ANALYTICAL STUDIES IN THE DETEaiON AND ESTIMATION OF COMPOUNDS

IN DRUG RNIMULATIONS

DlSSratTATION SUBMITTEO IN PARTIAL niinLMENT OF THE REQUIREMENTS

FOR THE A W A R Q O F Ttffi DEGREE OF

Utiatttt of ^l^aotfopiip w

lY • ^ ; I :^ V / 1 K

BY

RUMANA PARUQUI

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1994

DS2573

hcJedicated

TO tnij

/(Luntmij & J^apa

SAIDUL ZAFAR QURESHI M.Sc.,Ph.D..C.Chem. MRIC(London)

Professor of Analytical Chemistry

Phone c Off. 0571-25515

es 0571-20724

Department of Chemistry Aligarh Muslim University

Aligarh-202002 (INDIA)

Date

•to WHOM IT MAY CONCERN

This is to certify that the dissertation

entitled, "Analytical Studies in the Detection and

Estimation of Compounds in Drug Formulations" is the

original work of Miss. Rumana Faruqui and

suitable for submission for the award of Degree of

Master of Philosophy in Chemistry.

^ ^ y ^ p u ^

(Dr. (Mrs.) Iqbal Akhtar) (Prof. Sai Zafar Qureshi)

A C K N O W L E D G E M E N T

It gives me an inunense pleasrue to express/ with

great sense of gratitude, my thanks to Prof. Saidul Zafar

Qureshi/ my supervisor, whose able guidance and criticism

has been indispensible to the completion of this work. I

also.express my thanks to Co-supervisor Dr. (Mrs) Iqbal

Akhtar for her feverent supervision to the completion of

this task. I am also grateful to Prof. Nurul Islam,

chairman. Department of Chemistry for research facilities.

I won my particular gratitude to Dr. Nafisur Rahman

and Dr. Mubeen Ahmad Khan for their constant help and

fruitful suggestions.

I acknowledge my debt to my lab colleagues

Irshad Bhai, Ghazia and Sufia Apa for providing me a

considerable insight and cordial atmosphere. I feel very

happy to pay my sincere thanks to my sisters Shimail,

Tazeen and Asmina Apa as well as my freinds Lisa, Uzma

and Ummey Tamim.

An indebtness is owed to my Dada, Amman/ Mamu Sb.

and Mami without whose encouragement, and inspirations

this effort could not have been possible.

I pay my thanks to almighty Allah who led

me to this path of success.

( RUMANA FARUQUI )

CONTENTS

4 . CHAPTER - II

PAGE NO.

1. • LIST OF TABLES i

2. LIST OF FIGURES i i

3. CHAPTER - I

GENERAL INTRODUCTION 1

REFERENCES 18

5PECTR0PH0T0METRIC DETERMINATION OF THYROXINE SODIUM

INTRODUCTION 24

EXPERIMENTAL 26

RESULTS AND DISCUSSION 27

REFERENCES

LIST OF TABLES

PAGE NO.

30 Table 1. Effect of varying volume of HCl

(1.5M) on the formation of thyroxine

sodium complex.

Table 2. Effect of varying volume of NaN02 31

(0.2M) on the formation of

thyroxine sodium complex.

Table 3. Statistical analysis of the proposed 32

method with other spectrophotmetric

emthod.

Table 4. Interference of foreign substance 33

added for the estimation of

thyroxine sodium.

LIST OF FIGURES

PAGE NO,

Figure 1. Absorption spectrum of thyroxine 34

sodium complex at a temperature

ice-cold bath (0'5°C).

Figure 2. Effect of varying volume of HCl 35

(1.5M) on the formation of

thyroxine sodium complex,

temperature ice-cold bath (0-5°C)

Figure 3. Effect of varying volume of NaNO„ 36

(0.2M) on the formation of

thyroxine sodium complex at

temperature ice-cold water

bath (0-5°C)

Figure 4. Calibration curve of thyroxine ^7

sodium complex at 410 nm.

temperature ice-cold

bath (O-S^C).

CHAPTER - I

GENERAL INTRODUCTION

1

Analytical Chemistry is concerned with the

theory and practice of methods used to determine the compo­

sition of matter. The Science of Analysis can be divided

into areas called qualitative analysis and quantitative

analysis. It is concerned with what elements or compounds

are present in the sample. Quantitative anlaysis is

concerned with the determination of how much of particular

substance is present in the sample. The substance

determined, often referred to as the desired constituents

or analyte, may constitute small or large part of the

sample analyzed. A complete analysis actually consist of

five main steps: (1) sampling, that is , selection of

a representative sample of the material to be analyzed; (2)

dissolution of the sample; (3) conversion of anlayte into

a form suitable for measurments; (U) measurement; and

(5) calculation and interpretation of the measurement.

The importance of Analytical Chemistry can be

illustrated by considering its impact on clinical

anlaysis, in pharmaceutical research and quality control,

and in environmental anlaysis.

In the past, medical profession used clinical

results qualitatively and many of their diagnoses were

based on symptoms and/or X-ray examination. Currently,

over one billion laboratory tests are performed in

clinical laboratories per year. The major portion of

these test deal with blood and urine samples and

include the determination of glucos e, urea nitrogen,

protein nitrogen, soduim potassium, calcuim HCo3/H2C03,

uric acid and pH.

In the pharmaceutical industry the quality of

the manufactured drugs in tablet, solution and emulsion

form must be carefully controlled. Slight changes in

composition or in the purity of drug itself can affect

the therapeutic values. In other pharmaceutical studies,

it is necessary. to establish the properties and

therapeutic value of a drug before it is approved

and made available to the public. Establishment of the

permissible level of dosage of a drug requires the

determination of its decomposition product, its toxicity,

and its metabolite products at various stages.

It is an important task for the practicing

analytical chemist to choose an analytical method

which provides the best solution to the problem.

Methods of quantitative testing and quality control of

pharmaceuticals are controlled by FDA, Fedral Drug

Administration.

Pharmaceutical analysis, today, is on the

frontiers of Analytical Chemistry. Analysis in both

bulk and dosage forms is important. The matrix in

many cases may be extremely complex and therefore.

detection, determination and some form of separation is

usually required• Usually methods should be comparable

to or improvements over methods published from

standard (B.P., U.S.P., I.P.) pharmacopeia laboratory.

All the drugs in their nature are divided

into inorganic and organic. Both can be prepared from

natural sources, or artificially i.e. synthetically.

The enormous number of drugs used in medicine can be

classified into two ways.

1, PHARMACOLOGICAL CLASSIFICATION ; In this section,

drugs are divided on the bases of their action on an

organis m (the heart, brain, lymphatic system,

stomach, intestine etc.) Accordingly, drugs are

divided into such groups as narcotic s,, soporifics,

analgesi'cs diuertics and local anaesthetics.

2. CHEMICAL CLASSIFICATION : Here, the drugs are divided

according to their chemical structure and properties

regardless of their pharmacological action. It is

beyond the scope of this work to discuss all the

types of drugs, however, few important class of

compound used as drugs in medicine are discussed

below.

ALKALOIDS : Alkaloids are substances mostly of plants

and rarely of animal origin. In medicine alkaloids

are sucess fully used as drugs in the treatment of

cardiovascular, nervous, alimentary tract and many

other diseases (1) They are organic substances of basic

nature because their molecule always contain nitrogen.

The alkaloids are chiefly tertiary amines, and only

some of them are secondary amines or derivatives of

tetrasubstituted ammonium bases. Such class of durgs

can be grouped into the following heads.

(i) pyridine and piperidine derivatives (labeline

and its companions).

(ii) tropane derivatives (atropine, hyoscyamines,

cocaine etc)

(iii) guinoline derivatives (guinoHnet guidine etc),

(ly) isoguinoline derivatives (opium, alkaloids).

(v) indole derivatives (physostigmine, reserpine etc)

(vi) imidazol derivatives (pilocarbine, omeperazole).

(vii) purine derivatives (caffeine, theobromine,

theophyllin etc.)

ANTIBIOTICS : Antibiotics are very important class of

phrmaceuticals consisting of antibacterials, anti

infectives, antifungals, antiparasitics and

antimicrobials. Antibiotics are organic compounds in

their chemical nature. Antibiotics are chemical

compounds derived from or produced by living

organisms, which are capable, in small concentraions,

of inhibiting the life processes of microorganism. (2).

The chemistry of antibiotics is so varied

that a chemical classification is of little value.

However, some antibiotics are the products of

similar mechanisms in different organisms and that

these structurally similar products may exert their

activities in a similar manner. A number of

antibiotics have in common a macrolide structure,

that is, a large lactone ring. Erythromycin,

oleandomycin are included in this family. Tetracyclin

family represents a group of compounds very closely

related chemically. Streptomycins, Kanamycins,

neomycins, paramomycins and gentamycins are included

into a family of compounds containing closely related

amino sugar moities. The antifungal antibiotics

nystatins and amphotericins are examples of a group

of conjugated polyenes. The pencillins and cephalo

sporins are antibiotics derived from amino acids. A

large number of polypetide compounds that exhibit

antibiotic action include bacitracins, tyrothricins

and polymixins.

ANALGESICS : A drug which brings about insensibility to

pain without loss of consiousness is known as analgesic.

There are numerous drugs which , in addition to

possessing distinctive pharmacologic activities in other

area, may also posses analgetic properties (3). The

analgetic property may have direct or indirect effect.

Such class of drugs are grouped as sedatives (eg.

barbutrates); muscle relaxents (eg, mephenesin,

methocarbamol); tranquilizers (eg. meprobamate);

morphine and related compounds (morphine, hydromorphine

and codeine phosphate/sulphate etc); synthetic

analgesics (pentazocaine, methotrimeprazine, methadone

hydrochloride, nefopam etc. ) ; antipyretic analgesics (

salicylic acid derivatives, arylacetic acid

derivatives, aniline and p-aminophenol derivatives,

pyrazolone and pyrazolidinedione derivatives).

CENTRAL NERVOUS SYSTEM DEPRESSANTS : The agents

that produces depressent effect on the central

nervous system (U) as their principle pharmacological

action, include the general anesthetics, hypnotic

sedatives, skeletal muscle relaxents, tranguilizing

agents and anticonvulsants. The general anesthetics

(eg. ether) and hypnotic sedatives (eg. phenobarbital)

produce generalized or non-selective depression on

C.N.5. function. Many sedatives if given in large doses,

produce anesthesia. Certain. analgesics also produce

depression on central nervous system.

CENTRAL NERVOUS SYTEM STIMULANTS : Central nervous

system is a complex network of subunits which act as

conducting path ways between peripheral receptors and

effectors, enabling man to respond to his environment.

Drugs which have in common the property of

increasing the activities of various portions of the

central nervous system are called (5,6) central

nervous system stimulants (respiratory stimulants and

analeptics). Carbonic acid is a most effective

respiratory stimulant.

Analeptics, agents used to lessen narcosis

brought about by excess of depressant drugs often stimulate

a variety of nerve centres as well as many analeptic

drugs are also pressor durgs because their

stimulation of the vasomoter centre produce an increase

in vasoconstriction.

There are many drugs (cocaine, atropine,

amphetamines, ephidrine etc.) of varying pharmacologic

classes,in addition to, their desired effect elicit a

pronouneed stimulatory effect on central nervous

sytem.

CARDIOVASCULAR AGENTS : Agent used for their action

on the heart or on other parts of the vascular system so

that they modify the total output of the heart or the

distribution of blood to certain parts of the

circulatary system, are called cardiovascular agents

(7), This class include cardiotonic drugs vasodialators,

hypotensive drugs, antihyper cholesterolemic drugs and

sclerosing agents (8). Certain drugs have a direct

8

action on cardiovascular sytem, and in addition, this

calss of drug that affect certain constituents of blood.

The later include anticoagulant drugs, the hypoglycemic

agents the thyroid harmones and the antithyriod drugs.

In this dissertation we have developed a new

method for the determination of thyroxine sodium in drug

formulations, therefore,a more detailed study has been

done on the drug.

Thyroxine (9) is an active substance which is

isolated from thyriod gland (usually the sheep). It

is the tetra - iod - derivatives of amino acid.

^__>-CH2CH(NH2)COOH

V r THROXINE SODIUM

Thyroxine sodium is a c rude s a l t . It has the

several thousand times the activity of a fresh gland.

Def fated thyroid s u b s t a n c e has been used for many

years as a replacement theraphy in thyriod gland

defficiencies (10-12). The efficancy of the t h y r o i d

gland is known to depend on its thyroglobulin content

which is an i o d i n e containing globulin. Kendall in

1916 obtained thyroxine as a crystalline derivative and

found t h a t i t showed as such the same a c t i o n as the

whole thyroid substance. Recent studies show that even

more potent iodine containing harmone exist, which is

known as trii odothyronine. Thyroxine is supposed to be

the storage form of the harmone, while the triodothy-

ronine is the circulatory form. In the blood

thyroxine is more firmly bound to the globulin

fraction than is triodothyronine, which can then enter

the tissue cells.

LEVOTHYROXINE SODIUM U.S.P. : sodium L-3-[4-(4-hydroxy-

3, 5 diodophenoxy) -3, 5-diodophenyl] alanine, sodium

L-3-, 3, 5 5, -tetraiodothyronine. This compound is the

sodium salt of levosomer of thyroxine, which is an

active physiological principle obtained from the

thyriod gland of domesticated animals use for food by

man. It is also prepared synthetically. The salt is

light yellow tasteless odourless powder. It Is

hygroscopic but stable in dry air at room -iemperature.

It is soluble in alkali hydroxides, 1:275 in alcohol

and 1:500 in water to give a pH of ^^ 8.9.

m CH2-CHC00-Na

LEVOTHYROXINE SODIUM

Levothyroxine sodium i s used in r ep lacemen t t h e r a p y of

10

deceased thyroid function (hypothyroidism). In general,

lOOJU^g of levo thyroxine sodium is clinically equivalent

to thyroxine 30-60 mg of thyroid U.S.P.

LIOTHYRONINE SODIUM U.S.P. CYTOMEL Sodium L-3 [A~(

4- hydroxy -3-iodophenoxy) -3,5-diodophenyl ] alanine is

the sodium salt of L-3,3,5, triiodo thyronine. It

occurs as a light tan, odourless, crystalline powder

slighitly soluble in water or alcohol and has a

specific rotation of +18° to +22° in acid (HCl)

alcohol.

- + H2CHNH2-COO.Na

LIOTHYRONINE SODIUM

Liothyronine occurs in vivo together with levothyroxin e

it has the some qualitative activities as thyroxine

but is more active. It is absorbed readily from the

gastrointestinal tract, is cleared rapidly from the

blood stream and is bound more closely to the plasma

proteins than the thyroxine, propbably due to the less

acidic phenolic hydroxyl group.

It is used same as that of levothyroxine,

including treatment of matsbolic in sufficiency, male

infertility and certain avmcoloaical disorder.

11

ANALYSIS OF DRUG : The methods for analyzing medicinal

agents are divided into physical, chemical, physico-chemical

and biological once.

Physical methods of analysis involve the studying

of the physical properties of a substance. They include

the determination of solubility transparency and the

degree of turbidity, colour, density or specific

gravity, metting, boiling and freezing points.

Chemical method used for the analysis of durgs

are based on chemical reactions. They include the

determination of ash content, the hydrogen ion

concentration (pH) of medium and characterstic numerical

indices of oils and fats. Quantitative and qualitative

analysis of durge are generally carried out with

respect to functional groups.

Physcochemical methods are used to study the

physical phenomenon that occur as a result of chemical

reaction. Among the physiocochemical method of analysis,

photometry including photocolorimetry and

spectrophotometry, electrochemical and chromatographic

methods. Recently various kinds of chromatography

(column, paper, thin layer, gas, gas-liquid) and of

photometric methods based on the absorption of light by

the analyte have found there use in pharmaceutical

analysis. NMR, PMR, and mass spectrometry with gas chromatography

12

are the most important powerful analytical methods

used for the analysis of drug.

Biological method of anlaysis characterizes

the pharmaceu tical effect of the drug. Biological

tests are conducted on animals and less frequently

separate isolated organs or part of organs of animals.

The effect of durg is expressed in A.U. (activity units)

and is determined by the comparision with the activity

of a reference substance. The biogical methods are very

sensitive. Alkalo ids, vitamins and antibiotics, still

reveal activity when diluted millions of times.

Photocolorimetric methods of analysis are

based on measuring the absorption of non-monochromatic

light by coloured compounds, in the visible part of

the spectrum. If the analytes are colourless, they are

converted into coloured compounds by reaction with

suitable reagent. In the visual method, called

colorimetry, the intensity of the colour of analyte

solutions is compared to that of standard solutions

if the concentration of substance is known.

In the recent years, pharmacentical analysis has

been chracterized by the use of methods such as flame

photometry and diffrential spectrophotometry in the

qualitative analysis of drugs. These methods enable

one to determine the large amount of individual

components of a mixture because the absorbance of the

13

analyte solution is measured not rela.tive to the

pure solvent (or a solution of reagent), but relative

to a reference solution containing a known amount of

the analyte.

For the determination of specific pencillin in

pharmaceutical products spectrophotometric methods have

been developed. Spectrophotometry has been used for

the determination of amoxycillin (13, lU, 15).

Chromatograhic procedure have appeared for ampicillin,

amoxycillin and pencillin G (16). A diazotization

spectrophotometric method has been used to chracterize

amoxycillin by reaction with aromatic amines and

amino acids in bulk drugs and also in capsules,

tablets and syrups. A polymer immoblized enzyme

optrode for pencillin has been introdused for

determination of pencillin (17).

H' NMR has been applied most frequently to

the analysis of common analgesices (18,19) B- androgenic

blockers (20), and the antineoplastic ziechlorethamine

hydro chloride (21) and carbachol (22). Near IR

analysis has been used to assay tablets containing

acetaminophen in combination with caffeine (23). The

procedure was adopted to cough cold syrups. Sulpuar

based cardiovascular drugs, antiallergy or anti

inflammatory durgs and phenothizine have been

analyzed by 'H^ and C NMR spectroscopy (2^, 25).

14

A variety of cololimetric procedures have been

developed for analgesics, antipyretics and

antiinflammatories. Acetaminophen has been measured by

itself (26, 27) and in conbination with other compounds

such as salicylamide and oxyphenbutazone (28, 29).

Methods have also ben published for aspirin (30, 31)

Novalgin (32, 33) dipyrone (3U), indomethacin (35),

naproxen (36), and oxypheubutazone (37).

Iron reagents have been used to assay the

chlorpheniramine and pheniramine (38), meperidine

(39), nifedipine (40), nitrazepam (41), and nitroxazephine

HCl (42). A simple and sensitive ion-exchange method

has also been published for the detection of

chlorpromazine in human urine (43). Other types of

colorimetric procedures have also been developed for

the analysis of pharmaceuticals. Oureshi and

co-workers have recently developed. s pectrophotometric

methods for the determination of paracetomol (44^ and

acetaminophen (45) .

During the last 5 years a variety of

electroanalytic, chromatographic and spectroscopic assay

have appeared for the selective determination of

vitamin C. Spectroscopic methods are based on the

oxidation of ascorbic acid. A method based on

15

inhibition of peroxides activity has been described

(46). An extraction spectrophotometric procedure have

also been developed for the determination of vitamin C

(1*7) in drug formulations, urine and fruit guices with

potassium iodate.

Calorimetric procedures have been found based on

either the formation of charge transfer complex

between the active and iodine (48, 49), nitrosation of

active (50, 51) or reaction of or, active with a

diazotized reagent (52, 53). Recently a review on the

analysis of pharmaceuticals and related drugs have

been appared (54), representing a survey of

pharmaceutical analysis and related methodology.

The determination of iodin^ted thyronines is

of great importance. As the concentration of iodinated

thyronines in plasma are very low; only trace method

can be applied for the determination. Radioimmunoassay

(54, 55), fluorescence immunoassay (57), enzyme

immunoassay (58) and competitive protein bin-ding assay

(59), are most frequently used for the determination

of thyroxine sodium. A selective determination of

enatiometric thyromines is not possible with these

techniques. The reaction of L-thyroxine antibodies

with D-tbyroxine for a radioommunoassay was found to

be 100^ (60).

16

Recently the determination of D.L-triiodothyronine

and D.L-thyroxine by derivatization liquid chromatography

has been described (61). The D.L-thyronines are

derivatized with tertiarybutyloxy -L- leucine -N- hydroxy

succinimide ester and the resulting diasteromeric peptides

are separated by ion pair chromatorgraphy on reveresed

phase columns. This method was successfully applied to

the control of purity of pharmaceuticals and its

applications in human plasma seemed to be promising.

Chromatographic separations of thyroid barmons can be

carried out using gel chromatography (62, 63),

ion-exchange chromatography (6^, 65), gas chromatography

(6U), high performance liquid chroomatography HPLC (65).

Van der shiji and I, verms et al (66) have

reported the effect of temperature on free thyroxine

measurements and its analytical, and clinal consequences.

Throxine and 3,5,3, - triiodoothyroxine has been

determined by sub and super equivalance isotope dilution

analysis (67) at trace level (160 and 20 mg/ml) and

radioimmunoassay (68), for measurement of free

thyroxine in serum. New homogenious enzyme immunoassay

for the determination of theyroxine and thyroxine uptake

have been developed recently (69). A multicentre evaluation

of a two step enzyme immunoassay (70) of free thyroxine

have also been developed.

17

In this dissertation, we describe the

determination of thyroxine sodium in pure and dosage

forms using well known diazotization reaction. The

method is based on the reaction of amino group with

nitrous acid in an ice-cold medium.

18

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C H A P T E_Jl__- .JJ.

SPECTROPHOTOMETRIC DETERMINATION

OF

THYROXINE SODIUM

24

INTRODUCTION

The human thyroid gland produces L-isomers

of thyroxine sodium and D-isomer is being produced

synthetically. D-thyroxine has been found to reduce

the cholestrol level significantly, by increasing the

rate of degradation and oxidation of cholestrol (1-4).

D-thyroxine has been described to cause the supression

of thyrotropine.

Levothyroxine sodium i.e. thyroxine sodium

(L-3-3', 5-5'-tetraiodothyronine), which is an active

physiologic principle obtained from the thyroid gland of

domesticated animals used for food by man (5). It is- used

in replacement therapy of decreased thyroxine function

(hypothyroidism).

The British pharmacopoeia 1980 estimated (6)

thyroxine sodium by applying the oxygen-flask combustion

for iodine. Several other analytical procedures have

been reported for the determination of thyroxine

sodium, include polarography (7), gas chromatography

(8,9), thin layer chromatography (10,6) and high

performance liquid chromatography (11, 12) and isotope

dilution analysis (13). Radioimmunoassay (14, 15 , 16),

enzyme immunoassay (17,18), colourimetrie (19) and

fluorometric (20) procedure for the determination of

thyroxine sodium have also been developed. An

spectrophotometric method (21) for the determination of

25

thyroxine sodium with p-benzoquinone have been

appeared recently.

In this investigation a spectropbotometric

method for the determination of thyroxine sodium based

on the reaction of nitrous acid with functional group

of thyroxine sodium, in an ice-cold medium (0-5°C)

has been described.

26

EXPERIMENTAL

Reagents and Apparatus : A 1 mg/ml solution of

thyroxine sodium (Glaxo India) was prepared in a

alcohol-dimethyl sulphoxide mixture (50:50, v/v) . A 0.2

M sodium nitrite (freshly prepared daily) and 1.5 M

hydrochloric acid solutions were prepared in high

purity conductivity water. All other chemicals used

were of analytical grade.

A Bausch & Lomb 5pectronic-20 spectrophotometer

was used for absorptiometric measurements.

Procedure : To a precooled solutions of 3.5 ml sodium

nitrite and 1.5 ml HCl in a 10 ml volumetric flasks,

added accurately measured volume of thyroxine sodium

(0.1-1,6 ml) and mixture was then diluted upto the mark

with 50:50 alcohol : DMSO mixture (v/v). The flask were

then kept for 15 minutes in ice-cold bath for the

complete development of yellow colour and absorbances were

read at 410 nm against a reagent blank. Calibration

graph is shown in figure. 3

RESULTS AND DISCUSSION

Nitrous acid has been proved to be useful

reagent for the determination of amines and amino acids.

The reaction is carried out by adding aqueous solution of

sodium nitrite to a cold aqueous solution of amine or

amino acids in dilute mineral acids (22).

Thyroxine sodium with structural formula

sodium-L-3 l^-(U-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenly]

alanine, or sodium L-3, 3', 5,5'-tertraiodothyroxine is the

levoisomer of thyroxine.

LEVOTHYROXINE SODIUM

The reaction of thyroxine sodium with nitrous

acid involves the possible kinetic mechanism of the

following functional groups.

1. Involvement of aliphatic primary amine -NH^ group.

2. Carboxylic group

3. Hydroxy group

4. Iodide group

In the above groups the most effective activation

involves the action of nitrous acid on -NH- group. The

rest of the groups do not have the condusive conditions to

be activated. We therefore decided to discuss a tentative

mechanism of aliphatic primary amines with nitrous acid.

However, the product formed is extremely unstable and there-

28 fore the possibility of such reaction is ruled out. In

another approach the formation of nitrosamine which is

characteristically pale yellow appears to be more positive.

Although it is also unstable but the possibility of making

it stable under experimentalconditions can not be ruled out.

The reaction is quantitative one, involving the

measurement of intensity of yellow colour using alcohol :

DM50 (1:1); DMSO has a boosting effect on increasing colour

intensity. The effect of solvent on equilibrium constant

(K), the heat of formation, and the molar extinction

coefficient ( X ^ ^^s been studied on the formation of

•complexes of nitro compounds. Dimethylsuplphoxide plays

an important role to enhance the colour intensity (23) of

these compounds and the most versatile example of the use

of DMSO, is the detection of nitro compounds, where the

sensitivity of the identification is increased

tremendously (24).

Nitrous acid is unstable and is usually prepared

by mixing a freshly prepared solution of NaNo (sodium

nitrite) with appropriate concentration of hydrochloric o

acid at 0^5C . These conditions provide a source of NO

which is transferred readily to the nucleophilic nitrogen

of amine. _ ^^

HONO -h He " Hp + : N = 0

— N : + : N = 0 » -(Ji^=^N=0 In ' the reaction of primary aliphatic aaine

present in throxine sodium, formation of N-nitrosaaine

takes place as follows.

29

STUDY OF THE PROPOSED EXPERIMENTAL CONDITIONS

Under the proposed experimental conditions 0.6

ml of 1.5 M HCl and 2.0 ml of 0.2M sodium nitrite (Table 1

and 2) was found sufficient to produce high intensity

coloured product for 0.5 ml of drug. The results are

shown in Fig. 2 and 3. Beer's law is obeyed in the

concentration range of 10 ug/mt- 160 ug/ml of thyroxine 3

sodium with apparent molar absorptivity of 3.56 x 10 Z

-1 -1 mol cm

To test the reproducibility of the procedure 10

replicate determinations of 0.5 ml and 1.0 ml thyroxine

sodium were done. The relative standard deviation was

found to be 0. 65h and 0.85% for 0.5 ml and 1.0 ml of

thyroxine sodium respectively. Statistical analysis (Table

3) of the result obtained by proposed method and by other

spectrophotometric method (21) showed no significant

difference between the performance of the two methods.

Interferences of various ingradients used for

the preparation of thyroxine sodium tables were checked.

The results are shown in table 4 and no interference was

found for these ingradients.

The work is under progress and its application

for the analysis of conmercial thyroxine sodium tablets is

under study.

30

TABLE 1. Effect of varying volume of HCl on the formation of

thyroxine sodium complex.

Amount of Amount of Amount of Absorbance at Drug taken HCl (1.5 M) NaNO^ (0.2M) 410 nm

(ml) (ml) ?ml)

0.5 0.2 3.5 0.10

0.5 0.4 3.5 0.18

0.5 0.6 3.5 0.26

0.6 0.8 3.5 0.27

0.5 1.0 3.5 0.27

0.5 1.2 3.5 0.27

0.5 1.4 3.5 0.27

0.5 1.5 3.5 0.27

Temperature ice cold bath (0-5°C).

31

TABLE 2. Effect of varying volume of NaNO on the formation

of thyroxine sodium complex.

Amount of Amount of Amount of Absorbance at drug taken NaNO (0.2M) HCl (1.5M) 410 nm

("^^ (ml) (ml)

0.5 0.5 1.5 0.22

0.5 1.0 1.5 0.24

0.5 1.5 1.5 0.26

0.5 2.0 1.5 0.27

0.5 2.5 1.5 0.27

0.5 3.0 1.5 0.27

0.5 3.5 1.5 0.27

Temperature - ice cold bath (0-5°C).

32

TABLE 3. Statistical analysis of proposed method and other

spectrophotometric method.

Method Beer's law obeyed apparent molar Relati^B S.D. in cone, range absorptivity

3 Proposed 10 - 160 ug/ml 3.56x10 +_0.65',

,--i -1 1 mol cm

3 Reference 40 - 200 ug/ml 2.9x10 +_0.36'

(21) 1 mol'-^cm''''

33

TABLE 4. Interferences of foreign substances for the

estimation of thyroxine sodium (50 ug/ml)

S.NO. Foreign Substance Maximum tolerable amount (mg)

32.1*28

36.432

72.464

68.46

8.01

12.0

32.48

1.

2.

3.

4.

5.

6.

7.

Glucose

Fructose

Lactose

Sucrose

Ca'^

''3

Starch

34

Absorbance o

H CI

o-0) r t a-~ s

o 1 m

0

n •

(n o a M. B a

o o 3

Oi rt-

Ot

r t re a

t1 Ol

c

n

0

o o KM

Sk

o-(n o •1 T3 r t M.

o a

(n •o m 0 r t

B a

o H)

«• a-

o X a (D

35

C o

JO

o

<

U.J

0.2

0.1

-

1 1 1 1 1

-9

I . 1 J 0.2 O.A 0.6 0.8 1.0 1.-2

Volume of HCl

l.A 1.6

FIG. Effect of varying volume of HCl (1.5M)

on the formation of thyroxine sodium

complex, temperature ice-cold bath

(O'S^C).

0.3

36

0;

^ 0.2 o

o

(A

<

0.1

0.5 1.0 1.5 2.0 2.5 3.0 Volume of No N02

3.5

FIG 3 zEffect of varying volume of NaNO (0.2M)

on the formation of thyroxine sodium

complex at temperature ice-cold water

batb (O-S^C).

37

H

n Ql

o o

u

o fs)

1

Absorb o LJ

1

a n c e p j>

1

p t j i

1

o b> 1

o ^

1

IS}

C

a 3 •

r t (D

13 It <1 SI r t S

It

n (0 1 n o )» a

d-Bl r t

1 01 0

n

0-1 Q) f t

O a

0

e h

0 Hi

r t a-*>s h 0 X Kh a 0

(a Q

a c SI

>^ «3

« w

3

o •« -i

- J

o X mm-

3 0)

CO o a M *

C 3

*» o

C7>

o

CD

o

^J^

o o

o

o

o

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