The document discusses various analytical reagents and principles used in pharmaceutical analysis, including ninhydrin, para-dimethylaminobenzaldehyde (PDAB), 2,6-dichloroquinone chlorimide (Gibbs reagent), 1,2-napthaquinone-4-sulfonate, and 2,4-dinitrophenylhydrazine (DNPH). Each reagent's chemical principles, applications, and procedures for quantitative estimation of functional groups in drugs are outlined, highlighting their significance in detecting specific compounds and reactions. The document serves as a comprehensive guide for the application of these reagents in different analysis methods.

1. ADVANCED
PHARMACEUTICAL
ANALYSIS
M. Pharm 1st Semester
2019-
19
L. SANATHOIBA SINGHA

2. [1]
CHAPTER-2
Analytical principle, procedure and applications of the following reagents
(a) Ninhydrin:
(refer notes)
(b) 3-Methyl-2-benzthiazoline hydrazone (MBTH):
(refer notes)
(c) Folin-Ciocaltau (FC):
(refer notes)
(d) Para-dimethylaminobenzaldehyde (PDAB):
CHO
N
CH3H3C
Synonyms- Ehrlich’s reagent, p- formyl dimethyl aniline
PRINCIPLE-
The mechanism of aldehydes which condenses with the aromatic amines involves the condensation
of the aldehydes to release the oxygen molecule and then it combines with the amine group to form
the yellow Schiff's base in the presence of acidic medium such as HCl or H2SO4.

3. [2]
R2H2N
R1 CH O R1
H
C O
+ -
+
..
R1
H
C O
NH2R2
R1 C
H
OH
N
H
R2
R1 CH NR2
- H2O
Sciffs base
APPLICATIONS of Para-dimethylaminobenzaldehyde (PDAB)-
 It is used for the determination of urobilinogen in the urine, feces and bile. The urine sample
must be fresh because if the urine sample is left to oxide in air to form urobilin, the reagent
will not detect the urobilinogen.
 It can be used to detect the presence of indole alkaloids.
 It is also used as a stain in TLC.
 It may also be used for determination of hydrazine.
USES-
Nitazoxanide
reduce nitazoxanide
Zn granules with 5N HCl
in Methanol at RT
Prepare 100 mcg/ml of
reduced nitazoxanide
From the above soln
pipette out 0.5-2.5 ml
into 10 ml std vol flask
Add 5 ml of PDAB
reagent & heat 60-70 0
C
for 10 min
Dil upto 10ml with methanol
and measure the absorbance
at 559 nm

4. [3]
S
N
NH C
O
H3COCO
O2N
Zn/ HCl
S
N
NH C
O
H3COCO
H2N
+
CHO
N
H3C CH3
S
N
NH C
O
H3COCO
N
C
H
N
H3C
H3C
(e) Para-dimethylaminocinnamaldehyde (PDAC):
N
CH
H3C
H3C
O
Synonyms- Renz and Loew reagent, 4-Dimethylaminocinnamaldehyde (DMAC)
PRINCIPLE-

5. [4]
REAGENT PREPARATION :
APPLICATIONS of Para-dimethylaminocinnamaldehyde (PDAC) -
 It is used to detect indoles.
 It is used for the rapid identification of bacteria containing tryptophanase enzyme
systems.

6. [5]

7. [6]
(f) 2,6- Dichloroquinone chlorimide:
N
O
Cl Cl
Cl
Synonyms- Gibbs Reagent, 2,6- Dichloroquinone-4-chlorimide
PRINCIPLE-
 When Phenolic compounds reacts with Gibbs reagent, coupling reaction may occur.
 The first step of reaction is formation of the corresponding quinine imines.
 Quinone imines are condensation products of quinone chlorimines with phenols in
aqueous alkaline media.
 Imides portion of Gibbs reagent reacts with phenolic compounds gives corresponding
products.
NClO
Cl
Cl
+ OH
N
O
OH
ClCl
Gibbs reagent
Phenol Complex

8. [7]
 Because of unstability (protect from light & moisture) of the colour and the reagent
has limited its use to qualitative detection rather than quantitative analysis.
 The main difficulties are caused by the instability of the reagent, several precautions,
have been recommended.
 The rate decomposition increases with increasing pH
 The reagents, when purified by crystallisation from n-heptane and kept in the dark at
room temperature, are stable.
 Stock 0.01M solutions in ethanol or acetone are stable for at least 6 months if stored
in a refrigerator.
 The optimum pH for the colour reaction with phenols is 8.5. Borate, phosphate or
Britton-Robinson buffers are suitable, but not ammonia or glycol buffers
APPLICATIONS of 2,6- Dichloroquinone chlorimide -
 It is used in thin layer chromatography to produce color spots, for example: Sulphur
containing compounds show colored spots when sprayed with Gibbs reagent.
 It is used to identify octopamine in mammals.
 It is used for the detection of anti oxidants, 1o
,2o
aliphatic amines, 2o
,3o
aromatic amines ,
aromatic hydrocarbons ,phenoxy acetic acid herbicides.
 It is a very good reagent for Vitamin B6.
 It is used for the identification of un-substituted and p-alkoxy phenols.
USES-
Methyldopa
Alequots (0.5 to 2.5 ml) Transfer to 10 ml
std vol flask
Then add 2, 1.5, 1, 0.5 and 0
ml of water
Then add 1 ml of Na acetate
buffer and 1 ml og GIBB' s
reagent
Measure the absorbance at
400 nm against blank

9. [8]
HO
HO
NH2
COOH
CH3
+
O
N
ClCl
Cl
O
O
NH
COOH
CH3
N
N
O
ClCl
N
O
Cl Cl
O
Cl
Cl
Methyldopa
Gibbs reagent
(g) 1,2- Napathaquinone-4-sulfonate:
SO3Na
O
O
Synonyms- Folin’s Reagent
PRINCIPLE-
Amines and amino acids react with 1, 2- Napathaquinone-4-sulfonate at alkaline pH to form orange
colored complex.

10. [9]
R-NH2
O
O
NHR
NaOH
O
SO3Na
+
RN-H
CH3
O
O
SO3Na
Orange colored complex
Alkaline medium
O
O
N CH3R
+
O
APPLICATIONS of 1,2- Napathaquinone-4-sulfonate -
 It is used as a derivatizing agent to measure levels of amines and amino acids.
 It can be used with an acidic secondary reagent to distinguish MDMA (a recreational
drug) and related compounds from PMMA (a psychedelic drug). MDMA reacts with
the reagent to give a pink color.
 Used as fluorogenic agent.
 Used as chromogenic agent.
USES-
Cefatoxime
20-140 μg/ml , 475 nm, 0.5% 1 ml NQS reagent & 1 ml 1 N NaOH
N
S
O
O
O
H
N
S N
N
O
H
NH2
O
+
O
O
SO3Na
N
S
O
O
O
H
N
S N
N
O
H
O
O
O
Cefatoxime NQS
0.5% 1 ml NaOH
Orange colour complex

11. [10]
(h) 2,3,5- Triphenyltetrazolium:
Synonyms- TTC, Triphenyl tetrazolium chloride
PRINCIPLE-
It is a colourless compound, but in the presence of reducing substances red triphenylformazan
(TPF) is produced, which is the basic principle involved in colorimetric determination of drugs
and also useful in biological applications.
APPLICATIONS of 2, 3, 5- Triphenyltetrazolium:
 TTC test or tetrazolium test is used to differentiate between metabolically active and
inactive tissues. The white compound is enzymatically reduced to red TPF
(Triphenylformazan) in living tissues due to the activity of various dehydrogenases, while
it remains in its unreacted state in areas of necrosis since these enzymes have either
denatured or degraded.
 TTC has been employed in autopsy pathology to assist post-mortem identification of
myocardial infarctions. Healthy viable heart muscle will stain deep red from the cardiac
lactate dehydrogenase, while areas of potential infarctions will be more pale.
 Determination of traces of silver in synthetic samples.

12. [11]
 Used for analyzing pasteurized milk.
 Used for monitoring & controlling biomass in bioreactors (Fermentors).
USES-
Estimation of Silver in Synthetic Samples
PRINCIPLE
The silver content in synthetic samples can be analyzed by reducing the TTC in presence of
semi carbazide HCl, Nitric acid & NaOH.
PROCEDURE
Preparation of standard solution
 0.15 g of dried AgNO3 dissolved in water containing1ml of conc HNO3
 Solution made up to the mark with water in 100 ml standard flask.
 Aliquot of a standard solution containing 0.5-8.5g of silver is transferred to 25ml of
calibrated flask.
 To this add 0.5ml of 0.1% TTC solution+3ml of 0.5%NaoH solution
 Dilute it upto the mark with 0.8% HNO3
 Mix well and measure the absorbance at 510nm against a freshly prepared reagent
blank.

13. [12]
Preparation of sample solution of synthetic samples
 Take known amounts of silver nitrate samples and dilute it with conc. HNO3 till lower
concentrations has reached.
 Add the required amount of sample to this+0.5ml of 0.1%TTC+1ml of 0.5% semi
carbazide hydrochloride+3ml of 0.5% NaoH.
 Dilute it with 0.8% HNO3.
 Mix well & measure the absorbance at 510nm and find out the amount of silver present
in a given synthetic sample.
(i) 2,4- Dinitrophenylhydrazine (DNPH):
Synonyms- Brady’s reagent, Borche’s reagent
PRINCIPLE:
DNPH is relatively sensitive to shock and friction; it is a shock explosive so care must be taken
with its use. To reduce its explosive hazard, it is usually supplied wet.
2,4-Dinitrophenylhydrazine can be used to qualitatively detect the carbonyl
functionality of a ketone or aldehyde functional group. A positive test is signaled by a yellow or
red precipitate (known as a dinitrophenylhydrazone):
RR'C=O + C6H3(NO2)2NHNH2 → C6H3(NO2)2NHNCRR' + H2O
This reaction can be described as a condensation reaction, with two molecules joining together
with loss of water. It is also called addition-elimination reaction: nucleophilic addition of the -NH2
group to the C=O carbonyl group, followed by the removal of a H2O molecule.
The formation of this precipitate is a positive test for the carbonyl group of ketones and aldehydes.
The ketone or aldehyde is converted to its hydrazone by reaction with dinitrophenyl hydrazine.

14. [13]
The mechanism for the reaction between 2,4-DNPH and an aldehyde or ketone is shown below.
SYNTHESIS OF 2,4-DNP:
2,4-Dinitrophenylhydrazine is commercially available usually as a wet powder. It can be prepared
by the reaction of hydrazine sulfate with 2,4-dinitrochlorobenzene.
APPLICATIONS of 2,4-Dinitrophenylhydrazine (DNPH)-
 It is used for qualitative test of carbonyl groups associated with aldehydes and ketones.
 It has also been used to make photographic developer, explosives, and pesticides.
 It has been used at times to make dyes, other organic chemicals, and wood preservatives.
 It is also used as detecting agent in TLC.
Eg: Carbonyl containing compounds, such as vanillin, can be visualized by spraying with 2,4
DNPH.

15. [14]
USES-
Salbutamol
 Suitable volume of salbutamol sulphate is taken from stock solution(2.5-17µg/ml) in 10ml
volumetric flask
 Add 1 ml of 2,4-DNP(0.04%) + 2mlof KIO3.
 Made alkaline by adding 1ml of NaOH(10 N)
 Red colour complex was formed.
 Further diluted with water.
Absorbance is measured
(j) Bratton-Marshall Reagent:
NH2CH2CH2NH2
Synonyms- N-(1-Napthayl) ethylene diamine
PRINCIPLE-
 The amine present in the compound is first diazotized with an aqueous solution of
nitrous acid at 0-5 0
c
Ar-NH2 +HNO2 → Ar-N≡N + 2H2O
 The colorless diazotized salt is very reactive and when treated with a suitable coupling
agent like BMR ( Ar’-H) undergoes an Electrophilic substitution reaction to produce
azo derivative
Ar-N≡N +Ar –H → Ar –N=N-Ar + H+
 The azo derivatives are colored and consequently have absorption maxima in the
visible region. The azo derivatives are colored and consequently have an absorption
maxima in the visible region
Diazo compound + BMR → Pink colored complex
APPLICATIONS of BMR-
 It is one of the real landmark in the development of drug metabolism and
pharmacokinetics. It is widely used for determination of drugs and pharmaceuticals
containing free primary aromatic amino group.

16. [15]
 At present, it is employed for the determination of sulpha drugs and local anaesthetics.
USES-
Estimation of Sulphanilamides
Sulphanilamide undergoes diazotizations reaction and coupling reaction. Hence sulphanilamide
is treated with NaNO2 and HCl, where nitrous acid is produced, in turn diazotizes with
sulphanilamide. Diazonium salt formed is treated with BMR which undergoes coupling reaction
to give a red colored complex. The intensity of red color formed is directly proportional to the
amount of sulphanilamide present .
NaNO2+HCl → NaCl+HNO2
0.2-1 mcg/ml of standard
drug solution
Add 5ml of 0.5 N HCl
5ml 0.1% of sodium nitrite
mix & cool 5ml of ammonium sulphamate
was added to neutralize
excess of nitrous acid formed
5ml of BMR and measure at
545nm

17. [16]
(k) 3,5- Dinitrosalicylic acid (DNSA):
Synonyms- DNS, DNSA
DNSA reagent composition:
• Its composition is 1% 3,5-dinitrosalicylic acid (DNSA), 30% sodium potassium tartrate, and
0.4 M NaOH.
PRINCIPLE-
DNSA is an aromatic compound that reacts with reducing sugars and other reducing molecules
to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540nm. It was first
introduced as a method to detect reducing substances in urine and has since been widely used,
for example, for quantifying carbohydrate levels in blood.
(yellow) (orange-red)
SYNTHESIS OF DNSA-
DNSA can be prepared by nitration of salicylic acid.
APPLICATION OF DNSA-
 It is used in colorimetric determination of reducing sugars. It was first introduced as a
method to detect reducing substances in urine and has since been widely used, for
example, for quantifying carbohydrate levels in blood.
 It has been used assay of α-amylase.

18. [17]
USES-
Estimation of glucose
 Equal volumes of sample and the reagent are mixed and heated in a boiling water bath for 10
minutes.
 3,5-dinitrosalicylic acid is reduced to 3-amino,5-nitrosalicylic acid
 Measure the concentration of glucose by detecting the reducing end of the
monosaccharide.
 This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced
form which absorbs at 540nm.
 Amount of reduced DNS proportional to amount of glucose

19. [18]
CHAPTER-3
Principles and procedure involved in quantitative estimation of following functional groups in
drugs and elements
HYDROXYL GROUP:

20. [19]

21. [20]

22. [21]
AMINE GROUP:

23. [22]

24. [23]

25. [24]
CARBOXYL GROUP:

26. [25]

27. [26]

28. [27]
CARBONYL GROUP:
ESTER GROUP:

29. [28]

30. [29]
METHOXY GROUP:

31. [30]
SODIUM:

32. [31]
POTASSIUM:

33. [32]
CALCIUM:
By substitution titration

34. [33]

35. [34]
HALOGENS:
(X- Fluorine, Chlorine, Bromine, Iodine and Astatine)
Estimation of halogen is done by following two methods:
i. Carius Halogen Method
ii. Piria & Schiff’s method
i. Carius Halogen Method:
 In this method a known weight of the sample containing halogen (X) is heated
with fuming nitric acid in the presence of AgNO3 in a hard glass tube called
Carius tube.
 Silver halide (AgX) is precipitated, which is filtered, washed, dried and
weighed.
% of halogen (X) =
Atomic wt of X x Wt of AgX
Mol wt of AgX x Wt of sample
x 100

36. [35]
ii. Piria & Schiff’s method
 Known amount of the sample containing halogen (X) is fused with CaO + Na2CO3 in
Pt or Ni crucible.
 The product obtained is dissolved in excess of HNO3 and halogen is precipitated with
AgNO3 solution in the form of AgX.
 Silver halide (AgX) is precipitated is filtered, washed, dried and weighed.
% of halogen (X) =
Atomic wt of X x Wt of AgX
Mol wt of AgX x Wt of sample
x 100
PHOSPHORUS:
Colorimetric method for estimation of phosphorus
PRINCIPLE:
When ammonium molybdate solution is added to a solution of phosphate containing conc. H2SO4
it produces a yellow crystalline precipitation of ammonium phospho-molybdate. Phospho-
molybdate reacts with amino-naphthol-sulphonic acid and produces a molybdenum complex
which forms a blue- coloured solution.
The appearance of colour is due to the formation of a coloured complex in the reaction mixture.
The intensity of the colour against known concentration is plotted on a graph paper to prepare a
standard curve. By comparing the intensity of the colour of unknown samples with the standard
curve, the conc. of phosphate in the unknown sample can be estimated.
METHOD:
Preparation of standard phosphate solution:
11 µg KH2PO4 salt is dissolved in dist. water and the final volume is made up to 250 in a
volumetric flask. The phosphate concentration of the solution is 10 µg/ml.
Preparation of standard calibration curve of phosphate solution:
 10ml aliquots of the phosphate solution in the concentration range of 10-50 g/ml are
prepared while adding 0.4ml of NH2SO4, 0.8ml of 2.5% ammonium molybdate and
0.4ml of amino naphthol sulphonic acid to each.
 A control blank set is also prepared without the addition of phosphate soln.
 The intensity of the colour of the reaction mixture is measured by colorimeter at 570 nm
and a standard calibration curve is plotted

37. [36]
Estimation:The concentration of phosphorus in phosphate soln. of unknown concentration can
be determined by developing coloured solutions and estimating their intensities by Colorimeter.
With the help of the standard curve the conc. of such soln. is also calculated.
SULPHUR:
Estimation of Sulphur can be done by Carius Method.
 In this method a known amount of the Sulphur containing sample is heated with fuming
nitric acid or sodium peroxide (Na2O2) in a sealed tube called carius tube.
 Carbon and hydrogen are oxidized to CO2 and H2O.
 While Sulphur present in the sample is oxidized to sulphuric acid which is then
precipitated as barium sulphate (BaSO4) by adding excess of barium chloride (BaCl2)
solution.
 Barium sulphate (BaSO4) is precipitated is filtered, washed, dried and weighed.
H2SO4 + BaSO4 → BaSO4 + 2HCl
% of Sulphur =
𝐴𝑡𝑜𝑚𝑖𝑐 𝑤𝑡.𝑜𝑓 𝑠𝑢𝑙𝑝ℎ𝑢𝑟 (32) 𝑥 𝑤𝑡. 𝑜𝑓 BaSO4
𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑡.𝑜𝑓 BaSO4 (233) 𝑥 𝑤𝑡. 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝑥 100

38. [37]
CHAPTER 4
ADSORBED TETANUS VACCINE:
Tetanus Vaccine (Adsorbed) is a preparation of tetanus formol toxoid adsorbed on mineral
carrier. The formol toxoid is prepared from the toxin produced by the growth of Clostridium
tetani.
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally srerilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.

39. [38]
Abormal toxicity-
For antisera and vaccines- Unless otherwise prescribed in the individual monograph
inject intra-peritoneally one human dose but not more than 1.0ml into each of five healthy
mice, weighing 17 g to 22 g, and one human dose but not more than 5.0 ml into each of
two healthy guinea pigs weighing 250 g to 350 g. The human dose is that stated on the
label or in the accompanying information leaflet of the preparation under examination.
The preparation passes the test if none of the animals dies or shows signs of ill-health in 7
days following the injection. If more than one animal dies, the preparation fails the test. If
one of the animals die or show signs of ill health, repeat the test. The preparation passes
the test if none of the animals in the second group dies or show signs of ill health in the
time interval specified.

40. [39]

41. [40]
ADSORBED DIPHTHERIA VACCINE:
Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid with a mineral
adsorbent. The formol toxoid is prepared from the toxin produced by the growth of
Corynebacterium diphtheriae.

42. [41]
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.

43. [42]

44. [43]
Biological assay of adsorbed diphtheria vaccine:

45. [44]
HUMAN ANTI HAEMOPHILIC VACCINE:
Human anti haemophilic vaccine or Human coagulation factor VIII is a preparation of a
plasma protein fraction that contains the glycoprotein coagulation factor VIII together with varying
amounts of von Willebrand factor, depending on the method of preparation.
Production-
Dried human antihaemophilic fraction may be prepared from human plasma so obtained by
precipitation under controlled conditions of pH, ionic strength and temperature with organic
solvents, or by freezing and thawing. The precipitate may be washed by extraction with suitable
solvents, dissolved in a solution of sodium citrate adjusted to a pH of 6.8-7.2, which may also
contain sodium chloride. The solution is sterilized by filtration through a membrane filter,
distributed in sterile containers and dried from the frozen state. The air is removed or replaced by
oxygen free nitrogen and the containers are sealed so as to exclude microorganisms. No
antibacterial preservative is added but an antiviral agent may be added provided that it can be
demonstrated to have no deleterious effect on the final product in the amount present and to cause
no adverse reaction in man. Heparin may be used.
For the following tests, where it is directed that a solution is to be used, dissolve the contents of
sealed container in a volume of appropriate solvent equal to the volume of water for injection
stated on the label.
Description-
A white or pale yellow powder or friable soluble.

46. [45]
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Abormal toxicity-
For antisera and vaccines- Unless otherwise prescribed in the individual monograph
inject intra-peritoneally one human dose but not more than 1.0ml into each of five healthy
mice, weighing 17 g to 22 g, and one human dose but not more than 5.0 ml into each of
two healthy guinea pigs weighing 250 g to 350 g. The human dose is that stated on the
label or in the accompanying information leaflet of the preparation under examination.

47. [46]
The preparation passes the test if none of the animals dies or shows signs of ill-health in 7
days following the injection. If more than one animal dies, the preparation fails the test. If
one of the animals die or show signs of ill health, repeat the test. The preparation passes
the test if none of the animals in the second group dies or show signs of ill health in the
time interval specified.
Assay of Human Coagulation Factor VIII:
Human coagulation factor VIII is assayed by its biological activity as a cofactor in the activation
of factor X by activated factor IX (factor IXa) in the presence of calcium ions and phospholipid.
The potency of a factor VIII preparation is estimated by comparing the quantity necessary to
achieve a certain rate of factor Xa formation in a test mixture containing the substances that take
part in the activation of factor X, and the quantity of the International Standard, or of a reference
preparation calibrated in International Units, required to produce the same rate of factor Xa
formation.

48. [47]

49. [48]
RABIES VACCINE:
Rabies Vaccine for Human use is a freeze-dried or liquid (adsorbed) preparation of a suitable
approved, strain of fixed rabies virus grown in an approved cell culture/ embryos of
duck/chicken and inactivated by a validated method.
The freeze-dried vaccine is reconstituted immediately before use as stated on the label to give a
clear or slightly opalescent solution/suspension. It may be coloured owing to the presence of a
pH indicator.

50. [49]
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Abormal toxicity-
For antisera and vaccines- Unless otherwise prescribed in the individual monograph
inject intra-peritoneally one human dose but not more than 1.0ml into each of five healthy
mice, weighing 17 g to 22 g, and one human dose but not more than 5.0 ml into each of
two healthy guinea pigs weighing 250 g to 350 g. The human dose is that stated on the
label or in the accompanying information leaflet of the preparation under examination.

51. [50]
The preparation passes the test if none of the animals dies or shows signs of ill-health in 7
days following the injection. If more than one animal dies, the preparation fails the test. If
one of the animals die or show signs of ill health, repeat the test. The preparation passes
the test if none of the animals in the second group dies or show signs of ill health in the
time interval specified.
Pyrogen:
The test involves measurement of the rise in body temperature of rabbits following the
intravenous injection of a sterile solution of the substance under examination. It is designed
for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg
injected intravenously within a period of not more than 10 minutes.
Test animals- Use healthy, adult rabbits of either sex, preferably of the same variety,
weighing not less than 1.5 kg, fed on a complete and balanced diet and not showing loss of
body weight during the week preceding the test. House the animals individually in an area
of uniform temperature (± 2°), preferably with uniform humidity, and free from
disturbances likely to excite them.
Main test- Carry out the test using a group of three rabbits.
Preparation of the sample- Dissolve the substance under examination in, or dilute with,
pyrogen-free saline solution or other solution prescribed in the monograph. Warm the
liquid under examination to approximately 38°C before injection.
Procedure- Record the temperature of each animal at intervals of not more than 30
minutes, beginning at least 90 minutes before the injection of the solution under
examination and continuing for 3 hours after the injection. Not more than 40 minutes
immediately preceding the injection of the test dose, record the "initial temperature" of
each rabbit, which is the mean of two temperatures recorded for that rabbit at an interval
of 30 minutes in the 40-minute period. Rabbits showing a temperature variation greater
than 0.2° between two successive readings in the determination of "initial temperature"
should not be used for the test. In anyone group of test animals, use only those animals
whose "initial temperatures" do not vary by more than 1° from each other, and do not use
any rabbit having a temperature higher than 39.8° and lower than 38°.
Inject the solution under examination slowly into the marginal vein of the ear of each rabbit
over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The
amount of sample to be injected varies according to the preparation under examination and
is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml
per kg and not more than 10 ml per kg of body weight. Record the temperature of each
animal at half-hourly intervals for 3 hours after the injection. The difference between the
"initial temperature" and the "maximum temperature" which is the highest temperature
recorded for a rabbit is taken to be its response. When this difference is negative, the result
is counted as a zero response.

52. [51]
Interpretation of results- If the sum of the responses of the group of three rabbits does
not exceed 1.4° and if the response of any individual rabbit is less than 0.6°, the preparation
under examination passes the test. If the response of any rabbit is 0.6° or more, or if the
sum of the response of the three rabbits exceeds 1.4°, continue the test using five other
rabbits. If not more than three of the eight rabbits show individual responses of 0.6° or
more, and if the sum of responses of the group of eight rabbits does not exceed 3.7°, the
preparation under examination passes the test.

53. [52]

54. [53]
TETANUS ANTITOXIN: (Antiserum)
Tetanus Antitoxin is a preparation containing the specific antitoxic globulins or their derivatives
obtained by purification of hyperimmune serum of horses or other suitable animals and have the
specific activity of neutralising the toxin formed by Clostridium tetani. The liquid preparation may
contain a suitable antimicrobial preservative.
Description- A clear, colourless or pale yellow liquid or a freeze dried, cream-coloured powder
or pellet.
Identification- Specifically neutralises and renders the toxin formed by C. tetani harmless to
susceptible animals or by any other suitable in vitro test.
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.

55. [54]
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Abormal toxicity-
For antisera and vaccines- Unless otherwise prescribed in the individual monograph
inject intra-peritoneally one human dose but not more than 1.0ml into each of five healthy
mice, weighing 17 g to 22 g, and one human dose but not more than 5.0 ml into each of
two healthy guinea pigs weighing 250 g to 350 g. The human dose is that stated on the
label or in the accompanying information leaflet of the preparation under examination.
The preparation passes the test if none of the animals dies or shows signs of ill-health in 7
days following the injection. If more than one animal dies, the preparation fails the test. If
one of the animals die or show signs of ill health, repeat the test. The preparation passes
the test if none of the animals in the second group dies or show signs of ill health in the
time interval specified.
Biological Assay of Tetanus Antitoxin:
The potency of tetanus antitoxin is determined by comparing the dose necessary to protect mice
against the paralytic effects of a fixed dose of tetanus toxin with the dose of the Standard
Preparation of tetanus antitoxin necessary to give the same protection. For this purpose the
Standard Preparation of tetanus antitoxin and a suitable preparation of toxin, for use as a test toxin,
are required. The test dose of the toxin is determined in relation to the Standard Preparation and
the potency of the preparation under examination is then determined in relation to the Standard
Preparation using the test toxin.
Standard Preparation- The Standard Preparation is the 2nd International Standard for Tetanus
antitoxin, equine, established in 1969, consisting of freeze-dried hyperimmune horse serum
(supplied in ampoules containing 1400 Units), or another suitable preparation the potency of which
has been determined in relation to the International Standard.
NOTE - The severity of the tetanic paralysis to be regarded as the end-point is such that the
paralysis is readily recognised but not sufficiently extensive to cause significant suffering. For
humane reasons the animals should be examined at least Mice a day and should be killed as soon
as the end-point is reached.
Test animals- Use healthy mice, weighing between 17 to 22 g, from the same stock.
Preparation of test toxin- Prepare tetanus toxin from a sterile filtrate of an 8 to 10 day culture of
C. tetani. Test toxin may be prepared by adding this filtrate to glycerin in the proportion of 1
volume of the filtrate to 1 or 2 volumes of glycerin. This solution of test toxin is stored below 0°.
Test toxin may also be prepared in stable form by saturating the filtrate with ammonium sulphate,
collecting the resulting precipitate, drying it over phosphorus pentoxide at a pressure of 1.5 to 2.5

56. [55]
kPa and reducing it to a fine powder. The powder so obtained is preserved in the dry condition at
a low temperature either in sealed ampoules or over phosphorus pentoxide at a pressure of 1.5 to
2.5 kPa.
Determination of test dose of toxin (Lp/l0 dose)- First determine the Limes paralyticum/10
(Lp/10) dose of the test toxin. This is the smallest quantity of the toxin which when mixed with
0.1 Unit of the Standard Preparation and injected subcutaneously into mice causes tetanic paralysis
within 4 days. Prepare a solution of the Standard Preparation in a suitable liquid such that 1 ml
contains 0.5 Unit. Accurately measure or weigh a quantity of the test toxin and dilute it with, or
dissolve it in, a suitable liquid. Prepare mixtures such that each contains 2.0 ml of the solution of
the Standard Preparation (1 Unit); one of a series of graded volumes of the solution of the toxin,
and sufficient of a suitable liquid to give a final volume of 5.0 ml. Allow the mixtures to stand at
room temperature, protected from light, for 60 minutes and then inject 0.5 ml of each mixture
subcutaneously into mice, six mice being used for each mixture, and observe the mice for 4 days.
The test (Lp/10) dose of the toxin is the amount present in 0.5 ml of that mixture that contains the
smallest amount of toxin sufficient to cause tetanic paralysis in all six mice injected with it within
4 days.
Determination of potency of the antitoxin- Prepare a solution of the Standard Prepration in a
suitable liquid such that it contains 0.5 Unit per ml. Accurately measure or weigh a quantity of the
test toxin and dilute it with, or dissolve it in, a suitable liquid so that 1.0 ml contains five times the
Lp/10 dose as previously determined. Prepare mixtures such that each contains 2.0 ml of the
solution of the test toxin and one of a series of graded volumes of the preparation under
examination, and sufficient of a suitable liquid to give a final volume of5.0 ml. Prepare similar
mixtures containing 2.0 ml of the test toxin and one of a series of graded volumes of the solution
of the Standard Preparation centred on that volume (2.0 ml) that contains 1 Unit. Allow the
mixtures to stand at room temperature, protected from light, for 60 minutes and then inoculate 0.5
ml of each mixture subcutaneously into each mouse, six mice being used for each mixture, and
observe the mice for 4 days. The mixture that contains the largest volume of the preparation under
examination that fails to protect the mice from paralysis contains 1 Unit. The test is not valid unless
all the mice injected with mixtures containing 2.0 ml or less of the solution of the Standard
Preparation show paralysis and all those injected with more do not. Calculate the potency of the
preparation under examination in Units per ml.
OXYTOCIN:
Oxytocin is a cyclic nonapeptide hormone obtained by a process of fractionation from the posterior
lobe of the pituitary gland of healthy oxen or other mammals or by synthesis that has the property
of stimulating contraction of the uterus and milk ejection in receptive animals. It may be presented
as a solid or as a solution in a solvent containing an appropriate antimicrobial preservative such as
0.2 per cent w/v solution of chlorbutol.

57. [56]
Description- When presented as a solid, a white or almost white powder. When presented as a
liquid, a clear colourless liquid.
Identification:
Vapour phase hydrolysis- This is one of the most common acid hydrolysis procedures,
and it is preferred for microanalysis when only small amounts of the sample are available.
Contamination of the sample from, the acid reagent is also minimised by using vapour
phase hydrolysis. Place vials containing the dried samples in a vessel that contains an
appropriate amount of hydrolysis solution. The hydrolysis solution does not come in
contact with the test sample. Apply an inert atmosphere or vacuum (less than 200 μm of
mercury or 26.7 Pa) to the headspace of the vessel, and heat to about 110° for a 24 hours
hydrolysis time. Acid vapour hydrolysis the dried sample. Any condensation of the acid in
the sample vials is to be minimised. After hydrolysis, dry the test sample in vacuum to
remove any residual acid.
For analysis-Past-column ninhydrin derivatization- Ion exchange chromatography with
post-column ninhydrin derivatisation is one of the most common methods employed for
quantitative amino acid analysis. As a rule, a lithium-based cation-exchange system is
employed for the analysis of the more complex physiological samples, and the faster
sodium based cation-exchange system is used for the more simplistic amino acid mixtures
obtained with protein hydrolysates (typically containing 17 amino acid components).
Separation of the amino acids on an ion-exchange column is accomplished through a
combination of changes in pH and cation strength. A temperature gradient is often
employed to enhance separation.
When the amino acid reacts with ninhydrin, the reactant has a characteristic purple or
yellow colour. Amino acids, except imino acid, give a purple colour, and show an
absorption maximum at 570 nm. The imino acids such as proline give a yellow colour, and
show an absorption maximum at 440 nm. The post-column reaction between ninhydrin and
amino acids eluted from the column is monitored at 440 nm and 570 nm, and the
chromatogram obtained is used for the determination of amino acid composition.
The detection limit is considered to be 10 pmol for most of the amino acid derivatives, but
50 pmol for the proline derivative. Response linearity is obtained in the range of 20-500
pmol with con-elation coefficients exceeding 0.999.
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.

58. [57]
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Assay-
Determined by liquid chromatography
Test solution- Dissolve about 25 mg of the substance under examination in 100 ml of mobile
phase A.
Reference solution- A 0.025 per cent w/v solution of oxytocin RS in mobile phase
Chromatographic system-

59. [58]
HEPARIN SODIUM IP:
Heparin Sodium is the sodium salt of sulphated glycosaminoglycans present as a mixture of
heterogeneous molecules varying in molecular weights. It is present in mammalian tissues and is
usually obtained from the intestinal mucosa or other suitable tissues of domestic mammals used
for food by man. The sourcing of heparin material must be specified in compliance with applicable
regulatory requirements. It is purified to retain a combination of activities against different
fractions of the blood clotting sequence. It is composed of polymers of alternating derivatives of
D-glucosamine (N-sulphated, O-sulphated, or N-acetylated) and uronic acid, L-iduronic acid or
D-glucuronic acid) joined by glycosidic linkages. The component activities of the mixture are in
ratios corresponding to those shown by the Heparin Sodium reference standard. Some of these
components have the property of prolonging the clotting time of blood. This occurs mainly through
the formation of a complex of each component with the plasma proteins antithrombin III and
heparin cofactor II to potentiate the inactivation of thrombin. Other coagulation proteases in the
clotting sequence, such as activated factor X, are also inhibited.
Category- Anticoagulant.
Description- A white or grayish-white powder; odourless; moderately hygroscopic.
Identification-
A. It delays the clotting of freshly shed blood.
B. Gives reaction of sodium salts- Dissolve 0.1 g of the substance under examination in 2ml
of water or use 2 ml of the prescribed solution. Add 2 ml of a 15 per cent w/v solution of
potassium carbonate and heat to boiling; no precipitate is produced. Add 4 ml of a freshly
prepared potassium antimonate solution and heat to boiling. Allow to cool in ice and if
necessary scratch the inside of the test-tube with a glass rod; a dense, white precipitate is
formed.

60. [59]
Sterility:
The test is designed to reveal the presence of microorganisms in the samples used in the
test; interpretation of the results of testing is based on the assumption that all units of an
article or the entire bulk product or the contents of every container of the filled product in
a lot or batch, had they been tested, would also have given the same results. Since all the
units or the bulk or all the containers cannot be tested, a sufficient number of samples of
units or of containers should be examined to give a suitable degree of confidence in the
results of the tests.
The following culture media have been found to be suitable for the test:
 Fluid Thioglycollate Medium
 Soyabean-casein Digest Medium
Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria. Soyabean-casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
Direct Inoculation- Remove the liquid from the test containers with a sterile pipette or
with a sterile syringe or a needle. Transfer the quantity of the preparation under
examination prescribed in Table 4 directly into the culture medium so that the volume of
the preparation under examination is not more than 10 per cent of the volume of the
medium, unless otherwise prescribed. When the quantity in a single container is
insufficient to carry out the tests, the combined contents of two or more contains are to be
used to inoculate the media.
If the preparation under examination has antimicrobial activity, carry out the test after
neutralising this with a suitable neutralising substance or by dilution in a sufficient quantity
of culture medium. When it is necessary to use a large volume of the product it may be
preferable to use a concentrated culture medium prepared in such a way that it takes
account of the subsequent dilution. Where appropriate, the concentrated medium may be
added directly to the product in its container.
Incubate the inoculated media for not less than 14 days. Observe the cultures several times
during the incubation period. Observe the containers of media periodically during the 14
days of incubation. If the test specimen is positive before 14 days of incubation, further
incubation is not necessary. For products terminally sterilised by a validated moist heat
process, incubate the test specimen for not less than 7 days.
Assay:
Determine the potency of heparin sodium by comparing the concentration necessary to prevent the
clotting of sheep or goat or human plasma with the concentration of the reference solution of
heparin sodium necessary to give the same effect under the condition of the following method of
assay.

61. [60]
Test solution- Dissolve an accurate weighed quantity containing about 25 mg of heparin
sodium, in sufficient saline to produce a concentration of 1 mg per ml, and dilute
quantitatively to a concentration estimated to correspond to that of the reference solution.
Reference solution- Determine by preliminary trial, if necessary, approximately the
minimum quantity of heparin sodium RS which, when added in 0.8 ml of saline, maintains
fluidity in 1ml of prepared plasma for 1 hour after the addition of 0.2 ml of calcium chloride
(1 in 100). This quantity is usually between 1 and 3 Heparin Units. On the day of the assay
prepare a reference solution such that it contains, in each 0.8 ml of saline, the above-
determined quantity of the reference standard.
Preparation of plasma- Collect blood from sheep directly into a vessel containing about
8 per cent of sodium citrate in the proportion of one volume to each 19 volumes of blood
to be collected. Mix immediately by gentle agitation and inversion of the vessel. Promptly
centrifuge the blood, and pool the separated plasma. To a 1 ml portion of the pooled plasma
in a clean test tube, add 0.2 ml of calcium chloride (1 in 100), and mix. Consider the plasma
suitable for use if a solid clot forms within 5 minutes. To store plasma for future use,
subdivide the pooled lot into portions not exceeding 100 ml in volume, and store in the
frozen state, preventing even partial thawing prior to use. For use in the assay, thaw the
frozen plasma in a water bath at a temperature not exceeding 37°. Remove particulate
matter by straining the thawed plasma through a coarse filter.
Procedure- To meticulously clean 13 mm x 100mm test tubes add graded amounts of the
reference solution selecting the amount so that the largest does not exceed 0.8 ml and so
that they correspond roughly to a geometric series in which each step is approximately 5
per cent greater than the next lower. To each tube so prepared add sufficient saline to make
the total volume 0.8 ml. Add 1.0 ml of prepared plasma to each tube. Then add 0.2 ml of
calcium chloride (1 in 100), note the time, immediately insert a suitable stopper in each
tube, and mix the contents by inverting three times in such a way that the entire inner
surface of the tube is wet.
In the same manner set up a series using the test solution, completing the entire process of
preparing and mixing the tubes of both the reference solution and the test solution within
20 minutes after the addition of the prepared plasma. One hour, accurately timed, after the
addition of the calcium chloride, determine the extent of clotting in each tube; recognizing
three grades (0.25, 0.50, and 0.75) between zero and, full clotting (1.0). If the series does
not contain 2 tubes graded more than 0.5 and 2 tubes graded less than 0.5, repeat the assay,
using appropriately modified reference solution and test solution.
Convert to logarithms the volumes of reference solution used in the successive 5 or 6 tubes
that bracket a grade of clotting of 0.5, including at least 2 tubes with a larger and 2 tube
with a smaller grade than 0.5. Number and list the tubes serially, and tabulate for each the
grade of clotting observed in each tube. From the log-volumes, x, and separately from their
corresponding grades of clotting, y, compute the paired averages xi and yi of Tubes 1,2,
and 3, of Tubes 2, 3, and 4, of Tubes 3, 4, and 5, and, where the series consists of 6 tubes,

62. [61]
of Tubes 4, 5, and 6, respectively. If for one of these paired averages the average grade, yi,
is exactly 0.50, the corresponding xi is the median log-volume of the reference solution xS.
Otherwise, interpolate xS from the paired values of yi, xi and yi+1, xi+1 that fall
immediately below and above grade 0.5 as
xS = xi + (yi 0.5)(xi+1 xi) / (yi yi+1)
From the paired data on the tubes of the test solution, compute similarly its median log-
volume xU.
The log potency of the test solution is
M = xS-xU + 10gR
Where R =vS /vU is the ratio of the heparin Units (vS) per ml
of the reference solution to the mg (vU) of heparin sodium per
ml of the test solution.
ANTIVENOM:
Antivenoms are pharmaceutical preparations containing specific antibodies for the venom of
interest. They are obtained from serum/plasma of hyperimmunised animals with venom provided
by one or many species.
Assay:
The antivenom activity is assayed following the reaction generated by injection of well-defined
mixes of venom and antivenom in testing animals.
The potency assay aims at determining the required neutralizing dose (Effective Dose 50%) to
protect susceptible animals against the lethal effects of a fixed dose of a reference venom.
Determination of the LD50 of the venom-
Firstly, reconstitute the lyophilized venom at a determined concentration by volume with
0.85% saline (w/v). Make dilutions in geometric progression with the same diluent, using
a constant dilution factor of not more than 1.5 and equalize the final volumes. Inoculate,
by intraperitoneal route, a volume of 0.5 ml per mouse of each dilution in groups of at least
10 Swiss albino mice from 18 g to 22 g. Observe the animals up to 48 hours after
inoculation and record the number of deaths at each dilution. Calculate the LD50 using a
proven statistical method. The response range (percentage of deaths) should be between
the highest and the lowest dilution used, forming the regression curve that must present a
linear correlation. Confidence intervals should not be large, indicating better accuracy of
the test, the smaller the limits. Express the result in micrograms of venom per 0.5 mL.

63. [62]
Determination of antivenom potency-
Carry out progressive dilutions of the sample in saline at 0.85% (w/v) using a constant
dilution factor of not more than 1.5, so that the final volume after mixing with the challenge
dose is identical across all test tubes. Reconstitute and dilute the reference venom with
0.85% (w/v) saline solution and add constant volume to each tube so that each dose to be
inoculated per animal contains 5xLD50. Homogenize and incubate the mixture at 37°C for
60 minutes. Inoculate intraperitoneally with a volume of 0.5 ml per mouse of each mixture
in groups of at least eight Swiss albino mice of 18 g to 22 g. Observe the animals up to 48
hours after inoculation and record the number of live in each mixture. Calculate the 50%
Effective Dose (ED50) in microliters, using a proven statistical method. The response
range produced (percentage of survival) should be between the highest and the lowest
dilution used, forming the regression curve that should present a linear correlation.
Confidence intervals should not be large, indicating better accuracy of the test, the smaller
the limits. Calculate the power in milligrams per milliliter, according to the expression:
Antivenom Potency (mg/ml) = (Tv-1/ED50) x venom LD50
Where,
Tv = number of LD50 used per mouse in the venom test dose.
Antivenom potency was expressed in milligrams of venom neutralized by 1 mL of the
sample.

64. [63]
CHAPTER-5
STABILITY ZONES:
The climate is different in all the countries in the world. Stability study of the pharmaceutical drug
should be done according to the climatic conditions of the country. According to the climate, the
world is divided into five different zones:
These zones are created due to the difference in temperature and humidity in different parts of the
world. These zones have different stability conditions for pharmaceutical products. Following are
the climatic conditions for these zones.