The most prominent manifestation of insulin activity, an abrupt decrease in blood glucose, was the basis for biologic assay from the time of its first clinical use. The procedure, although relatively cumbersome, has the great merit of accurately reflecting the effect on the diabetic patient. The advent of practical yet sophisticated physicochemical methods (e.g., liquid chromatography) to measure insulin potency quantitatively has resulted in a more accurate and precise compendial test for insulin and insulin products. However, the bioidentity of insulin and insulin products cannot be assessed by these methods. Thus, a qualitative test in rabbits is included in this chapter, and its use is called for in the appropriate monographs.

The Rabbit Blood Sugar Method—Quantitative is used to determine the potency of Insulin Reference Standards, for the validation of the stability of new insulin preparations, and to determine the specific activities of insulin analogs.

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

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 Preparations 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.

Group	Differences	Individual Response (y)	Total Response (T)	Standard Deviations of Differences (S)
1	Standard Solution 2 – Assay Solution 1	y1	T1	S1
2	Assay Solution 2 – Standard Solution 1	y2	T2	S2
3	Assay Solution 2 – Standard Solution 1	y3	T3	S3
4	Standard Solution 2 – Assay Solution 1	y4	T4	S4

Proceed as directed for Rabbit Blood Sugar Method—Quantitative with the following modifications:

This version of Fieller's Theorem is for the case where the numerator and denominator are uncorrelated. The equation assumes the numerator and denominator are normally distributed and the groups of rabbits are equal-sized.

Then, the 95% confidence interval for the ratio is:

where f (degrees of freedom in the standard errors) = 4(k – 1), where k is the number of rabbits in a group, t is the upper 97.5 percentile of the t-distribution with f degrees of freedom, and

If g 1, the denominator is not significantly different from 0 and the formula does not work.