707 707 JP XV Ocial Monographs / Human Chorionic Gonadotrophin

System performance: When the procedure is run with 10

mL of the standard solution under the above operating condi-
tions, gonadorelin and the internal standard are eluted in this
order with the resolution between these peaks being not less
than 3.
System repeatability: When the test is repeated 6 times with
10 mL of the standard solution under the above operating
conditions, the relative standard deviation of the ratio of the
peak area of gonadorelin to that of the internal standard is
not more than 1.5%.
Containers and storage ContainersTight containers.
StorageLight-resistant.
Human Chorionic Gonadotrophin
Chorionic Gonadotrophin

Human Chorionic Gonadotrophin is a dried preparation

of gonad-stimulating hormone obtained from the urine of
healthy pregnant women after the manufacturing process to
remove or inactivate the virus.
It contains not less than 2500 human chorionic gonadotro-
phin Units per mg, and contains not less than 3000 chorionic
gonadotrophin Units per mg protein.
It contains not less than 80z and not more than 125z of
the labeled human chorionic gonadotrophin Units.
Description Human Chorionic Gonadotrophin occurs as a
white to light yellow-brown powder.
It is freely soluble in water.
Identication Calculate b by the following equation, using
Y
3
and Y
4
obtained in the Assay: b is not less than 120.
b( E/I)
E(Y
3
Y
4
)/f
f: Number of test animals per group.
Ilog (T
H
/T
L
)
Purity (1) Clarity and color of solutionDissolve 0.05 g
of Human Chorionic Gonadotrophin in 5 mL of isotonic so-
dium chloride solution: the solution is clear and colorless or
light yellow.
(2) EstrogenInject subcutaneously into each of three
female albino rats or albino mice ovariectomized at least two
weeks before the test, single dose of 100 units according to
the labeled Units dissolved in 0.5 mL of isotonic sodium
chloride solution. Take vaginal smear twice daily, on the
third, fourth and fth day. Place the smear thinly on a slide
glass, dry, stain with Giemsa's TS, wash with water, and
again dry: no estrus gure is shown microscopically <5.01>.
Loss on drying <2.41> Not more than 5.0z(0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.03 EU/unit.
Abnormal toxicity Dilute Human Chorionic Gonadotro-
phin with isotonic sodium chloride solution so that each mL
of the solution contains 120 Units, and use this solution as
the sample solution. Inject 5.0 mL of the sample solution
into the peritoneal cavity of each of 2 or more of well-
nourished, healthy guinea pigs weighing about 350 g, and ob-
serve the conditions of the animals for more than 7 days: all
the animals exhibit no abnormalities.
Specic activity When calculate from the results obtained
by the Assay and the following test, the specic activity is not
less than 3000 human chorionic gonadotrophin Units per mg
protein.
(i) Sample solutionTo an exactly amount of Chorionic
Gonadotrophin add water to make a solution so that each
mL contains about 500 Units of human chorionic gonadotro-
phin according to the labeled amount.
(ii) Standard solutionWeigh accurately about 10 mg of
bovine serum albumin, and dissolve in water to make exactly
20 mL. To a suitable volume of this solution add water to
make four solutions containing exactly 300, 200, 100 and 50
mg of the albumin per mL, respectively.
(iii) ProcedurePipet 0.5 mL each of the sample solu-
tion and the standard solutions, put them in glass test tubes
about 18 mm in inside diameter and about 130 mm in length,
add exactly 5 mL of alkaline copper TS, mix, and allow the
tubes to stand in a water bath at 309C for 10 minutes. Then
add exactly 0.5 mL of diluted Folin's TS (1 in 2), mix, and
warm in a water bath at 309C for 20 minutes. Determine the
absorbances of these solutions at 750 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24> using a solution
obtained in the same manner with 0.5 mL of water as the
blank.
Plot the absorbances of the standard solutions on the
vertical axis and their protein concentrations on the horizon-
tal axis to prepare a calibration curve, and determine the pro-
tein content of the sample solution from its absorbance by us-
ing this curve. Then calculate the amount of the protein in the
sample.
Assay (i) Test animalsSelect healthy female albino rats
weighing about 45 to 65 g.
(ii) Standard solutionDissolve a quantity of Human
Chorionic Gonadotrophin Reference Standard in bovine se-
rum albumin-isotonic sodium chloride solution to prepare
four kinds of solutions, having 7.5, 15, 30 and 60 Units per
2.5 mL, respectively. Inject these solutions into four groups
consisting of ve test animals each, and weigh their ovaries,
as directed in procedure of (iv). Inject bovine serum albumin-
isotonic sodium chloride solution to another group, and use
this group as the control group. According to the result of
this test, designate the concentration of the reference stan-
dard which will increase the masses of the ovaries about 2.5
times the mass of the ovaries of the control group as a low-
dose concentration of the standard solution, and the concen-
tration 1.5 to 2.0 times the low-dose concentration as a high-
dose concentration. Dissolve a quantity of Human Chorionic
Gonadotrophin Reference Standard, in bovine serum al-
bumin-isotonic sodium chloride solution, and prepare a high-
dose standard solution S
H
and a low-dose standard solution
S
L
whose concentrations are equal to those determined by the
above test.
(iii) Sample solutionAccording to the labeled units,
weigh accurately a suitable quantity of Human Chorionic
Gonadotrophin, dissolve in bovine serum albumin-isotonic
sodium chloride solution, and prepare a high-dose sample so-
lution T
H
and a low-dose sample solution T
L
having Units e-
qual to the standard solutions in equal volumes, respectively.
708 708 JP XV Human Chorionic Gonadotrophin for Injection / Ocial Monographs
(iv) ProcedureDivide the test animals at random into 4
groups, A, B, C and D, with not less than 10 animals and
equal numbers in each group. Inject subcutaneously 0.5 mL
of S
H
, S
L
, T
H
and T
L
in each group for 5 days. On the sixth
day, excise the ovaries, remove the fat and other unwonted
tissues attached to the ovaries, and remove the adhering
water by lightly pressing between lter paper, and immediate-
ly weigh the ovaries.
(v) CalculationDesignate the mass of ovaries by S
H
, S
L
,
T
H
and T
L
as y
1
, y
2
, y
3
and y
4
, respectively. Sum up y
1
, y
2
, y
3
and y
4
on each set to obtain Y
1
, Y
2
, Y
3
and Y
4
.
Units per mg of Human Chorionic Gonadotrophin
antilog M(units per mL of S
H
)(b/a)
MIY
a
/Y
b
Ilog (S
H
/S
L
)log (T
H
/T
L
)
Y
a
Y
1
Y
2
Y
3
Y
4
Y
b
Y
1
Y
2
Y
3
Y
4
a: Mass (mg) of sample.
b: Total volume (mL) of the high dose of the test solution
prepared by diluting with bovine serum albumin-isotonic
sodium chloride solution.
F? computed by the following equation should be smaller
than F
1
against n when s
2
is calculated. And compute L (P
0.95) by the following equation: L should be not more than
0.3. If F? exceeds F
1
, or if L exceeds 0.3, repeat the test in-
creasing the number of the test animals or arranging the assay
method in a better way until F? is smaller than F
1
or L is not
more than 0.3.
F?(Y
1
Y
2
Y
3
Y
4
)
2
/4fs
2
f: Number of test animals per group.
s
2
Sy
2
(Y/f)/n
Sy
2
: The sum of the squares of each y
1
, y
2
, y
3
and y
4
.
YY
1
2
Y
2
2
Y
3
2
Y
4
2
n4(f1)
L2 (C1)(CM
2
I
2
)
CY
b
2
/(Y
b
2
4fs
2
t
2
)
t
2
: Value shown in the following table against n used to
calculate s
2
.
n t
2
F
1
n t
2
F
1
n t
2
F
1
1 161.45 13 4.667 25 4.242
2 18.51 14 4.600 26 4.225
3 10.129 15 4.543 27 4.210
4 7.709 16 4.494 28 4.196
5 6.608 17 4.451 29 4.183
6 5.987 18 4.414 30 4.171
7 5.591 19 4.381 40 4.085
8 5.318 20 4.351 60 4.001
9 5.117 21 4.325 120 3.920
10 4.965 22 4.301 3.841
11 4.844 23 4.279
12 4.747 24 4.260
Containers and storage ContainersTight containers.
StorageLight-resistant, and in a cold place.
Human Chorionic Gonadotrophin
for Injection
Chorionic Gonadotrophin for Injection

Human Chorionic Gonadotrophin for Injection is a

preparation for injection which is dissolved before use.
It contains not less than 80z and not more than
125z of the labeled human chorionic gonadotrophin
Units.
Method of preparation Prepare as directed under Injections
with Human Chorionic Gonadotrophin.
Description Human Chorionic Gonadotrophin for Injec-
tion occurs as a white to light yellow-brown powder or mass-
es.
Identication Proceed as directed in the Identication un-
der Human Chorionic Gonadotrophin.
pH <2.54> Prepare a solution so that each mL of isotonic
sodium chloride solution contains 2 mg of Human Chorionic
Gonadotorophin for Injection: the pH of this solution is be-
tween 5.0 and 7.0.
Loss on drying <2.41> Not more than 5.0z (0.1 g, in vacu-
um, phosphorus (V) oxide, 4 hours).
Bacterial endotoxins <4.01> Less than 0.03 EU/unit.
Uniformity of dosage units <6.02> When calculate the ac-
ceptance value using the mean of estimated contents of the u-
nits tested as M, it meets the requirements of the Mass varia-
tion test.
Foreign insoluble matter <6.06> Perform the test according
to the Method 2: it meets the requirement.
Insoluble particulate matter <6.07> Perform the test accord-
ing to the Method 1: it meets the requirement.
Sterility <4.06> Perform the test according to the Membrane
ltration method: it meets the requirement.
Assay Proceed as directed in the Assay under Human
Chorionic Gonadotrophin. The ratio of the assayed Units to
the labeled Units should be calculated by the following equa-
tion.
The ratio of the assayed Units to the labeled Units
antilog M
Containers and storage ContainersHermetic containers.
StorageLight-resistant, and in a cold place.
Human Menopausal Gonadotrophin

Human Menopausal Gonadotrophin is a dried

preparation of gonad-stimulating hormone obtained