RABBIT BLOOD SUGAR METHOD—QUANTITATIVE
Diluent—
Prepare an aqueous solution containing 0.1% to 0.25% (w/v) of either cresol or phenol, 1.4% to 1.8% (w/v) of glycerin, and sufficient hydrochloric acid to produce a pH between 2.5 and 3.5, unless otherwise directed in the individual monograph.
Standard Stock Solution—
Dissolve either a suitable quantity of accurately weighed
USP Insulin RS or a vial of lyophilized
USP Insulin RS of the appropriate species in
Diluent to make a
Standard Stock Solution containing 40 USP Insulin Units per mL and having a pH between 2.5 and 3.5, unless otherwise directed in the individual monograph. Store in a cold place, protected from freezing, and use within 6 months.
Standard Solutions—
Dilute portions of the Standard Stock Solution with Diluent to make two solutions, one to contain 1.0 USP Insulin Unit per mL (Standard Solution 1), and the other to contain 2.0 USP Insulin Units per mL (Standard Solution 2).
Assay Stock Solution—
Proceed as directed under
Standard Stock Solution except use a suitable quantity of the preparation under test in place of
USP Insulin RS. The
Assay Stock Solution contains about 40 USP Insulin Units per mL.
Assay Solutions—
Dilute portions of the Assay Stock Solution with Diluent to make two dilutions of the preparation under test, one of which may be expected, on the basis of the assumed potency, to contain 1.0 USP Insulin Unit per mL (Assay Solution 1), and the other to contain 2.0 USP Insulin Units per mL (Assay Solution 2). In the case of neutral insulin injection, adjust to a pH of 2.5 to 3.5 prior to making the dilutions.
Doses of the Solutions To Be Injected—
Select on the basis of trial or experience the dose of the dilutions to be injected, the volume of which usually will be between 0.30 mL and 0.50 mL. For each animal the volume of the Standard Solution is the same as that of the Assay Solution.
Preparation of Animal—
Select suitable, healthy rabbits each weighing not less than 1.8 kg. Keep the rabbits in the laboratory for not less than 1 week before use in the assay, maintaining them on an adequate uniform diet, with water available at all times.
Procedure—
Divide the rabbits into four equal groups of preferably not less than six rabbits each. On the preceding day, approximately 20 hours before the assay, provide each rabbit with an amount of food that will be consumed within 6 hours. Follow the same feeding schedule before each test day. During the assay, withhold all food until after the final blood specimen is taken. Handle the rabbits with care in order to avoid undue excitement, and inject subcutaneously the doses indicated in the following design (see
Table 1), the second injection being made on the day after the first injection, or not more than 1 week later. The time between the first and second injection is the same for all rabbits.
Table 1
| Group |
First Injection |
Second Injection |
| 1 |
Standard Solution 2 |
Assay Solution 1 |
| 2 |
Standard Solution 1 |
Assay Solution 2 |
| 3 |
Assay Solution 2 |
Standard Solution 1 |
| 4 |
Assay Solution 1 |
Standard Solution 2 |
Blood Samples—
At 1 hour ± 5 minutes and 2½ hours ± 5 minutes after the time of injection, obtain from each rabbit a suitable blood specimen from a marginal ear vein. Blood can also be collected effectively from the central auricular artery.
Dextrose Determination—
Determine the dextrose content of the blood specimens by a suitable procedure that is adapted to automated analysis. The following procedure may be used.
Anticoagulant Solution—
Dissolve 1 g of edetate sodium and 200 mg of sodium fluoride in 1 L of water, and mix.
Dextrose Standard Preparations—
Transfer known concentrations of
USP Dextrose RS to suitable vessels, and dilute quantitatively and stepwise with
Anticoagulant Solution (1:9) to obtain a range of
Dextrose Standard Solutions containing between 20 and 100 mg per 100 mL, having known concentrations similar to the concentrations in the rabbit blood samples.
Test Preparations—
Pipet into separate, suitable vessels 0.1 mL of each Blood Sample and 0.9 mL of Anticoagulant Solution.
Procedure—
Subject the Test Preparations to dialysis across a semipermeable membrane for a sufficient time so that the dextrose passes through the membrane into a saline TS solution containing glucose oxidase, horseradish peroxidase, 3-methyl-2-benzothiazolinone hydrazone hydrochloride TS, and N,N-dimethylaniline. The absorbances of the Test Preparations are determined at 600 nm in a recording colorimeter. The absorbances of the Dextrose Standard Preparations are similarly determined at the start and the end of each run.
Calculation—
Calculate the response of each rabbit to each injection from the sum of the two blood-sugar values, and subtract its response to
Standard Solution 1 from that to
Standard Solution 2, disregarding the chronological order in which the responses were observed, to obtain the individual differences,
y, as shown in the accompanying table.
When the number of rabbits, f, carried through the assay is the same in each group, total the y's in each group and compute Ta = –T1 + T2 + T3 – T4 and Tb = T1 + T2 + T3 + T4. The logarithm of the relative potency of the test dilutions is M¢ = 0.301Ta / Tb. The potency of the injection in USP Units per mL equals the antilog (log R + M¢), where R = vS / vU, in which vS is the number of USP Units per mL of the Standard dilution, and vU is the number of mL of injection per mL of the Assay dilution.
Determine the confidence interval of the log-relative potency
M¢ (see
Confidence Intervals for Independent Assays under
Design and Analysis of Biological Assays 
111
). If the confidence interval is more than 0.082, which corresponds at
P = 0.95 to confidence limits of about ±10% of the computed potency, repeat the assay until the combined data of the two or more assays, redetermined as described in
Combination of Independent Assays under
Design and Analysis of Biological Assays 111, meet this acceptable limit.
| Group |
Differences |
Individual Response (y) |
Total Response (T) |
| 1 |
Standard solution 2 – Assay solution 1 |
y1 |
T1 |
| 2 |
Assay solution 2 – Standard solution 1 |
y2 |
T2 |
| 3 |
Assay solution 2 – Standard solution 1 |
y3 |
T3 |
| 4 |
Standard solution 2 – Assay solution 1 |
y4 |
T4 |
Bioidentity Test
Proceed as directed for Rabbit Blood Sugar Method—Quantitative with the following modifications:
Procedure—
Divide the rabbits into four equal groups of two rabbits each.
Calculation—
Proceed as directed for Calculation under Rabbit Blood Sugar Method—Quantitative, but do not determine the confidence interval of the log-relative potency, M¢.
Interpretation—
If the potency value obtained is not less than 15 USP Units per mg, the Bioidentity Test requirement is met. If the potency value is less than 15 USP Units per mg, repeat the test using eight more rabbits. If the average potency of the two sets of tests is not less than 15 USP Units per mg, the requirement of the test is met.
