Assay for luteinizing hormone—
Diluent—
Dissolve 10.75 g of dibasic sodium phosphate, 7.6 g of sodium chloride, and 1.0 g of bovine serum albumin in 1 L of freshly distilled water. Adjust with 1 N sodium hydroxide or dilute with 20% phosphoric acid to a pH of 7.2 ± 0.2.
Standard preparations—
Dissolve an accurately weighed quantity of
USP Menotropins RS in the
Diluent to obtain solutions having known concentrations of about 8.75, 17.5, and 35.0 USP Luteinizing Hormone Units per mL.
Assay preparations—
Following the procedure given under the Standard preparations, use Menotropins in place of the USP Reference Standard to obtain similar solutions.
Control solution—
Use the
Diluent as the control solution. Store all solutions at 5 ± 3
for the duration of the assay and properly dispose of any unused portions.
Test animals—
Select 20- to 21-day old male rats with weights within a 10 g range of each other. House the animals under uniform conditions of temperature, light, food, and water. Mark the animals for identification, and divide them at random into 7 groups of the same number, having not less than 6 animals per group. Assign one group to each Standard preparation, one group to each Assay preparation, and one group to the Control solution.
Dose determination trial—
Use the method described for Procedure to determine a 3-dose range in which the lowest dose produces a definite response in some of the the rats in the low-dose group (as compared with the control group) and the highest dose produces a submaximal to maximal response in the high-dose group. Doses must be established in a geometric progression. The normal dose response range will occur between 3.5 and 28 USP Luteinizing Hormone Units total dose per rat. Useful dose ranges will vary with the sensitivity of the rat strain selected.
Procedure—
Inject each rat of each group subcutaneously in the dorsal area with 0.2 mL of the solution to which it was assigned. For the Dose determination trial only, similarly inject each rat in the control group with 0.2 mL of the Control solution. Repeat these injections at approximately the same time of day after 24, 48, and 72 hours. Twenty-four hours after the last injection, weigh each rat, sacrifice the animals, and carefully dissect out the seminal vesicle of each rat, removing any fat and fibrous tissue. Thoroughly dry the vesicles by pressing against absorbent paper, avoiding damage to the vesicles, and immediately weigh them to the nearest 0.2 mg, using a suitable balance.
Calculation—
Tabulate the observed seminal vesicle weights (y) for each dosage group of f rats. For apparently outlying seminal vesicle weights, an attempt may be made to correct the organ mass relative to the mass of the rat from which it was taken. For the y-value in question, calculate for each of the f rats in the appropriate group the ratio of seminal vesicle weight to total body weight. Reject the y-value if its corresponding ratio differs from the rest of the group by more than 1.5 standard deviations.
If the data from one or more rats are missing, adjust to groups of equal size by suitable means (see
Replacement of Missing Values under Design and Analysis of Biological Assays 
111
). Total the values of
y in each group, and designate each total as
T, using subscripts 1 to 3 for the three successive dosage levels and subscripts
S and
U for the Standard and the material under test, respectively. Test both the agreement in slope of the dosage–response lines for the Standard and for the material under test, and the lack of curvature as directed for a 3-dose balanced assay (see
Tests of Assay Validity under
Design and Analysis of Biological Assay 111). If the combined discrepancy as measured by
F3 exceeds its tabular value in
Table 9, repeat the assay.
Determine the logarithm of luteinizing hormone potency of the Menotropins taken by the formula:
M = (4iTA / 3TB) + log R,
in which
TA =
S(
TU 
TS);
TB =
S(
T3 T1);
i is the interval between successive log doses of both the
Standard preparation and the
Assay preparation; and
R =
vS /
vU is the ratio of the high dose of the Standard in USP Luteinizing Hormone Units (
vS) to the high dose of the Menotropins in mg (
vU). Compute the log confidence interval (see
Design and Analysis of Biological Assays 111).
Replication—
Repeat the entire determination at least once. Test the agreement among the two or more independent determinations, and compute the weight for each (see
Combination of Independent Assays 111). Calculate the weighted mean log-potency
M and its confidence interval,
LC (see
Confidence Intervals for Individual Assays 111). The potency,
P*, is satisfactory if
P* = antilog
M is not less than 80% and not more than 125% of the labeled potency and if the confidence interval does not exceed 0.18.
Assay for follicle-stimulating hormone—
Diluting solution—
Using the Diluent under Assay for luteinizing hormone, dissolve an accurately weighed quantity of USP Human Chorionic Gonadotropin RS to obtain a solution having a concentration of 70 USP Chorionic Gonadotropin Units per mL, readjusting the pH, if necessary, to 7.2 ± 0.2.
Standard preparations—
Dissolve an accurately weighed quantity of
USP Menotropins RS in the
Diluting solution to obtain solutions having known concentrations of about 2.5, 5.0, and 10.0 USP Follicle-Stimulating Hormone Units per mL.
Assay preparations—
Following the procedure given under the Standard preparations, use Menotropins in place of the USP Reference Standard to obtain similar solutions.
Control solution—
Use the
Diluting solution as the control solution. Store all solutions at 5 ± 3
for the duration of the assay, and properly dispose of any unused portions.
Test animals—
Select 20- to 21-day old female rats with weights within a 10-g range of each other. Proceed as directed under Test animals in the Assay for luteinizing hormone beginning with “House the animals.”
Dose determination trial—
Use the method described for Procedure to determine a 3-dose range in which the lowest dose produces a definite response in some of the rats in the low-dose group (as compared with the control group) and the highest dose produces a submaximal to maximal response in the high-dose group. Doses must be established in a geometric progression. The normal dose response range will occur between 0.5 and 6.0 USP Follicle-Stimulating Hormone Units total dose per rat. Useful dose ranges will vary with the sensitivity of the rat strain selected.
Procedure—
Inject each rat of each group subcutaneously in the dorsal area with 0.2 mL of the solution to which it was assigned. For Dose determination trial only, similarly inject each rat in the control group with 0.2 mL of the Control solution. Repeat these injections at approximately the same time of day after 24 hours and 48 hours. Twenty-four hours after the last injection, weigh each rat, sacrifice the animals, and carefully dissect out the ovaries of each rat, removing any fat and fibrous tissue. Thoroughly dry the ovaries by pressing against absorbent paper, avoiding damage to follicles on the ovary surface, and immediately weigh them to the nearest 0.2 mg, using a suitable balance.
Calculation—
Tabulate the observed ovarian pair weight for each rat designated by the symbol
y, for each dosage group of
f rats. For apparently outlying ovarian weight gain, an attempt may be made to correct the organ mass relative to the mass of the rat from which it was taken. For the
y-value in question, calculate for each of the
f rats in the appropriate group the ratio of ovarian weight to the total body weight. Reject the
y-value if its corresponding ratio differs from the rest of the group by more than 1.5 standard deviations.
If the data from one or more rats are missing, adjust to groups of equal size by suitable means (see
Replacement of Missing Values under
Design and Analysis of Biological Assays 111). Total the values of
y in each group, and designate each total as
T, using subscripts 1 to 3 for the three successive dosage levels and subscripts
S and
U for the Standard and the material under test, respectively. Test both the agreement in slope of the dosage-response lines for the Standard and for the material under test, and the lack of curvature as directed for a 3-dose balanced assay (see
Tests of Assay Validity under
Design and Analysis of Biological Assays 111). If the combined discrepancy as measured by
F3 exceeds its tabular value in
Table 9 (see
Combination of Independent Assays under
Design and Analysis of Biological Assays 111), regard these data as preliminary, and repeat the assay.
Determine the logarithm of follicle-stimulating hormone potency of the Menotropins taken by the formula:
M = (4iTA / 3TB) + log R,
in which
TA =
S(
TU TS);
TB =
S(
T3 T1);
i is the interval between successive log doses of both the
Standard preparation and
Assay preparation; and
R = vS /
vU is the ratio of the high dose of the Standard in USP Units
(vS) to the high dose of the Menotropins in mg
(vU). Compute the log confidence interval (see
Design and Analysis of Biological Assays 111).
Replication—
Repeat the entire determination at least once. Test the agreement among the two or more independent determinations, and compute the weights/mean log-potency
M and its confidence interval,
LC (see
Confidence Intervals for Individual Assays under
Design and Analysis of Biological Assays 111). If this exceeds 0.18, repeat the assay until the confidence interval of the combined results is 0.18 or less.
The potency P* is satisfactory if P* = antilog M is not less than 80% and not more than 125% of the labeled potency and if the log confidence interval does not exceed 0.18.
