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1-The following procedure was done during the determination of the

Potency of a Clostridium Antitoxin Preparation for veterinary use:
- The Reference antitoxin preparation was reconstituted and diluted to
2.0 I.U./ml,
- The dry test clostridium toxin preparation was reconstituted and
diluted for final use to 0.04 mg/ml.
- The antitoxin preparation under examination was diluted 100 times to
get the assumed potency; from the minimum on the label.
- Mixtures of the solution of the reference preparation and the test toxin
solution were prepared such that each contained 2.0 ml of the test
toxin solution, one of a graded series of volumes of the reference
preparation solution and sufficient of the diluent to bring the volume
up to 5.0 ml.
- Also prepared were mixtures of the solution of the antitoxin
preparation under examination and the test toxin solution such that
each contained 2.0 ml of the test toxin solution, one of a graded series
of volumes of the solution of the antitoxin preparation under
examination and sufficient of the diluent to bring the volume up to the
same, 5.0 ml.
- After an appropriate duration of neutralisation, for each mixture, a
dose of 0.25ml was injected into each mouse in the group. The analysis
was done in triplicate and the volumes of the prepared Reference
antitoxin dilution in the mixtures that prevented 50% deaths in the
groups of mice were 1.97ml, 1.99 ml and 2.04ml whilst the
corresponding volumes for the diluted antitoxin preparation under
examination were 1.72 ml, 1.75ml and 1.75 ml.

(a)- What is the Estimated potency of the Clostridium antitoxin

preparation under examination (as supplied) ? (5
Marks).

(b)- If it was labelled that for use in a certain species, the antitoxin has
to be diluted 1:50 times before administration of a specific volume.

(i)- After determining the estimated potency, under normal situation,

as a pharmacist would you worry about this dilution factor? Give
reasons for your answer. (3 Marks).

(ii) - In the event of scarcity, as a pharmacist, what would you

recommend to be the dilution factor basing on the estimated
potency? (2 Marks).

(c)- If the injected 0.25ml of the mixture was expected to have

contained one Test dose of the toxin, what is the potency of the
given dry toxin preparation? Give your answer in L+/y Dose/mg
and give the value of y.(10 Marks).

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Answering:

(a)- What is the potency of the Clostridium antitoxin

preparation under examination (as supplied) ? (Total 5
Marks).

The total sums are {2.04 + 1.99 + 1.97}ml (Ref) and {1.72 + 1.75
+ 1.75}ml ( Diluted Test Prepn)
= 6.00ml (Ref) and 5.22ml (Diluted
Test Prepn). (1)

The amount of the antitoxin in 6.0ml of the Reference dilution

That of the Diluted prepn under examination in 5.22ml.

→ 5.22 ml of the diluted prepn = 6.00ml x 2.0 I.U/ml = 12.00

I.U.(1)

 the Potency of the diluted prepn is 12.000 I.U.  5.22ml

= 2.30 I.U. of antitoxin /ml,.(1).

Put dilution factor (100), the Minimum Potency is therefore

230I.U. of Antitoxin / ml. ans.(2).

8 / –4 – 5 Marks.

(b)- If it was labelled that for use in a certain species, the

antitoxin has to be diluted 1:50 times before administration of a
specific volume.

(i)- After determining the estimated potency, under normal situation,

as a pharmacist would you worry about this dilution factor? Give
reasons for your answer. (3 Marks).

No. Because the overall aim according to BP is not to have less

than the Minimum stated Potency. (3)

(ii) - In the event of scarcity, as a pharmacist, what would you

recommend to be the dilution factor basing on the estimated
potency? (2 Marks).

= (50 x 6)  5.22 = 57. (1)

Therefore dilution is (1 : 57). (1).

4 / – 3-5 Marks.

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(c)- If the injected 0.25ml of the mixture was expected to

have contained one Test dose of the toxin, what is the potency of
the given dry toxin preparation? Give your answer in L+/y
Dose/mg and give the value of y. (10 Marks).

Given the reference mixture. 1.97ml , 1.99ml& 2.04ml gave the

concerned deaths.

 Average vol. Ref. antitoxin of used in the 5.0ml mixture was

= {(1.97 + 1.99 + 2.04) 3} = 2.00 ml.(1)

 Amount of antitoxin in that desired mixture (5.0ml)

= 2.00 ml X 2.0 IU/ml} = 4.0 IU.(1)

The amount of antitoxin in 0.25ml of the mixture is

(0.25ml x 4 IU)  5.0ml = 1.0IUxml 5.0ml = 0.2 IU (2)

the value of y is 1  0.2 = 5. (3)

the Test Dose was L+/5 Dose.(1)

Each mixture contained 2.0ml of the Toxin solution i.e. 5 ml of

mixture contained 2.0ml of Toxin solution.

0.25ml of the mixture contained (2.0 5.0) X 0.25 ml of Toxin

solution
= 0.1ml of Toxin solution Test Dose. (2)

NB: 1.0ml of the Toxin solution had 0.04mg of Dried Toxin

Test Dose of the Toxin solution had (0.1 X 0.04mg of Dried

Toxin
= 0.004mg.(2)

i.e. One Test Dose L+/5 Dose of the toxin  0.004mg of Dried
Toxin

1mg of Dried  (1  0.004) = 250 L+/5 Dose(2)

Answer is 250.0 L+/5 Dose/mg.(1)

15 / – 10-15 Marks.

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2-In an assay of European Viper Venom Antiserum, the standard test

venom was reconstituted and diluted for final use to 28 LD50./ml .
Serial dilutions of the antiserum preparation were done using its
recommended diluent.
Venom-antiserum mixtures were made for injection in the mice, such
that each mixture contained 2.5 ml of the final diluted reconstituted
venom and 2.5 ml of one of the dilutions of the antiserum, thoroughly
mixed.
0.5 ml of each of the venom-antiserum mixture was injected into each
of the mice allocated for each treatment.
The mixture found to have the PD50 was initially derived from 0.15 ml
of the antiserum preparation. What is the potency of the antiserum?
(12 Marks).

Answers:
Potency = (Tv – 1)/PD50

Tv?
- Volume of mixture was (2.5 + 2.5) = 5.0ml
- Volume of Reconstituted Venom used was 2.5ml
- Therefore Total Amount of Venom in the mixture was
2.5ml X 28 LD50 = 70 LD50(2)
- Amount of Venom per ml of mixture was
(70 LD50 ÷ 5.0ml) = 14 LD50/ml
- Therefore Amount of Venom injected in each mouse was
- (0.5ml X 14 LD50/ml ) = 7 LD50
- i.e theTv was 7 LD50.(2).

PD50?
- Volume of Antiserum with the PD50 was 0.15ml
- I.e. the Total Amount of Antiserum in the mixture was 0.15ml
- The Amount of antiserum per ml of the mixture was
(0.15ml ÷ 5.0ml)= 0.03(0.03ml/ml???). (2)
(= 0.03 ml of Antiserum per ml of the mixture).
- Therefore Amount of Antiserum injected in each mouse was
- (0.03ml/ml X 0.5ml) = 0.015ml
- i.e thePD50 was 0.015ml.(2)
Potency = (Tv – 1)/PD50 = {(7 – 1)} LD50 ÷ 0.015ml (2) = 400(1)
LD50/ml.
Correct Units stated(1).
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8/ –8 -- 20 Marks.

3- The table below contains information about Bovine Tuberculin Purified

Protein Derivative Preparations.NB: T.U. is Tuberculin Units.

Stated Potency
Item
Preparation T.U. /
No mg/ml
mg
Reference Bovine Tuberculin
1 4 6,250
Purified Protein Derivative.

Test Bovine Tuberculin Purified

2 5 4,000
Protein Derivative Preparation

Bearing in mind in what form it is administered in diagnosis, what is the

Potency Ratio and give explanations where necessary? (8 Marks).

Answers:
Because the preparation is administered as a liquid preparation ,
we have to use volumes of equipotency. (2).
The T.U, is a biological Unit and is the one used for getting the
equipotent volumes. (1).

∴Potency Ratio = Vol of Test / Equipotent Vol of Refertence.

Or

Potency Ratio = Potency of Reference in T.U. / Potency of Test

in T.U. (2).

= (4 X 6,250) ÷ (5 X 4,000) (2).

= 1.25, ans. (1).

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4- In the assay of streptokinase injection, four serial dilutions were done

for both the reference preparation and the test preparation. The
dilutions were in triplicate for both preparations.
The absorbance of the blank was 0.100 AU. The average absorbances
were as tabled below.

Dilution Average absorbances

(IU/mL) Reference Test
2.0 0.400 0.380
1.0 0.367 0.347
0.5 0.333 0.313
0.25 0.300 0.280

NB: the injection was made from reconstituting the whole content of the
vial to 10mLs initially, and then it was diluted 8,000 times to make the first
dilutions based on its stated potency.
a) Plot this assay report on a reasonable graph as per the B.P. (9 Marks).
b) Determine the potency ratio (Estimated Potency). (5 Marks).
c) Calculate the stated potency. (3 Marks).
d) Does the preparation pass the potency test given that the authority
requires the estimated not to be less than 80% and not more than 115%
of the stated. (3 Marks).

Answers:
(a)-

Dilution Log 10 of Average Absorbance

(IU/mL) Dilutions Reference Test
2.0 0.3010 0.400 0.380
1.0 0.0000 0.367 0.347
0.5 -0.3010 0.333 0.313
0.25 -0.6021 0.300 0.280
(2).

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Absorbances Vs Log10 dilutions

0.42
(2) - Axes 0.4
f(x) = 0.110952373966045 x + 0.366701106091239
0.38
f(x) = 0.110952373966045 x + 0.346701106091239
Absorbance

0.36 Reference
Linear
0.34 (Ref-
er-
0.32 ence)
0.3
(3) - Trend 0.28
0.0000
Lines 0.26
5 5 5 5 25 15 9 05 15 25 5
.6 .5 .4 .3 . . 99
0. 0. 0. .3
-0 -0 -0 -0 -0 -0 9 0
0.1801
99
9 99
9
9 99
49
0.0
-

Log10 Dilutions (2) Labels

.

(b)- Potency Ratio =

EquiPotent Dilution of Test / EquiPotent Dilution of Reference
(2).
AntiLog (log Test Dilut – Log Ref Dilut)
= AntiLog (0.18018 – 0.00000 ) (2) .
= AntiLog 0.18018

= 1.5142, ans. (1) .

(Tot 5 Marks).

(c)- Stated Potency = 2.0IU/mL X 8,000 (2) = 16,000 IU/mL. (1).

answer.
(Tot 3 Marks).

(d)-
- Since at dilution Stated Potency becomes the Assumed
Potency and

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- Dilution is done to achieve equipotency with the Reference

dilution,

It means the Estimated Potency of the Test = The Potency of the

Reference ÷ The Potency Ratio {of the Estimated Potency Test to the
Reference Test (Stated Potency)} i.e.

In percentage:
Estimated Potency of the Test = 100% ÷ The Potency Ratio {of the
Estimated Potency Test to the Reference Test (Stated Potency)}

= {(1 ÷ 1.5142) X 100 % = 66.042 %. (2),

And 67 % is less than 80%, the Lower Pharmacopoeia Limit. So it fails

the test. (1).
(Tot 3 Marks).

5-In the determination of the potency of a preparation of Avian Tuberculin

Purified Protein Derivative (ATPPD), the first dilution of the reference
preparation was prepared to be 10 times dilute as the stated potency of the
preparation under test. Then subsequent dilutions were 5 folds dilutions.
The labelled potency was 24,000IU/mL. Assume linearity over the accepted
diameters.
(a)- What is the subsequent estimated potency?
(b)- What is the potency ratio?
(c)- Does the preparation pass the Potency test (Limits from the stated potency
is 75% and 133%). Explain.

Diameter of lesions in mm
Dilution Standard test
1st 20 22
2nd 15 17
3rd 10 12
4th 0 2

Answers
NB: (i)– Apply graph and Slope mathematics.
(ii) – Note the 4th dilutions are out of linearity.

Diameter of lesions in mm Concn IU/mL Log(10)

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Dilution Standard test Standard Standard

1st 20 22 2,400
2nd 15 17 480
3rd 10 12 96
4th 0 2 19.2

(a)- What is the subsequent estimated potency?

Slope = (Log S1 – LogS3)/(20 – 10) = (LogU1 – Log S1)/(22 – 20)
=> (Log U1 – Log 2,400)/2 = (Log 24,00 – Log 96)/10
=> 10Log {(U1)/2,400} = 2log(2400/96)
=> 10Log {(U1)/2,400} = 2log25
=> Log {(U1)/2,400} = (2 X 1.397940008672)/10
=> Log {(U1)/2,400} = 0.2795880017344
=> {(U1)/2,400} = AntiLog 0.2795880017344
=> {(U1)/2,400} = 1.9037
=> U1 = 1.9037 X 2,400 = 4,568.77 IU/mL
=> Estimated Potency = U1 X Initial Dilution factor = 4,568.77 X 10
=> Estimated Potency = 45,687.70 IU / mL., Ans.

(b)- What is the potency ratio (Estimated)?

Potency Ratio (P.R.) = Potency of Reference / Potency of Estimated
NB: Reference Potency is made to assume the Stated Potency
=> Potency Ratio = Stated Potency / Estimated Potency
=> Potency Ratio = 24,000 / 45,687.70 = 1 / 1.9037
=> Potency Ratio = 0.5253, Ans.

(c)- Does the preparation pass the Potency test (Limits from the stated
potency is 75% and 133%). Explain.
Percent Estimated Potency to Stated Potency = 100% / P.R.
=> Percent Estimated to Stated Poteency = 100% / 0.5253
=> Percent Estimated to Stated Poteency = 190.37%.
Fails Potency Test because Estimated Potency is higher than the upper
limit.

END. Good Luck.

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