Supplement I to the Japanese Pharmacopoeia Fourteenth Edition

1394 O‹cial Monographs for Part I
Supplement I, JP XIV
sample solution: both spectra exhibit similar intensities of
absorption at the same wavelengths.
(2) Determine the infrared absorption spectrum of Benzylpenicillin
Potassium as directed in the potassium bromide
disk method under the Infrared Spectrophotometry, and
compare the spectrum with the Reference Spectrum or the
spectrum of Benzylpenicillin Potassium Reference Standard:
both spectra exhibit similar intensities of absorption at the
same wave numbers.
(3) Benzylpenicillin Potassium responds to the Qualitative
Test (1) for potassium salt.
Optical rotation [a] 20
D : +270 – +3009(1.0 g calculated on
the dried basis, water, 50 mL, 100 mm).
pH The pH of a solution obtained by dissolving 1.0 g of
Benzylpenicillin Potassium in 100 mL of water is between
5.0 and 7.5.
Purity (1) Clarity and color of solution-A solution
obtained by dissolving 1 g of Benzylpenicillin Potassium in
10 mL of water is clear, and colorless or light yellow.
(2) Heavy metals—Proceed with 2.0 g of Benzylpenicillin
Potassium according to Method 4, and perform the test.
Prepare the control solution with 2.0 mL of Standard Lead
Solution (not more than 10 ppm).
(3) Arsenic—Prepare the test solution by incinerating
1.0 g of Benzylpenicillin Potassium according to Method 4,
and perform the test. In the incineration, use a crucible of
porcelain, and after addition of 10 mL of a solution of
magnesium nitrate hexahydrate in ethanol (95) (1 in 10) add
1 mL of hydrogen peroxide (30), then burn the ethanol (not
more than 2 ppm).
(4) Related substances—Dissolve 40 mg of Benzylpenicillin
Potassium in 20 mL of water, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
add water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with 20 mL eachof
the sample solution and the standard solution as directed
under the Liquid Chromatography according to the following
conditions, and determine each peak area by the automatic
integration method: the area of the peak other than
benzylpenicillin obtained from the sample solution is not
more than the peak area of benzylpenicillin from the standard
solution, and the total area of the peaks other than benzylpenicillin
from the sample solution is not more than 3
times the peak area of benzylpenicillin from the standard
solution.
Operating conditions—
Detector: An ultraviolet absorption photometer (wavelength:
254 nm).
Column: A stainless steel column 4.6 mm in inside diameter
and 25 cm in length, packed with octadecylsilanized
silica gel for liquid chromatography (7 mm in particle diameter).
Column temperature: A constant temperature of about
259C.
Mobile phase: A mixture of a solution of diammonium
hydrogen phosphate (33 in 5000) and acetonitrile (19:6),
adjusted the pH to 8.0 with phosphoric acid.
Flow rate: Adjust the ‰ow rate so that the retention time
of benzylpenicillin is about 7.5 minutes.
Time span of measurement: About 5 times as long as the
retention time of benzylpenicillin.
System suitability—
Test for required detection: Pipet 10 mL of the standard
solution, and add water to make exactly 100 mL. Conˆrm
that the peak area of benzylpenicillin obtained from 20 mL
of this solution is equivalent to 7 to 13z of that from 20 mL
of the standard solution.
System performance: Dissolve 40 mg of Benzylpenicillin
Potassium in 20 mL of water. Separately, dissolve 10 mg of
methyl parahydroxybenzoate in 20 mL of acetonitrile. To
1 mL of this solution add water to make 20 mL. Mix 1 mL
each of these solutions, and add water to make 100 mL.
When the procedure is run with 20 mL ofthissolutionunder
the above operating conditions, benzylpenicillin and methyl
parahydroxybenzoate are eluted in this order with the resolution
between these peaks being not less than 8.
System repeatability: When the test is repeated 5 times
with 20 mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak
area of benzylpenicillin is not more than 2.0z.
Loss on drying Not more than 1.0z (3 g, reduced pressure
not exceeding 0.67 kPa, 609C, 3 hours).
Assay Perform the test according to the Cylinder-plate
method as directed under the Microbial Assay for Antibiotics
according to the following conditions.
(1) Test organism—Staphylococcus aureus ATCC 6538
P
(2) Culture medium—Use the medium iii in 3) Medium
for other organisms under (1) Agar media for seed and base
layer.
(3) Standard solutions—Weigh accurately an amount of
Benzylpenicillin Potassium Reference Standard, equivalent
to about 40,000 units, dissolve in phosphate buŠer solution,
pH 6.0 to make exactly 100 mL, and use this solution as the
standard stock solution. Keep the standard stock solution at
not exceeding 59C and use within 2 days. Take exactly a suitable
amount of the standard stock solution before use, add
phosphate buŠer solution, pH 6.0 to make solutions so that
each mL contains 2 units and 0.5 units, and use these solutions
as the high concentration standard solution and the
low concentration standard solution, respectively.
(4) Sample solutions—Weigh accurately an amount of
Benzylpenicillin Potassium, equivalent to about 40,000
units, and dissolve in phosphate buŠer solution, pH 6.0 to
make exactly 100 mL. Take exactly a suitable amount of this
solution, add phosphate buŠer solution, pH 6.0 to make
solutions so that each mL contains 2 units and 0.5 units, and
use these solutions as the high concentration sample solution
and the low concentration sample solution, respectively.
Containers and storage
Containers—Tight containers.