Results of Two Analysis 15? Days Apart.... 51+
32 Recovery of Iproniazid From Gastrointestinal Tract of Rats Following the Oral Administra tion of 0.98 mg. Iproniazid Phosphate...... 56 33 Weight of Iproniazid Found in Body Tissues of Rabbits Receiving 3-25 mg. Iproniazid Phosphate Per Kg. Body Weight Daily for Four Days 58 LIST OP FIOURES
Figure Page
1 Structural Formula of Iproniazid 8
2 Absorbance Spectra of Iproniazid In Acidic, Basic and Neutral Solutions (2£ Gramma per ml.)...... 16
viii INTRODUCTION
Investigators ore presently directing a great deal of attention to mood changing drugs. However, substances affecting the moods have been known since the beginning of recorded history.
The production of alcoholic beverages dates back to early Biblical days, for Noah Is reported to have made wine and to have consumed it in excess. The pioneer wine manu facturers probably had no idea of any therapeutic effects to be derived from their product but they must have noticed some of the effects produced when varying quantities were inbibed.
The ancient Chinese who obtained alcohol by distilling fer mented rice observed the mental depression caused by alcohol.
Thrill seekers, certain authors searching for new Ideas and persons discontented with their normal lives are among the many who consumed cannabis, cocaine or morphine for the state of euphoria experienced. This practice has been going on in some of the Oriental countries for many centuries. .
The modem gaseous anesthetics produce a state of ex citement before the patient sinks into a stupor. This was
1 2 particularly noted in the case of nitrous oxide, commonly known as "laughing gas."
In the early part of the Nineteenth Century, bromides were introduced for the treatment of epilepsy, since they served to reduce the number and Intensity of the attacks.
During World War II, bromides were used to quiet animals, particularily dogs, In areas subjected to frequent bombing attacks.
After the Introduction of the bromides, a host of or ganic compounds were Introduced for use as hypnotics and soporifics. The best known members of the group include such drugs as the barbiturates, chloral hydrate, and paral dehyde. All of these compounds are mental depressants.
The advent of the tranquilizing agents, such as mepro bamate, chloropromazine, and the Rauwolfia alkaloids brought about a great improvement in the treatment of mental diseases.
These drugs are also very beneficial in the treatment of many anxiety states.
Before the Introduction of insulin shock therapy, psychiatrists had to rely on psychotherapy for the treatment of mental depressions. Electroshock therapy was later added and In many cases was both useful and successful. For a long time these were the only means of treating mental de pressions. Recently some organic compounds have been synthe sized which have been shown to possess mood changing 3 properties. These have proved to be very useful in the treatment of individuals with mental depressive states, One of these compounds is iproniazid.
4 HISTORICAL
In 19^1 pox (l), working on the synthesis of new com pounds for the treatment of tuberculosis, prepared isonico tinic acid hydrazide as an intermediate in an attempt to synthesize a pyridine analog of the German compound tibione.
This intermediate was tested as a tuberculostatic agent In mice by Grunburg and Schnitzer (2) and In human beings by
Robitzek, Sellkoff and Ornstein (3 )* It was found to be effective against tuberculosis in both mice and humans and thus It provided the parent structure for a large number of compounds which were investigated for potential tuberculo static properties.
One of the compounds synthesized by Pox and Gibas (ij.) was l-Isonlcotlnyl - 2-isopropyl hydrazine, the generic name of which is iproniazid. These authors stated that stimulation of the central nervous system was one of the side effects produced in patients to whom the compound was given. This was considered an undesirable side effect and since it was more marked with iproniazid than with other hydrazine derivatives in the dosages used for the treatment of tuberculosis, Iproniazid was abandoned for that use. 5 Bosworth and coworkers (5>)» (6 ) felt that iproniazid had great potentialities and continued to use the drug. They stated that it had valuable properties in addition to its bacteriostatic effect in the treatment of tuberculosis. In
1955 Bosworth and coworkers (7 ) reported that they had found the iproniazid improved the patient’s general outlook and thus aided the healing process.
Studies of the effect of iproniazid on mentally de pressed patients were carried out almost simultaneously and independently by three groups of Investigators. One of these
Investigators, Crane (8 ), (9) reported on the use of Ipronia zid in depressed patients, most of whom had been hospitalized for tuberculosis. Not all of these patients improved and some undesirable reactions were noted but the moods of most of the depressed persons were Improved by the treatment.
Loomer, Saunders, and Kline (10) used Iproniazid in the treatment of severely depressed and regressed hospital psy chotic patients. They found that some chronically depressed patients, who had not been helped by any other therapy, returned to a normal state of mind. In other cases the improvement was very slow In manifesting itself, while still others were not benefitted at all.
The third group of investigators, Scherbel, Schuchter and Harrison (11), pointed out that recovery from any chronic disease may be limited by depression, exhaustion and anxiety on the part of the patient. They found iproniazid to be of
definite value in treating the depression of patients with
rheumatoid arthritis.
As the clinical value of iproniazid for depressed
patients became evident, information concerning its mode of
action became of Interest. Zeller and coworkers (1?), (13),
(llj.) discovered that iproniazid was an inhibitor of monoamine
oxidase in the brain and liver mitochondria. Monoamine
oxidase disappeared completely from the brain of adult rats
two and one half hours after the injection of ten milligrams
of iproniazid. Other investigators then attempted to
discover what biochemical effects were produced in the brain
by this drug.
TJdenfriend, Weirsbach and Bogdanski (15) studied the
conversion of serotonin in brain tissue to the excretion product 5-hydroxy-indole acetic acid by the enzyme mor^amine oxidase. They found that the administration of iproniazid to animals caused a rapid rise in serotonin content of the brain which was maintained for an extended time interval.
Previously, Pletscher, Shore and Brodie (16 ) had shown that reserpine caused a release of serotonin from the brain and thus lowered the concentration in the brain. A number of other compounds known to affect the central nervous system were also investigated with respect to their capacity for releasing serotonin. Pletscher et al. concluded that this property was limited to those Rauwolfia alkaloids which were capable of exerting a tranquilizing effect.
Thus the concept was formulated that a tranquilizer, such as reserpine, lowered the level of serotonin in the brain and an energizer, such as iproniazid, increased the concentration of serotonin in the brain. PREPARATION AND CHEMICAL PROPERTIES OP IPRONIAZID
CH, I 3 C—H I CH3
H— (H .C— H
Pig. 1 - Structural Formula of Iproniazid
Iproniazid is synthesized by the reaction of isonico- tinyl hydrazine with propanol-2, and acetone followed by reduction with hydrogen according to a patented procedure
(18). The product is crystallized from benzene and puri fied by recrystallizatlon from a mixture of equal parts ligroin and benzene. The resulting product is a white needle-shaped crystalline compound which melts at 112° to
113°C. This compound is readily soluble in water, alcohol, and chloroform, slightly soluble in carbon tetrachloride
8 and ethylene dichloride, and very slightly soluble in acetone and ether.
Chemically the compound is rather inert. All of its chemical reactions are those of the nyridine nucleus, snecif- ically of the nitrogen. Cyanogen bromide will react with the nitrogen which then can be coupled with an amine to give a colored product. This is the Konig reaction (19).
Q CH, ch3 0 «■I || C— N— K — A — H C—N— H—C—H c— N — N--C—H H 1 1 ' H H Ah 3 H ch5 ch5 J * 4 k H- CNBr H
«C—K—M—C—H f 3 I P -H
CHj h - d :
H + 2 RHH2 R-N — L=n-r -(- ck-nh2 ^Br H
Fusion of inroniazid with 2, i^-dinitrochlorobenzene yields a pyridinium quaternary salt (20), which on hydrol ysis yields a derivative of glutaconic aldehyde. 10 0 CH-5 0 CH-* i — N — N — i - H H H CH3 f-rrb
NaOH
—N-N—C—H C—N—»—C—H H H
—OH
ft ft J
— OH
The stability of iproniazid suggests that Its activity in vivo is not due to a scission of the parent compound as might be expected, but rather is a function of the entire compound. GENERAL METHODS FOR THE DETERMINATION OF
CARBOXYLIC ACID DERIVATIVES
Since iproniazid is a relatively new compound, very few methods for determining it have been reported. General methods for the determination of pyridine carboxylic acids which might be applicable to iproniazid are therefore included here.
A survey of methods of determining hydrazine and hydra zine derivatives of pyridine carboxylic acids discloses pro cedures based on the reaction of the amino group with p-dimethyl aminobenzaldehyde or with salicylaldehyde. How ever, since iproniazid does not have a free hydrazine group, it must be hydrolyzed before these reactions can be employed.
The reactions involving the pyridine part of the mole cule are those utilizing cyanogen bromide or 2 , ij.-dinitro- chlorobenzene• Several methods have been proposed, based on the Konig (1 9 ) reaction of pyridine-type compounds with cyanogen bromide. Rubin, Drekter, Scheiner, and DeRitter
(21 ) (22) reported a colorimetric method for the determina tion of Iproniazid in blood plasma based on the reaction of isonicotinic acid with cyanogen bromide followed by a couplii^j
11 with ammonia. Since this reaction occurs with both isonico tinic acid and nicotinic acid, a determination of the con centration of nicotinic acid or of its derivatives was also necessary in order to estimate the amount of iproniazid by difference. Rubin et al. devised a method for the determina tion of nicotinic acid by treating the reaction product formed by cyanogen bromide with Ketol (p-methylamenophenol sulfate). Isonicotinic acid yields no color with this re agent. The amount of iproniazid could thus be determined by difference. Jacobs (23 ) later reported that this procedure was very cumbersome and difficult to carry out.
Swaminathan (2lj.), determining the nicotinic acid content of food stuffs, used aniline to develop a color after the
Konig reaction. Sweeney (2£) also using the Konig reaction for the determination of nicotinic acid in pharmaceutical preparations and food products employed successively sulfonic acid and then 2-naphthylamine sulfonic acid as color re agents.
Vilter, Spies, and Mathews (20) used 2, J|-dinltrochloro- benzene to determine nicotinic acid and nicotinamide in human urine. The reaction had been originated by Zincke (26), for the determination of pyridine. In the method devised by
Vilter et al., nicotinic acid in urine was fused with 2 , lj.-dinitrochlorobenzene to form a quaternary pyridinium salt.
The addition of alkali caused the decomposition of the fused 13 compound to glutaconaldehyde, a red-yellow colored substance.
Scott (27) successfully modified the reaction of nyri-
dine with 2, l^-dinitrochorobenzene described by Zincke to assay the isonicotinic acid hydrazide content of pharmaceut
ical preparations. This procedure also is not specific for iproniazid, since similar reactions are obtained with other isonicotinic acid derivatives and the derivatives of nico tinic acid.
Jacobs (23) proposed a colorimetric method based on the reduction of potassium ferricyanide in acid solution by iso nicotinic acid hydrazide. This causes the formation of
Prussian or Trumbull's blue and. in the acid solution, normal blood components do not interfere. However, this is an empirical method and close adherence to the prescribed pro cedure is necessary.
Herington (28) described the use of trisodium penta- cyanoaminoferrate as a completing reagent for isonicotinic acid. Glycerol was added to intensify to color and to keep the complexes in solution. The greatest problem was the relative instability of the aqueous solution of trisodium pentacyanoaminoferrate. Herington applied the procedure to isonicotinic acid and isonicotinic acid amide in aqueous solutions. He reported that isonicotinic acid and its amide could be determined in the presence of nicotinic acid and its amide by this method. EXPERIMENTAL PROCEDURES AND RESULTS
A. Attempts to Develop Colorimetric Procedures for the
Determination of Iproniazid
Attempts were made to adapt existing colorimetric pro
cedures for the determination of nicotinic acid derivatives
to the determination of iproniazid.
The Konig reaction of pyridine derivatives with cyanogen
bromide was first Investigated. When aniline (2lp) was used
to develop a color after completion of the reaction with
iproniazid, a colored compound was obtained which could not
be distinguished from the product formed when nicotinic acid
and nicotinamide were subjected to the same reactions.
Changes In the concentration of the reagents, the order df
addition, and the time of color development failed to yield
a means of differentiation between Iproniazid and either
The Iproniazid used throughout this investigation was obtained from Marsilid phosphate tablets (Hofftaan-LaRoche Inc., Nutley, New Jersey) purchased from local pharmacies since iproniazid in the pure form could not be obtained by us. The procedure for Isolating Iproniazid from the tablets was as follows. An aqueous solution of Marsilid phosphate prepared from ground tablets was made alkaline (pH>v*8 j with ammonium hydroxide and then extracted with chloroform. The residue from the evaporation of the chloroform extract was recrystallized from a solution of equal parts benzene and ligroin. Ik 15 nicotinic acid, nicotinamide, isonicotinic acid, or isonico tinamide.
Sulfonic acid or 2-naphthylamine sulfonic acid (25) used as color reagents after the Konig reaction with ipron iazid also gave rise to colored compounds which could not be distinguished from similar colors obtained with the same pyridine derivatives tested previously. A selectivity could not be obtained by changes in the concentration of the re agents, the pH of the phosphate buffer, or by a different type of buffer such as citric acid and sodium phosphate.
Variations in the order of the addition of the reagents and the time allowed forreaction and color development did not produce a means of determining iproniazid in the presence of the other pyridine derivatives being tested. Since these reactions failed to produce a satisfactory method for the determination of iproniazid and since cyanogen bromide is a dangerous chemical to handle, this phase of the investigation was dropped.
The method for the determination of nicotinic acid and nicotinamide described by Vilter et al. (20) was next applied to the determination of iproniazid. A colored product essentially the same as that obtained by treatment of nico tinic acid with 2, i^-dinitrochlorobenzene resulted. Changes in the quantity of 2 , i^-dinitrochlorobenzene either did not produce the maximum color intensity or the color of the reaction product was masked by the yellow color of the re agent Itself. In order to cause a reaction between ipronia zid and 2 , J^-dinitrochlorobenzene to occur, it was necessary to maintain the heat between 100-10f?°C. With less heat, fusion did not occur and with more heat, the reaction prod uct charred and was destroyed. The use of a more concen trated alcoholic sodium hydroxide solution did not seem to alter the color of the reaction product. It was hoped that a change in the concentration of the alcoholic sodium hy droxide would produce a different color either with ipronia zid or the nicotinic acid derivatives but the only effect was an increase in the intensity of the color when an in crease in the concentration of the alcoholic base was used.
A decrease in the intensity of the color followed a decrease in the concentration of the alcoholic base. Alcoholic potassium hydroxide produced a more intense color than the sodium hydroxide solution but no differentiation between iproniazid, nicotinic acid, and isonicotinic acid.
B. Spe ctrophotometric Procedures
When colorimetric procedures failed to yield a satis factory method for the determination of iproniazid, the ab sorption spectrum of iproniazid was next examined in the hope of developing a spectrophotometric method for its determina tion. Ultraviolet absorption studies carried out on an 17 aqueous solution of Iproniazid showed essentially one rather broad band of absorption between 265> and 270 Absorption studies on an acidic solution of Irroniazld or on an alka
line solution showed essentially the same absorption band as a neutral solution (Figure 2). However more absorption was obtained with the acid solution. Since the b?nd of absorp tion was rather broad, ranging from 26£ to 270 268 m p was chosen as the wavelength for measuring the intensity of the absorption produced by iproniazid. i. . bobne pcr fIrnai i cdc basic, inacidic, Iproniazid of spectra Absorbance - 2.Pig. ABSORBANCE n eta ouin. (2J> solutions. per ml.) gamma neutral and AE EGH (mp) LENGTH WAVE
19 C. Attempts to Separate Iproniazid from Pyridine Carboxyl!c
Acids and Their Derivatives
Since the presence of pyridine carboxylic acids and
their derivatives interfered In all methods proposed for the
determination of iproniazid, in the next phase of the invest
igation, an attempt was made to separate the iproniazid from
such substances. It was hoped that by extraction procedures,
either iproniazid or the interfering substances could be re
moved from the starting material.
One ml. portions of aaueous solutions of iproniazid,
isonicotinic acid and isonicotinic acid amide containing 2J?
gamma per ml. of the respective chemicals were extracted with
50 ml. portions of ether, chloroform, carbon tetrachloride,
benzene, and ethylene dichloride respectively. The absorbance
of each of the resulting solutions of iproniazid, isonico
tinic acid and isonicotinic acid amide in the respective
solvent was determined at ?68 tyy using the pure solvent as
the optical reference. Each of these values was then compared with the absorbance of aqueous standard solutions containing
the same amounts of the corresponding substance (iproniazid,
isonicotinic acid and isonicotinic acid amide). Table 1
shows chloroform to be the best solvent. However isonicotinic acid and isonicotinic acid amide also were soluable in this solvent. 20 An emulsion was formed whenever an attempt was made to
extract the aqueous solution with chloroform. Therefore the
mixture was transferred to a centrifuge tube and the emulsion
broken by centrifugation at 1500 times gravity for ten
minutes.
Table 1
The Amounts of Iproniazid, Isonicotinic Acid and Isonicotinic Acid Amide Extracted by Organic Solvents from 1 ml. Portions of Aqueous Solutions Containing 25? Gamma of the Respective Compounds Gamma Gamma Gamma Solvent Iproniazid Isonicotinic Isonicotinic Recovered Acid Acid Amide Recovered Recovered
Chloroform 16.0 18.3 16.8 Ether 6.k 8.2 8. ip Carbon te trachloride 13.0 14.0 13.2 Benzene 2.5 2.3 2.1* Ethylene dichloride 3.0 k-7 2.9 ’
In an effort to improve the selectivity of the chloro
form extraction, aqueous solutions of iproniazid were mad©
acidic or basic before adding the chloroform. Absorbance of
the resulting chloroform solutions showed that essentially
no iproniazid was extracted from an acid solution by chloro
form. The best recovery could be effected by a chloroform
extraction of a basic solution of iproniazid. When solutions
of isonicotinic acid and isonicotinic acid amide were made basic before extraction with chloroform, no absorbance due to 21
either of these two compounds could be detected in the chloro
form extracts. The addition of isonicotinic acid or isonico
tinic acid amide to a solution containing iproniazid did not
interfere with the recovery of iproniazid from the aqueous
solution.
Experiments with various bases and with various concen
trations of bases showed that 1 ml. of a 2* per cent solution
of ammonium hydroxide added to the solution of iproniazid
before extraction with 5>0 ml. portions of chloroform was the most effective (Table 2).
Table 2
The Amount of Iproniazid Extracted Prom 1 ml. of An Aqueous Solution Containing 2£ Gamma Iproniazid by £0 ml. Chloroform After the Addition of 1 ml. of Various Bases in the Concentrations Listed Gamma Iproniazid Recovered by Extraction Per Cent After the Addition of Various Bases Concentration Ammonium Sodium Potassium of Base Hydroxide Hydroxide Hydroxide 1 9.9 6.2* 7.9 2 17.9 13.0 16.2 3 20.1 11*. 6 17.0 h 22.0 1$.k 18.1 5 21.2* 15.5 19.3
In order to determine the time required for maximum extraction of iproniazid from basic aqueous solutions by chloroform, a series of experiments was carried out using various Intervals of time for the extractions. Table 3 shows maximum recovery of iproniazid in 20 minutes using
Kahn type mechanical shaker# 22 Table 3
Effect of Time of Extraction With Chloroform on Iproniazid Recovery Prom 1 ml. Basic Aqueous Solution Containing 25 Gamma Iproniazid Per ml. Extraction Time Gamma Iproniazid In Minutes Recovered
5 llj.. 6 10 21.3 15 21.8 20 22.1 2$ 22.0 30 22.0 60 22.1
DeterralnntIons were next made to find the minimum amount of chloroform necessary to extract Iproniazid from basic aqueous solutions. One ml. portions of iproniazid solution containing 200 gamma per ml. were employed In order to pro vide amounts as large as might be expected in fluids obtained from human subjects. Repeated tests showed that a volume of
35 ml. chloroform was sufficient to extract the iproniazid
(Table I}.). Table U
Amounts of Iproniazid Recovered Prom Basic Aqueous Solutions by Various Quantities of Chloroform Ml. Chloroform Gamma Iproniazid Recovered Prom Aqueous Used for Solution Containing 200 Gamma per ml. Extraction 25 li*2 30 170 35 176 1*0 176 kS 177 5o 176 23 As chloroform is quite volatile, the concentration of iproniazid in such a solution seemed likely to change due to evaporation of the solvent. It therefore seemed desirable to transfer the iproniazid to a less volatile solvent before making; spectrophotometrie measurements. Since iproniazid could not be extracted from acidified aqueous solutions by chloroform, there was a possibility that acids might be used to remove Iproniazid from chloroform. Table 5 shows the re sults obtained when $ ml. portions of hydrochloric acid solu tions of various normalities were used to extract 25 ml. aliquots of chloroform solutions containing 22 gamma ipronia zid. A concentration of 1 normal hydrochloric acid appeared to be the most satisfactory.
Table 5
Effect of Concentration of Hydrochloric Acid on the Amount of Iproniazid Recovered Prom 25 ml. Chloroform Solution Containing 22 Gramma Iproniazid Per ml. Normality of Gamma Hydrochloric Iproniazid Acid______Recovered 0.1 U.9 0.5 1 1 .1; 1.0 20.9 1.5 20.9 2.0 20.8
The length of time necessary for complete extraction of iproniazid from the chloroform solution by means of hydro chloric acid was next investigated* Results shown In Table
6 indicate that extraction Is most nearly complete in 20 min. 2k Table 6
Time Necessary to Extract Iproniazid Prom 25 ml* Chloroform With 5 ml* of IN Hydrochloric Acid Extraction Time Gamma Iproniazid Recovered Prom 25 ml. In Minutes Chloroform Solution Containing 22 gamma 5 10 a 15 18.0 20 20.9 25 20.9 30 20.9
D. The Determination of Iproniazid in Aqueous Solutions
After conditions had been established for the separation of iproniazid from Isonicotinic acid and isonicotinic acid amide as well as for the further removal of iproniazid from chloroform extracts, the quantitative estimation of ipron iazid In aqueous solutions was attempted. Colorimetric pro cedures for iproniazid previously investigated and discarded because of interference by pyridine carboxylic acids~and their derivatives were again considered. However, the amounts of iproniazid likely to be ^resent in body fluids proved too slight to be detected by such methods. Consequently, only the spectrophotometric method was available and the general procedure was as follows.
An aqueous solution of Iproniazid was prepared by placing
10 mg. iproniazid in a 100 ml. volumetric flask and diluting to volume with distilled water. Solutions containing 2.5, 5,
10, 15, 20, 25 gamma of Iproniazid were next prepared by 25 transferring 2.5, 5, 10, 1 5 , 20, 25 ml. respectively of the stock solution to 100 ml. volumetric flasks and diluting to volume with distilled water. A part of each of these diluted solutions was kept for use as a standard later. One ml. of each solution was transferred to bottles containing 1 ml. of ij. per cent ammonium hydroxide and 35 ml. chloroform, and the mixture shaken mechanically for 20 minutes. Then the con tents of the bottles were centrifuged at 1500 times gravity for 10 minutes after which the aqueous supernatant fluid was discarded. Twenty-five ml. of respective chloroform frac tions were next transferred to bottles containing 5 ml. of a
1 normal hydrochloric acid solution and the mixture shaken mechanically for 20 minutes followed by centrifugation for 5 minutes. After centrifugation, the aqueous layers were poured into absorption cells and the absorbance of the solu tions compared with the absorbance of the corresponding standard solutions. The average values of 10 determinations for each standard solution are listed in Table 7.
Since these results indicate an apparent recovery of approximately 80 per cent of the dissolved iproniazid, it was decided to treat the standard in the same fashion as the unknown in making future determinations in order to compen sate for losses due to solubility of the iproniazid. 26
Table 7
The Determination of Iproniazid In Aqueous Solutions Iproniazid In Standard Iproniazid Pound Solutions {Gamma) (Gamma) 2.5 2.1 5.0 h.2 10.0 B.k 15.0 12.5 20.0 16.7 25.0 20.9
Table 8 shows the values obtained for a series of Ipron iazid solutions of known concentrations when compared with an iproniazid standard treated In the same manner as the solu tions of known concentrations.
Table 8
The Amount of Added Iproniazid Pound in 1 ml. Solution When the Iproniazid Standard Was Extracted In the Same Manner Gamma Iproniazid Added Gamma Iproniazid Pound To 1 ml. Water In 1 ml. Aqueous Solution 2.0 1.9 3.0 3.0 5.0 5.1 8.0 8.1 10.0 10.0 12.0 11.9 15.0 l)+.8
E. Quantitative Estimation of Iproniazid Added to Fooled
Serum
In the next phase of the investigation, measured amounts of iproniazid were added to pooled human serum and quantita tive estimations made according to the procedure just outlined. 27 However, before such a procedure could be adopted, It was necessary to determine whether or not substances were norm ally present In serum which would show absorbance at 268
Therefore samples of normal serum were extracted and examined at 268 m . Table 9 shows the results obtained when serum samples from ten different individuals were so tested. A small but rather constant absorbance was present correspond ing to approximately 0.1* gamma iproniazid.
Next, estimations of iproniazid added to serum were made.
For this nurpose a solution of 25> gamma iproniazid per ml. serum was prepared and 1 ml, aliquots of this solution were extracted with chloroform and 1+ per cent ammonium hydroxide.
Table 9
Absorbance At 268 m Shown by 1 ml. Samples of Sera Obtained From Ten Normal Persons Sample Number Absorbance Produced 1 0.007 2 0.007 3 0.007 k 0.006 5 0.007 6 0.006 7 0.007 8 0.007 9 0.007 10 0.007 Average 0.007
After shaking 20 minutes the mixture was centrifuged at 1^00 times gravity for 10 minutes after which the chloroform fraction containing the iproniazid was extracted with one 28
normal hydrochloric acid solution. The absorbance at 268 m
was then measured to determine the amount of iproniazid
nresent. Table 10 shows the values obtained.
Table 10
Amount of Iproniazid Recovered from 1 ml. Serum Samples Containing 25 Gamma Iproniazid_____ Sample Number Gamma Iproniazid Recovered 1 14.0 2 14.3 3 14.1 4 13.8
It may be readily seen that these quantities of ipro
niazid are lesa than those obtained when an aqueous solution
of the same concentration was similarly treated (Table 7,
page 26). Further experiments in which the extraction time was varied indicated that it was necessary to shake the serum with ammonium hydroxide and chloroform for 30 minutes to ob
tain maximum extraction of tbe iproniazid (Table 11).
Table 11
Effect of Time of Extraction With Chloroform On Iproniazid Recovery From 1 ml. Serum ______Containing 25 Gamma Per ml.______Extraction Time Gemma Iproniazid in Minutes______Recovered 15 12.8 20 14.2 2f> 16.2 30 17.5 35 17.4 5.0 16.9 45 17.1 60 17.0 29
Since the amount of iproniazid recovered still fluctu
ated to some extent, it was decided to add ammonium chloride
or ammonium sulfate to create a salting out effect. Added
ammonium salts should stabilize this extraction by competing with the iproniazid for the aqueous solvent. Ammonium sul
fate and ammonium chloride were added to respective serum samples containing iproniazid, followed by ammonium hydroxide
and chloroform and the extraction procedure performed as before. It was found that the addition of an ammonium salt produced a constant recovery of inroniazid from the senna
(Table 12).
Table 12
Stabilization of the Recovery of Iproniazid Prom Serum by the Addition of Ammonium Salts Gamma Iproniazid Gamma Iproniazid Gamma Iproniazid Recovered Without Recovered After Recovered After Addition of Addition of 3 Grams Addition of 3 Grami Ammonium Salt Ammonium Chloride Ammonium Sulfate 17.3 17. h 17.5 17.6 17. £ 17.5 17.2 17.5 17-1|. 17-14- 17.5 1 7 4 17. k 17.5 17.6 17.1 17.5 17.5
Varying amounts of ammonium chloride were added to a aeries of samples in order to determine the minimum amount of ammonium chloride necessary to produce a constant recovery of iproniazid from the serum (Table 13)* This amount was found to be approximately two grams. 30
Table 1 3
Effect of the Addition of Various Quantities of Ammonium Chloride on the Chloroform Extraction of Iproniazid Prom 1 ml. Serum Containing 25 Gramma per ml. Grams of Ammonium Gamma Iproniazid Chloride Added Recovered 0.5 17.1 1.0 17.0 1.5 17.2 2.0 17.5 2.5 17 J+ 3.0 17.5 3.5 17.5 lf.0 17.5
Even when the recovery of iproniazid from serum did not
fluctuate, the amount recovered was less than that recovered
from aqueous solutions to which corresponding concentrations
of iproniazid had been added. To determine if this recovery,
approximately 70 per cent, is the same for other amounts of
iproniazid added to serum, solutions of known concentration were prepared and analyzed by this method. The results are
shown in Table lif.
Table Ilf
Determination of Iproniazid in 1 ml. Pooled Serum Gamma Iproniazid Added Gamma Iproniazid Recovered Per ml. Serum______Per ml^. Serum ___ 1.25 0.9 2.50 1.8 3.75 2.6 5.00 3.5
Since only approximately 70 per cent of the iproniazid added to serum could be recovered, it was decided to add 31 iproniazid to the serum for use as the standard and to
subject the standard so nrepared to the same procedure as an unknown. This is essentially the same type of procedure
followed in the determination of iproniazid in aqueous solu
tion where the standard was treated in the same manner as the unknown.
Accordingly a stock solution of iproniazid was prepared by transferring 50 mg. iproniazid to a 1000 ml. volumetric flask and diluting to volume with pooled serum to produce a standard solution containing 50 gamma iproniazid per ml.
Working standards were prepared by diluting 5, 10, 15 and 20 ml. portions respectively of stock solution to a volume of
200 ml. with pooled serum. This resulted in standards con taining 1 .25, 2.50, 3.75, and 5-0 gamma iproniazid per ml.
Table 15 shows the values obtained for a series of sera samples to which known amounts of iproniazid had been added when compared with a standard of iproniazid dissolved in serum and treated in the same manner as the specimens under investigation. 32 Table
The Amount of Added Iproniazid Pound in 1 ml. Serum When Compared With a Standard Dissolved in Serum Gamma Iproniazid Added Gamma Iproniazid Pound to 1 ml. Serum in 1 ml. Serum 2.0 2.0 3.0 2.9 It-.O U.o 5.0 5.0 7.0 6.9 9.0 8.9 10.0 10.0
To find the smallest quantity of iproniazid that could be determined in serum by the proposed method, solutions containing 0.5, 0.75, 1.0, 1.25 and 1.5 gamma iproniazid per ml. serum were prepared. Ten determinations of the ipro niazid content of each of these solutions were made using 1 ml. of solution per determination. Table 16 shows the mini mum amount determined accurately was 1.0 gamma per ml. serum.
Table 16
Minimum Amount Iproniazid That Can Be Determined Accurately In 1 ml. Serum Gamma Iproniazid Average Gamma Iproniazid Added to 1 ml. Pound In 1 ml. Serum Serum o.5o 0.2 0.75 0.6 1.00 1.0 1.25 1.25 1.50 1.50
P. Quantitative Estimation of Iproniazid Added to Urine
Before attempting the devise a method for the estimation of iproniazid in urine, the extraction procedures previously 33 developed for the isolation of iproniazid from water and from
serum were employed to determine whether other substances having an absorption peak at 268 m p would be thus extracted
from urine. Five ml. samples of freshly voided urine when so
treated showed absorbance as given in Table 17.
Table 17
Absorbance at 268 my Produced by Extracts From Normal Urine Treated According to the Procedure for the Isolation of Iproniazid Volume Absorbance Absorbance Produced by Extraction Urine Produced by of 5 ml. Aqueous Solution Contain Used in ml. 5 ml. Urine ing 1 gamma Iproniazid Per ml. _ 57228 07l20 5 0.287 0.120 5 0.288 0.120 5 0.288 0.120
Only when the concentration of ammonium hydroxide added to urine was reduced below 2 per cent (Table 18), was there a decrease in the absorbance of the material extracted accord ing to the procedure for iproniazid. However, a concentration of at least 2 per cent ammonium hydroxide had previously been shown necessary for optimum extraction (Table 2, page 21).
Table 18
Effects of Various Concentrations of Ammonium Hydroxide Added to Urine Before Extraction With Chloroform______Concentration of Ammonium Absorbance at 268 vyy Produced Hydroxide in Per Cent______By Extract of $ ml. Urine 1 0.199 2 0.288 3 0.287 k 0.288 5 0.288 3k Likewise varying the amount of added ammonium chloride did not cause any significant change in the absorbance at
268 mp (Table 19).
Table 19
Effect of Various Amounts of Ammonium Chloride Added to Urine Before Extraction With Chloroform on the Absorbance at 268 ryj
Grams Ammonium Chloride Absorbance Produced By Added to 5 ml. Urine Extract of 5 ml. Urine
1.0 0.281 1.5 0.279 2.0 0.288 2.5 0.268 3.0 0.288 3.5 0.287 U.o 0.287
However when specimens of freshly voided urine from 57 individuals were extracted according to the procedure for iproniazid the absorbance was found to be essentially the same in each instance (Table 20). 35 Table 20
Absorbance at 268 iry/ Produced by 5 ml. Aliquots of 24 Hour Urine Prom 57 Individuals
Sample 2k Hour Urine Absorbance Produced by Number Volume (ml.) Extract of 5 ml. Urine
1 i5oo 0.267 2 960 0.287 3 2690 0.279 k 1200 0.282 5 1153 0.290 6 S70 0.293 7 2255 0.291 8 2300 0.280 9 860 0.290 10 1800 0.289 11 1970 0.289 12 1000 0.288 13 565 0.290 lit- 800 0.285 15 485 0.298 16 700 0.294 1I 2480 0.283 18 890 0.286 19 1450 0.289 20 990 0.284 21 750 0.291 22 - * 1440 0.284 23 540 0.281 21* 1345 0.287 25 1505 0.288 26 1460 0.286 2l 1415 0.287 28 1165 0.290 29 2240 0.289 30 1385 0.286 31 850 0.283 32 500 0.291 33 950 0.289 34 1320 0.290 35 1670 0.284 36 1290 0.286 37 1220 0.288 38 1130 0.286 39 450 0.299 40 2700 0.285 36 Table 20 (Continued)
Sample 24 Hour Urine Absorbance Produced by Number Volume (ml.) Extract of 5 ml. Urine
41 1350 0.289 42 1220 0.288 43 1130 0.289 1010 0.288 s 1460 0.289 1U20 0.287 i+7 1280 0.289 48 1100 0.288 660 0.286 % 1510 0.287 51 1250 0.287 52 2500 0.284 $; 1200 0.293 1200 0.267 I! 1435 0.281 56 690 0.290 57 1700 0.296 Average 0.288
A concentration of 25 gamma iproniazid per ml. was next added to a urine specimen and 10 separate determinations of iproniazid made with 5 ml* aliquots. Iproniazid values, listed in Table 21, were obtained by subtracting the absorb ance of the extracted urine itself from the absorbance of extracted urine to which the iproniazid had been added.
These values were then compared with the amount of iproniazid which had been added to the urine specimen. It is shown in
Table 21, that the amount of Iproniazid found in the urine was less than that added* 37 Table 21
Comparison of the Amounts of Iproniazid Pound In 5 ml. Urine With That Added
Gamma Iproniazid Gamma Iproniazid Per Ml. Added Per Ml. Pound in Extracts Prom 5 ml. Urine
25.0 17.1* 25.0 17 .It 25.0 17-3 25.0 17.1* 25.0 17.5 25.0 17.1* 25.0 17.1* 25.0 17.1* 25.0 17.1* 25.0 17.3
Solutions containing known amounts of iproniazid were prepared, extracted and analyzed by the proposed method to find out if the percentage of recovery was constant with various concentrations of iproniazid in urine. The results are listed in Table 22.
Table 22
The Amount of Added Iproniazid Pound In 5 ml. Urine Gamma Iproniazid Added Gamma Iproniazid Pound to 5 ml. Urine In 5 ml. Urine 25 17.5 50 35.0 75 51*. 5 100 70.5 125 87.0 i5o 105.5
Since only about 70 per cent of the iproniazid added to urine could be found (the same per cent as was found in serum 38
to which iproniazid had been added), it was decided to use
urine as the solvent for the iproniazid standard and to sub
ject the standard to the same extraction procedure as the
urine in which the amount of iproniazid was to be determined
Accordingly, standards for the determination of ipronia
zid in urine were prepared as follows: 100 mg. iproniazid
were dissolved in sufficient urine to make 200 ml. of solu
tion. One ml. of this stock standard contained 500 gamma
iproniazid in urine. Working standards were prepared by
diluting 5, 10, 15, 20, 25, 30 ml. portions of the concen
trated standard solution to 500 ml. with urine. The result
ing standards thus contained 5, 10, 15, 20, 25, and 30 gamma
iproniazid per ml.
Table 23 shows the amount of added iproniazid found in
urine specimens when the standard used for comparison con
sisted of iproniazid dissolved in urine instead of water.
Table 23 The Amount of Added Iproniazid Pound in 5 ml. Urine Specimens When the Standard Consisted of Iproniazid Dissolved in Urine Gamma Iproniazid Added Gamma Iproniazid Found To 5 ml. Urine In 5 i'll. Urine 20 19.6 40 39.4 60 58.7 80 80.1 100 100.2 120 118.9 lil-0 139.0 160 157.3 39
Isoniazid if present as a metabolite of iproniazid would
also be extracted by chloroform. Since the absorbance pattern
of isoniazid is very similar to that of iproniazid, any iso niazid present would be determined as iproniazid by this method. This would, of course, give high iproniazid values.
Therefore, attempts were made to determine isoniazid in the urine of a rabbit thc'.t had received 12 mg. iproniazid phos phate for five days. When the procedure of Feigl (2 9 ) for the determination of iproniazid was followed the presence of the drug was not detectable.
Or. Procedure Finally Adopted for the Quantitative Deter
mination of Iproniazid in Blood and Urine
1. Reagents
(a) Ammonium Chloride, granular reagent grade,
Allied Chemical and Dye Corporation
(b) Ammonium hydroxide, ^ per cent, I4.35? ml. Dupont
reagent grade ammonium hydroxide diluted to
a 2 liter volume with distilled water
(c) Chloroform, analytical reagent grade,
Mallinckrodt.
(d) Hydrochloric acid, 1 N l6f? ml. Dupont reagent
grade hydrochloride acid diluted to a 2 liter
volume with distilled water Procedure
One ml. serum or 5 ml. urine were transferred
to an 8 ounce wlde-mouth bottle containing approxi mately 2 grams ammonium chloride. One ml. of Ij. per
cent ammonium hydroxide and 3$ ml. chloroform were
next added. The top of the bottle was then covered
with a 3 by 3 inch square of aluminum foil and
closed tightly with a screw cap. The bottle was
shaken mechanically for 30 minutes in a Kahn type
shaker. Then the contents of the bottle were
transferred to a 50 ml. glass, round bottom centri
fuge tube and centrifuged at 1^00 times gravity for
10 minutes.
Three distinct layers became evident* The upper layer was a mixture of serum and ammonium hydroxide-ammonium chloride solution; the middle layer was comprised of precipitated protein, while the lower layer consisted of chloroform solution.
Some undissolved ammonium chloride remained in the bottom of the centrifuge tube.
After the aqueous layer had been removed by aspiration with a capillary pipette, a 2^ ml. pipette was introduced under the protein layer in order to withdraw samples of the chloroform ex
tract. This was best accomplished by tipping the centrifuge tube approximately degrees and tapping the side of the tube lightly with the finger. This caused the protein layer to float to one side of the tube so thot the pipette could be introduced directly into the chloroform layer.
A 2£ ml. aliquot of the chloroform extract was thus transferred to a clean 8 ounce wlde-mouth bottle to which 5 ml. of a 1 N solution of hydro chloric acid had been added previously. The top of the bottle was again covered with a 3 by 3 inch square of aluminum foil and closed with a screw cap. After the bottle had been shaken mechanically on a Kahn type shaker for 20 minutes, the contents of the bottle were again transferred to a round bottom, glass centrifuge tube and centrifuged at 1^00 times gravity for $ minutes.
Two distinct layers became evident. The upper layer consisted of the aqueous hydrochloric acid solution which contained the iproniazid while the lower layer was chloroform. The aqueous solu tion containing the iproniazid was then transferred to a Beckman silica absorption cell by means of an aspirator. The absorption of the solution at 268
151y was determined using water as the blank, which had been treated in the same manner as the sample. k2 A Beckman model DU spectrophotometer was used to
measure the ultraviolet absorption of the solution*
■E*10 Quantitative Estimation of Iproniazid in Rabbit
Serum Following Oral Adminls tratlon
Before attempting to determine iproniazid in the blood of patients, estimations were made using serum from rabbits given the drug* Since iproniazid is administered orally to people, a similar route was selected for the rabbits.
Iproniazid mixed with food may be fed to a rabbit or a solution of the drug may be administered by a stomach tube.
The disadvantages of feeding the drug in the food are (1) that the quantities of food eaten may vary from day to day and (2) the animal may detect the presence of the added substance in the food and refuse to eat the usual quantity.
Administration of a solution of iproniazid by a stomach tube has the advantage that the amount of the drug received can be controlled since rabbits cannot vomit.
A Marsilid tablet for oral use contains an excipient which is Insoluble in water* Therefore the excipient was removed before attempting to administer a solution of the drug to rabbits by stomach tube*
Four So mg* Marsilid phosphate tablets each containing
32*5 mg* iproniazid were powdered and dissolved in 20 ml. of distilled water* This suspension was quantitatively transftrred U3 to a 50 ml. centrifuge tube and after centrifugation the
yellow supernatant liquid was decanted into a 100 ml. volu metric flask. The residue was resuspended in l£ ml. of
distilled water and centrifuged after which the supernatant
liquid was added to the solution in the volumetric flask.
This washing of the residue was repeated twice and the resi
due discarded. The combined aqueous extracts were diluted to a 100 ml. with distilled water and the resulting solution
contained 1.3 mg. iproniazid phosphate.per 1 ml.
The recommended daily dose of Marsilid phosphate for human beings is $0 to 15?0 mg. This is approximately 1 to 2 mg. per kg. of body weight. For this experiment comparable doses were given to rabbits.
A Davol Robinson #10 France soft rubber catheter, pre viously chilled in ice water, was introduced into the stomach of a 2.6 kg. rabbit with the aid of a little glycerine, a hypodermic syringe attached and 1.3 ml. of iproniazid phos phate solution then administered. This was equivalent to 1 mg. Marsilid phosphate per kg. of body weight. Two hours after the administration of the drug, 6 ml. of blood were withdrawn by cardiac puncture and the serum prepared. -Dupli cate samples consisting of 1 ml. of serum were extracted by the proposed procedure for iproniazid and the absorbance at
266 m measured. The absorbance was almost the same as that produced by the control samples of other rabbit serum (Table 21j.), Table 2k
Absorbance at 268 mJJ Produced by Extracts Prepared Proan the Serum of a Rabbit According to the Procedure For the Isolation of Iproniazid Absorbance Produced by 1 Absorbance Produced by the Extract ml. Normal Rabbit Serum From 1 ml. Rabbit Serum Drawn 2 Hours After the Administration of 0.6^ mg. Iproniazid phosphate per kg, body weight 0.007 0.009 0.007 0.010
Since the difference between the absorbance produced by
the extract from the serum and by that produced from the
serum after the administration of iproniazid phosphete was
so small when 1 ml. samples of serum were used, It was
decided to increase the sample size to 2 ml. It was hoped
that there might be appreciable differences in the amounts
of absorbance in this volume. The extraction of the drug
from serum by the described method was not affected by In
creasing the volume from one to two ml. as was shown by
analysis of one and two ml. samples of serum each containing
5 gamma iproniazid.
Accordingly, 1.8 mg. Iproniazid was administered to a
second rabbit weighing 2.7 kg. and 2 hours later 10 ml. blood were withdrawn by cardiac puncture. Duplicate determinations
of iproniazid in the serum were then made according to the
fonaer procedure except that 2 ml. samples of serum were used. The absorbance and concentration of iproniazid found
in these samples are shown in Table 25# Table 25
Absorbance and Amount of Iproniazid in the Serum of a Rabbit 2 Hours After Receiving 1.8 mg. Iproniazid
Absorbance Produced by Gamma Iproniazid In Sample Extract of 2 ml. Serum 2 2 ml. of Serum 2 Hr s. Number Hrs. After Administration After Administration of 1.8 mg. Iproniazid Of 1.8 mg. Iproniazid 1 0 .021* 1.0 2 0.026 1.1
Since the blood of a rabbit comprises 1/llth to l/l2th of the total body weight, a 2.7 kg. rabbit could be expected
to have from 225 to 21*5 grams or approximately 210 to 230 ml. of blood. A concentration of 0.5 gamma iproniazid per ml. serum represents about 0.25 gamma per ml. in whole blood and therefore a total amount not over 58 gamma was present in the blood of the entire 2.7 kg. rabbit 2 hours after the drug was administered.
To determine when the maximum concentration of ipronia zid was present in the serum, samples of blood were obtained at various time Intervals after the administration of the drug. Since the amount of blood that can be withdrawn from a rabbit without causing injury is relatively small, several rabbits were used for this experiment. Blood was drawn in
10 ml. amounts every 4 to 7 days from the same animal. One and three tenths mg. iproniazid per kg. body weight was U6
administered orally and a blood sample withdrawn by cardiac
puncture at the time interval indicated in Table 26. Seven
rabbits were used and a different time interval was selected
in each instance. An eighth rabbit receiving water instead
of iproniazid served as the control. After appropriate
recovery periods for the rabbits, this experiment was re
peated twice. The results of the sera analysis shown in
Table 26 are average values obtained from the three experi ments .
Similar series of determinations were made after the
administration of 0.6$ mg. iproniazid and 2.6 mg. iproniazid
per kg. body weight. These values are also included in
Table 26.
Table 26
Iproniazid Content of Kabbit Serum Drawn at Various Intervals After Oral Administration of Iproniazid
Interval Between Iproniazid Content of Serum in Gamma per mL. Administration of 0.6$ mg. per 1.3 mg. per 2.6 mg. per Iproniazid and kg. Body k g . Body kg. Body Withdrawal of Weight Weight Weight Blood Sample Administered Administered Administered 1/2 hr. 1.1 1.1* 1.5 1 hr. 1.1 1.7 1.8 1-1/2 hr. 0.8 1.5 2.0 2 hr. 0.6 1.2 1.7 h hr. 0.$ 1.1 1.1* 8 hr. 0.0 0.7 1.0 2l\. hr. 0.0 o.$ 0.$ Table 26 shows that the maximum concentration of Ipron
iazid in the blood was reached within 1-1/2 hours after the administration of the Marsilid.
It was decided to study the effect of repeated doses of Marsilid on the serum concentration of iproniazid. A dose of 2.0 mg. iproniazid phosphate (1.3 mg. iproniazid) per kg. of body weight was administered daily to a series of seven rabbits in 1 mg. doses at 12 hour intervals. Twenty- four hours after the first dose, a 10 ml. sample of blood was withdrawn from the first rabbit and the concentration of iproniazid in the serum determined. Each day a different rabbit was bled and the iproniazid content of the serum de termined. Seven days after the original blood sample was withdrawn, a second sample was obtained from the same rabbit and corresponding samples drawn from the remainder of the rabbits from the ninth to the fourteenth days respectively.
On the fourteenth day the administration of the drug was stopped, but additional samples of blood were drawn daily until a third sample had been obtained from each rabbit after a second seven day interval. The concentration of iproniazid in the serum was determined. The results of these experi ments (Table 27) indicate that a serum level of 1.7 gamma per ml. was maintained during the entire 2 week period.
When the administration of the drug was stopped, the serum level decreased gradually over a 4 day period. kQ Table 27
Concentration of Iproniazid in Rabbit Serum Following The Daily Administration of 2.0 mg. Iproniazid Phosphate Per kg. Body Weight Rabbit Duration of Experi Gamma Iproniazid Found Number ment in Days In 1 ml. Serum 1 1 1.7 2 2 1.6 3 3 1.7 k k 1 *I 5 $ 1.8 6 6 1.7 7 7 1.7 1 8 1.7 2 9 1.7 3 10 1.6 k 11 1 *I 5 12 1.8 6 13 1.7 7 ih 1.7 Administration of Drug Discontinued 1 1$ 1.7 2 16 l.k 3 17 1.0 4 18 0.7 S 19 0.0 6 20 0.0 7 21 0.0
Similar experiment were conducted in which Ip.0 mg. iproniazid phosphate (2.6 mg, iproniazid) per kg. body weight were administered daily to seven rabbits in divided doses of 2.0 mg. per kg. Blood samples obtained in exactly the same manner as in the preceding experiment were analyzed for iproniazid content (Table 28). The results indicate that a serum concentration of 1.9 gamma iproniazid per ml. was maintained during period of drug administration and that the decrease in the serum content of the drug was again k9 gradual for a 5 -day period after the administration of the drug was discontinued.
Table 28
Concentration of Iproniazid in Rabbit Serum Following The Daily Administration of J4..0 mg. Iproniazid Phosphate Per kg. Body Weight Rabbit Duration of Experi Gamma Iproniazid Foun< Number ment in Days In 1 ml. Serum 1 1 1.9 2 2 1.9 3 3 2.0 k b 1.9 5 5 1.9 6 6 1.9 7 7 1.9 1 8 1.8 2 9 2.0 3 10 1.9 k 11 1.9 5 12 1.9 6 13 2.0 7 14 1.9 Administration of Drug Discontinued 1 15 1.8 2 16 1.6 3 1Z 1.3 k 18 0.9 5 19 o.5 6 20 0.5 7 21 0.0
I. Determination of the Amount of Iproniazid Excreted In
the Urine
The concentration of iproniazid in the serum accounts for only a small portion of the drug administered as was shown in the previous experiments. Since almost all drugs are excreted to some extent in the urinef it was decided to 50 determine whether iproniazid was being excreted in urine and
if so to what extent.
Rabbits were again employed for these studies. Twenty-
four hour urine samples were collected separately for three
days from two rabbits receiving no medication. Aliquots of
the six urine samples, which served as controls, were ex
tracted and the absorbance of each sample determined at
268 ny/. All six samples showed about the same absorbance,
which was practically the same as that previously found in
human urine. Therefore it was decided to follow the pro
cedure established for the determination of iproniazid in
human urine for this nart of the investigation..
Two rabbits were each given 2,0 rag. iproniazid phosnhate
per kg. of body weight daily for a 14 day interval and indi
vidual 24 hour urine samples collected during the period of
the drug administration and for seven additional days.
Aliquots of the daily urine samples of each rabbit were ex
tracted and analyzed in duplicate. The results are shown in
Table 29.
The average daily excretion of iproniazid over the 14
day period of the drug administration was 1.4 mg. with a
range of 0.9 to 1.6 mg. The iproniazid content of the 24 hour urine specimens remained about the same for three days
after administration of the drug was stopped, but the drug
was no longer detectable after the fifth or sixth day. 51 Table 29
Amounts of Iproniazid Found In 21+ Hour Urine Specimens Obtained From Two Rabbits That Had Received Daily Oral Doses of 2*0 mg. Iproniazid Phosphate Per kg. Body Weight Interval (in days) During Total mg. Iproniazid Found In Which the Urine Specimen 2 k Hour Urine Specimens Was Obtained Rabbit A Rabbit B (Weight (Weight 2.1+ kg, ) 2.7 kg.) 1 0.9 0.9 2 1.3 1.5 3 1.5 1.6 4 1.6 1.5 5 l.i+ 1.6 6 1.5 1.4 7 1.5 1.4 8 1.4 1.4 9 1.5 1.5 10 1.5 1.5 11 1.5 1.5 12 1.5 1.4 X? 1.4 1.5 I k 1.5 1.5 Average 1.4 1.4 Administration of Drug Stopped 15 1.5 1.5 16 1.5 1.5 17 1.5 1.3 18 1.2 1.4 19 0.9 0.6 20 o.5 0.0 21 0.0 o.o Total Amount Recovered 27.1 26.7 Total Amount Admini- stered 43.7 49.1 Per Cent Recovered 62.1+ 54.3
When this experiment was repeated after doubling the
dally dose of iproniazid, the daily excretion of Iproniazid
varied from 1.6 to 3.6 mg. with an average value of 3*1 m g. 52
(Table 30I Iproniazid could not be detected in the urine
beyond the sixth day after the administration was stopped.
Table 30
Amounts of Iproniazid Pound In 21; Hour Urine Specimens Obtained Prom Two Rabbits That Had Received Daily Oral Doses of 2.6 mg. Iproniazid Phosphate Per kg. Body Weight
Interval (in days) During Total mg. Iproniazid Pound In Which the Urine Specimen 21; Hour Urine Specimens Was Obtained Rabbit C Rabbit D (Weight (Weight 2.9 kg.) 2.1 k g . )
1 1.6 1.9 2 3.0 3.2 3 3.3 3.6 h 3.3 3.3 5 3.1 3.3 6 3.3 3.3 7 3.2 3.3 8 3.1 3.5 9 3.3 3.3 10 3.2 3.U 11 3.3 3.2 12 3.3 3.3 13 3.1 3.3 Ik 3.3 3.3
Average 3.1 3.1* Administration of Drug Stopped
l£ 3.2 3.1* 16 3.2 3.3 3.1; 3.1 16 3.0 3.0 19 2 .1; 2.1 20 0.6 0.7 21 0.0 0.0
Total Amount Recovered S9.2 63.8 Total Amount Received 10S.6 112.8 Per Gent Recovered S6.2 S 6 .6 5>3 J. Determination of Iproniazid in Serum and 2k Hour Urine
Specimens From Psychotic Patient3^
Since Marsilid phosphate has been used for the treat ment of mentally depressed patients, it was hoped that many samples of blood and urine from such patients would be avail
able for study. However, some recent toxicity has been attributed to the drug and its use has been restricted. Six
patients at Columbus State Hosnital were still receiving
2% mg. Marsilid phosphate (16.2 mg. iproniazid) twice daily after a period of 5 months. Blood and 2 k hour urine speci mens were obtained from these patients on two occasions l£ days apart. Aliquots of these specimens were analyzed, in duplicate, for inroniazid by the method previously described and the values obtained are shown In Table 31*
Iproniazid was not found in the serum of one of these patients who was known to frequently refuse to take medica tions. The iproniazid content of the serum of the remaining
5 patients ranged from 0.9 to 1.6 gamma per ml. and the 2I4. hour urine content from 5.7 to 13.0 mg.
^•Specimens for these were obtained from patients at the Columbus State Hospital (for the mentally ill) through the courtesy of Dr. Benjamin Kovitz. 5if Table 31
Concentration of Iproniazid in Serum and Urine of 6 Male Mental Patients Receiving £0 mg. Marsilid Phosphate (32 * Ip mg. iproniazid) Daily Results of Two Analysis l£ Days Apart
Serum Concen- Mg. Daily .. tration in Excretion of Pa Age Diagnosis1 Gamma Per ml. Iproniazid tient in Urine 1st 2nd 1st 2nd Anal Anal Anal Anal ysis ysis ysis ysis C.L.2 61 Psychosis with syph ilitic meningo encephalitis 0.0 0.0 «»«»«■
J.L. 61 Psychosis with syph ilitic meningo encephalitis 1.6 1.5 5.7 3.0* R.H. ^3 Psychosis with syph ilitic meningo encephalitis l.£ 1.5 13.0 5.2* N.M. lf2 Psychosis with syph ilitic Meningo encephalitis 1.2 1.2 12.6 11.9 P.M. 50 C.N.S. syphilis, meningo encephalitis 0.9 0.9 5.0* 1 1 .If R.N. 51 Psychosis with syph ilitic meningo encephalitis 0.9 1.0 0 .8* 1 1 .If
1Diagnoses furnished by Dr. Benj. Kovitz of Columbus State Hospital. p Patient refused to collect urine specimen. Does not take medication most of the time.
* Not the entire 21f hour specimen. K. Absorption of Iproniazid From the Gastrointestinal Tract
of a Rat
As shewn previously, when single doses of iproniazid phosphate were administered to rabbits the maximum serum concentration of iproniazid was found between one and two hours later. Thus it would seem that the drug might be rather rapidly absorbed from the gastrointestinal tract.
Accordingly an attempt was made to determine the approximate rate of absorption from the gastrointestinal tract using male white rats as the experimental animals.
To determine the amount of absorbance at 268 mj/, pro duced by the contents of the stomach and small intestine without added iproniazid, two male white rats weighing approx* imately 200 grams each were sacrificed by an overdose of chloroform. The stomach and small intestine were immediately removed and the contents were washed out with 20 ml, dis tilled water so that the final volume was approximately 2f> ml. One ml. of the resulting mixture of aqueous gastroin testinal contents was then extracted and examined at 268 in the usual manner. The absorbance in 1 ml. of such material was found to he 0.011*. which is equivalent to approximately
0.8 gamma iproniazid.
A solution containing 0.98 mg. iproniazid phosphate per ml. was then prepared and 1 ml. portions of this solution administered orally to 10 male white rats by using as the 56 stomach tube the shank of a Becton-Dickinson Frankfeldt
Hemorrhoidal Needle #!*93LRR size - 20 gauge 5/8”, which has had the needle removed.^" At definite time intervals after administration of the drug, pairs of rats were sacrificed and their stomachs and small intestines removed and washed out with 20 ml. distilled water* One ml. portions of the resulting aqueous gastrointestinal contents and washings were extracted and analyzed for iproniazid. The results in
Table 32 indicated that approximately two-thirds of the iproniazid administered had been absorbed in 2 hours.
Table 32
Recovery of Iproniazid Prom Gastrointestinal Tract of Rats Following the Oral Administration of 0.98 mg. Iproniazid Phosphate
Interval in Mg. Iproniazid Mg. Iproniazid Per Cent Minutes Recovered From Absorbed (by Absorbed After Drug Ga s trointes t inal Difference) Administered Contents 15 0.91* 0.01* l*.l 30 0.76 0.22 22.5 60 0.63 0.35 35.7 90 0.1*8 0.50 51.0 120 0.30 0.68 79.lt-
*The author Is indebted to Dr . John W. Nelson, Professor College of Pharmacy, Ohio State University, for suggesting this method for administering the drug. L, Determination of Iproniazid in Body Tissue
Two rabbits were fed 3*2£ rag* iproniazid phosphate per kg. body weight (by stomach tube) daily. After ij. days these
2 rabbits and 2 controls were sacrificed by an overdose of chloroform and the brain, lungs, liver and kidneys were immediately removed and weighed. For the determination of iproniazid these organs (with the exception of the liver where only 20 grams were used) were minced in a Virtis homo- genizer. Homogenates of the organs from the control animals served as standards, after the addition of measured amounts of iproniazid. The homogenates of the tissues from the test animals as well as those used as prepared standards were transferred to respective extraction bottles and about 2 grams ammonium chloride, 1 ml. Jp per cent ammonium hydroxide solution and 85 ml. chloroform were added to each bottle.
After shaking, the mixtures were centrifuged and 50 ml, of each of the chloroform extracts were transferred to other bottles. Following the addition of 10 ml, 1 normal hydro chloric acid solution, the mixtures were treated according to the usual method for determining iproniazid. The con centrations of Iproniazid found in these tissues are shown
In Table 33. 58
Table 33
Weight of Iproniazid Found in Body Tissues of Rabbits Receiving 3-25 mg* Iproniazid Phosphate Per Kg. Body Weight Daily for Four Days
Mg. Iproniazid Found Tissue Weight Rabbit E Rabbit F (Grams) (Weight (Weight 3.0 k g . ) I+.2 kg.)
Brain 8 .I4. 0.00 Brain 9.3 0.00 Liver 85.8 5.35 Liver llj.6.1 9.01
Lung 12.ii. 0.05 Lung 13*0 0.10
Kidney 15.9 0.80 Kidney 23.1 1.28
Total Iproniazid Found in these Tissues 6.2 10.39
Total Iproniazid Administered to Rabbit 39*0 5I+.6
Per Cent Found in These Tissues 15.9 19.0 DISCUSSION
After the Introduction of Marsilid for the treatment of mentally depressed oatients, toxicologists were occasionally asked to determine iproniazid in body fluids obtained from such patients. When a search of the literature failed to disclose an accer>table method for the estimation of ipronia zid the present investigation was begun.
Since the amount of iproniazid administered is small, it was hoped that an Inexpensive and satisfactory colorimetric method suitable for use in the small laboratory might be developed. Several colorimetric methods for the determina tion of nicotinic acid and its derivatives have been developed which, as these studies show, do not distinguish between nicotinic acid and isonicotinic acid. However, this usually presents no problem because nicotinic acid is a normal com ponent of the body while isonicotinic acid is not. Even when isonicotinic acid or one of its derivatives is being admin istered, the amount is small in comparison to the normal concentration of nicotinic acid. In fact, the quantity present in blood was so small that Iproniazid could not be
59 60
detected by the established colorimetric methods for nico
tinic acid and its derivatives.
When no colorimetric methods could be devised, the study was directed toward suectrophotometrie methods of analysis.
The absorption spectrum of isonicotinic acid and of its
derivatives was found to differ sufficiently from that of nicotinic acid and its derivatives to permit the use of the absorption spectrum for distinguishing members of these two series of pyridine carboxylic acids. However, since iso nicotinic acid amide, iproniazid, and isoniazid had approxi mately the same absorption pattern as the parent isonicotinic acid, an attempt was made to isolate iproniazid before measuring the absorption. A study of the solubilities of isonicotinic acid, Isonicotinic acid amide, and iproniazid in various solvents showed that iproniazid in an aqueous amraonia- oal solution could be extracted with chloroform while iso nicotinic acid and its amide could not be extracted. The addition of an ammonium salt to the ammoniacal solution before the addition of chloroform slightly increased the amount of iproniazid recovered and caused the extraction of a constant amount of iproniazid from solutions of known con centration. This procedure originally carried out with 1 ml. aliquots of aqueous solutions of iproniazid in concentrations ranging from 2.£ to 2£ gamma per ml. gave satisfactory results.
However when 1 ml. portions of serum from animals or patients 61 were used in place of the prepared aqueous solution, there was not enough iproniazid present to permit an accurate spectroohotometric measurement. Accordingly 2 ml. portions were used and these gave satisfactory results. However, this presented some difficulty when working with small ani mals where the total blood volume is small.
All of the solutions used for the administration of iproniazid to animals were prepared from Marsilid phosphate tablets so that the experimental animals received iproniazid in the same relative concentrations as human patients being treated with this drug. Since approximately 6£ per cent of the Marsilid phosphate tablet was iproniazid, an individual taking lf>0 mg. Marsilid phosphate daily actually received
97.5 mg. iproniazid.
Only a small portion of the iproniazid administered to animals or humans was found in the blood. In rabbits, the concentration of iproniazid in the serum was only slightly increased when the dose of Iproniazid administered was doubled. The concentration of iproniazid found in the serum of human patient, who had received smaller doses of ipronia zid was slightly less than that found in the rabbit serum.
The dally excretion of iproniazid by rabbits was over I4.0 per cent of the daily dose while humans excreted over 3£> per cent.
The excretion of iproniazid by the rabbit continued for six days after the administration of the drug was discontinued. The absorption of orally administered Marsilid vras next studied in order to determine the per cent of iproniazid absorbed a3 well as the rate of absorption. Rats were used for these experiments because of their convenient size. The stomach and small intestines could be easily removed and the contents fliished out with a small volume of water. The amount of inroniazid found in the gastrointestinal contents and wash ings represented the unabsorbed drug. By subtracting this value from the weight of drug administered, the amount of drug absorbed could be calculated. Iproniazid absorption in rats was 80 per cent complete in two hours while the drug had reached its highest level in the blood after one hour when only 50 per cent of the drug hnd been absorbed.
Approximately 18 per cent of the drug was found in the liver and kidneys. The iproniazid found in the kidney and liver together with that In the serum accounts for most of the drug found In the urine. Furthermore, the iproniazid in these tissues may explain the continued excretion of the drug in the urine for several days after the administration has been discontinued.
It could well be that some or all of the iproniazid is metabolized at the isopropyl group to give rise to a series of oxidation products having the same general absorption pattern as the parent compound. Further research Is needed 63 to settle this point. However, even if such compounds are produced and have been erroneously reported as Iproniazid they can only have had their origin in iproniazid. Thus in cases of toxicity due to iproniazid this method still offers the toxicologist a means for determining the amount of the drug that has been administered. SUMMARY
Attempts were made to develop a procedure for the
determination of iproniazid in body fluids. Various colori metric methods proposed for the determination of pyridine
carboxylic acids and their derivatives were investigated#
One of these methods was the reaction of the pyridinium group with cyanogen bromide followed by coupling with an amine which produced a colored product# Another method was the reaction of the pyridinium group with 2, lj.-dinitro- chlorobenzene to form a substituted pyridinium quaternary salt# The pyridine ring was cleaved by alkaline hydrolysis and the addition"of a dilute acid then produced the colored compound glutaconaldehyde. These procedures did not produce a method which could be used for the determination of ipro niazid in body fluids due to the low concentration of ipronia- zid present#
A spectrophotometrie procedure for the determination of iproniazid was next proposed. At 26f> to 270 rajj , isonico tinic acid and its derivatives produce a band of absorption which is not shown by nicotinic acid and its derivatives. By extracting from a basic solution with chloroform, iproniazid 65 could be separated from isonicotinic acid and isonicotin-
amide. The resulting chloroform solution of ioroniazid was
then extracted with 5 ml* of 1 S hydrochloric acid. The ab
sorbance of the solution resulting from the extraction could
then be related to the concentration of iproniazid in the solution.
Since the amount of added iproniazid found in blood or urine was only 70 ^er cent of the theoretical amount present when aqueous standards were used, known amounts of the drug were added to serum or urine for use in standards and these standards were treated in the same manner as unknowns.
The quantity of iproniazid in the blood of rabbits that had received the drug orally was determined and found to be small. The blood level reached its peak in about 1 to 1-j^ hours after the administration and the concentration amounted to approximately 5 per cent of the total dose.
The urinary excretion of inroniazid by the rabbit amount ed to approximately 50 per cent of the total dose and the drug could still be found in the urine 5 to 6 days after the administration was discontinued.
When the rate of absorption of iproniazid from the in- testional tract of the rat was studied, absorption was found to occur rather rapidly. Approximately 20 per cent of the dose was absorbed in 30 minutes and in 2 hours after admini stration approximately 80 per cent had been absorbed* The blood and urine concentrations of iproniazid of 5 mental patients who had been receiving 25 mg. Marsilid
phosphate tablets twice daily was determined. The blood
concentration here ranged from 0.9 to 1.6 gamma per ml. and the urinary excretion varied from 0.9 to 1.6 mg. per 2\± hours.
Approximately 18 per cent of the drug was found in the liver and kidneys of the rabbit. BIBLIOGRAPHY
1. Pox, H. H., Synthetic tuberculostats: III Isonicotin- aldehyde thiosemicarbazone and some related compounds., J. Org. Chera., 12, 555-562, 1952.
2. Grunberg, E., and Schnitzer, R. J., Studies on the activity of hydrazine derivatives of Isonicotinic acid in the experimental tuberculosis of mice., Quart. Bull. Sea View H0sp., 11, 3-11, 1952.
3. Robitzek, E. H., Selikoff, I. J., and Ornstein, G. G., Chemotherany of human tuberculosis with hydrazine derivatives of isonicotinic acid., 'Quart. Bull. Sea View Hosp., 13, 27-51, 1952. ij.. Pox, H. H., and Gibas, J. T., Synthetic tuberculostats: VII Mono-alkyl derivatives of isonicotinyl hydrazine., J. Org. Chem., 16, 9i|il-1002, 1953*
5. Bosworth, D. M., Wright, H. A., and Fielding, J. W., The treatment of bone and joint tuberculosis: Effect of 1-isonicotinyl - 2-isopropylhydrazine., J. Bone and Joint Surg., 3kA. 761-771, 1952.
6. Bosworth, D. M., Wright, H. A., Fielding, J. W., and Wilson, H. J., The use of iproniazid in the treatment of bone and joint tuberculosis., J. Bone and Joint Surg., £ A , 577-588, 1953.
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8. Crane, G. E., The psychiatric side-effects of iproniazid., Am. J. Psychlat., 112. lj.9i|-501, 1958.
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10. Loomer, H. P., Saunders, J. C., Kline, N. S., A clinical and pharmacodynamic evaluation of iproniazid as a psychic energizer., Psychiat. fies. Her. Wash., 8 , 129-11+1, 1957. 11. Scherbel, A. L., Schuchter, S. L., and Harrison, J. W., Chemotherapy in rheumatoid arthritis: A concept, Cleveland Clin. Quart., 2lj., 109-115, 1957-
12. Zeller, E. A., Barsky, J., Pouts, J. fi. Kirchheiner, W. P., and Van Orden, L. S., Influence of isonico- tinlc acid hydrazide (INH) and 1-isonicotinyl - 2-iso- proryl hydrazides (IIH) on bacterial and mammalian enzymes., Experentia, 819. 31+9, 1952.
13. Zeller, E. A., Barsky, J., Berman, E. fi. and Pouts, J. fi,, Action of isonicotinic acid hydrazides and re lated compounds on enzymes involved in the autonomic nervous system., J. Pharmacol and Exper. Therap., 106. 1+27-1+28, 1952.
llj.. Zeller, E. A., and Barsky, J., In vivo inhibition of liver and brain monoamine oxidase hydrazine., Proc. Soc. Exper. Biol, and Med., 81, 1+59-1+61, 1952.
15. Udenfriend, S., Weissbach, H., and Bogdanski, D, E., Biochemical findings relating to the action of sero tonin, Ann. New Y«**k. Acad. Sci., 6 6 , 602-608, 1957.
16. Pletscher, A., Shore, P. A., and Brodie, B. B., Sero tonin as a mediator of reserpine action in brain., J. Pharmacol, and Exper. Therap., 116. 81;, 1956.
1 7 . Brodie, B. B., Pletscher, A., and Shore, P. A., Poss ible role of serotinin in brain function and in reser pine action., Ann. New York Acad. Sci., 6 6 . 631-61+1, 1957. 18. Pox, H. H., (Hoffman-Lafioche Inc.) U. S, Patent 2,596,069, (May 6 , 1952).
19. Konig, W., Uber eine niue, vom pyridin derivlerende klasse von farbstoffen., J. Pract. Chem., 69. 105-137, 1901;. 20. Vilter, S. P., Spies, T. D., and Mathew, A. P., A Method for the determination of nicotinic acid, nico tinamide and possibly other pyridine-like substances in human urine., J. Biol. Chem., 125. 85-98, 1938. 69
21. Rubin, S. H., Brekter, L., Scheiner, J,, and De Ritter, E., Determination of blood plasma levels of hydrazine derivatives of isonicotinic acid., Dis. of Chest., 21. U39-i|i]-9, 1952.
22. De Ritter, E., Drekter, L., Scheiner, J., and Rubin, S. H., Urinary excretion of hydrazine derivatives of iso nicotinic acid in normal humans. Proc. Soc. Exp. Biol, and Med., 22, 65k-658, 19^2 .
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2I±. Swaminathan, M., A colorimetric method for the estima tion of nicotinic acid in foodstuffs., Nature, lkl. 8 3 0 , 1938.
25. Sweeney, J. P., Report on a chemical method for nico tinic acid., J, Assoc. Official Agr. Chemists., 3k. 380-387, 1951.
26. Zincke, T., Ueber dinitrophenylpyridiniumchlorid and dessen umwandlungsproducte., Ann., 330. 361-371+, 1901+.
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28. Herington, E. P. G., The use of inorganic coraplees in colour reactions of organic compounds. Part I the determination of isonicotinic acid., Analyst, 78 . 171+-176, 1953.
29. Peigl, P., Spot Tests in Organic Analysis. 5th Ed* Elvesier Publishing Co., New York, pp. 1+96-l}.97> 1956. autobiography
I, Carl Edward Moyer, was born in Dayton, Ohio,
December 2I4., 1926. I received my secondary school educa tion in the rublic school of Osborn (now Fairborn), Ohio.
After graduation from high school, I spent twenty months in the military service. I then completed three years of my undergraduate training at the University of Dayton after which I spent another twenty-three months in the military service. I then returned to the University of Dayton, from which I received the Bachelor of Science degree in 1953* 1 received the Master of Science degree in Physiological
Chemistry in 1957* During the years 1955 to 1958 I have been employed by the Department of Physiological Chemistry as an assistant.
70