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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23
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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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