Supplement I to the Japanese Pharmacopoeia Fourteenth Edition

1398 O‹cial Monographs for Part I
Supplement I, JP XIV
as the high concentration sample solution and the low concentration
sample solution, respectively.
Containers and storage
Bleomycin Sulfate
硫 酸 ブレオマイシン
Containers—Tight containers.
Change to read except the structural formula
and chemical name:
Bleomycin Sulfate contains not less than 1400 mg
(potency) and not more than 2000 mg (potency) per
mg. The potency of Bleomycin Sulfate is expressed as
mass (potency) of bleomycin A 2 (C 55 H 84 ClN 17 O 21 S 3 :
1451.00).
Description Bleomycin Sulfate occurs as a white to yellowish
white powder.
It is freely soluble in water, and slightly soluble in ethanol
(95).
It is hygroscopic.
Identiˆcation (1) To 4 mg of Bleomycin Sulfate add 5 mL
of copper (II) sulfate TS, and dissolve in water to make
100 mL. Determine the absorption spectrum of this solution
as directed under the Ultraviolet-visible Spectrophotometry,
and compare the spectrum with the Reference Spectrum:
both spectra exhibit similar intensities of absorption at the
same wavelengths.
(2) Determine the infrared absorption spectrum of
Bleomycin Sulfate as directed in the potassium bromide disk
method under the Infrared Spectrophotometry, and compare
the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave
numbers.
(3) Bleomycin Sulfate responds to the Qualitative Tests
(1)and(2)forsulfate.
pH The pH of a solution obtained by dissolving 10 mg of
Bleomycin Sulfate in 20 mL of water is between 4.5 and 6.0.
Content ratio of the active principle Dissolve 10 mg of
Bleomycin Sulfate in 20 mL of water, and use this solution
as the sample solution. Perform the test with 20 mL ofthe
sample solution as directed under the Liquid Chromatography
according to the following conditions, and determine
each peak area by the automatic integration
method: the peak area of bleomycin A 2 (the ˆrst principal
peak) is between 55z and 70z, thatofbleomycinB 2 (the
second principal peak) is between 25z and 32z, the total
peak area of bleomycin A 2 and bleomycin B 2 is not less than
85z, the peak area of demethylbleomycin A 2 (a peak having
the relative retention time of 1.5 – 2.5 against bleomycin A 2 )
is not more than 5.5z, and the total area of the rest peaks is
not more than 9.5z.
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
409C.
Mobile phase stock solution: Dissolve 0.96 g of sodium 1-
pentanesulfonate and 1.86 g of disodium dihydrogen
ethylenediamine tetraacetate dihydrate in 1000 mL of water
and 5 mL of acetic acid (100), and adjust the pH to 4.3 with
ammonia TS.
Mobile phase A: A mixture of the mobile phase stock
solution and methanol (9:1).
Mobile phase B: A mixture of the mobile phase stock solution
and methanol (3:2).
Flowing of the mobile phase: Control the gradient by
mixing the mobile phases A and B as directed in the following
table.
Time after injection
of sample (min)
Mobile phase
A(z)
Mobile phase
B(z)
0–60 100→ 0 0 → 100
60–75 0 100
Flow rate: About 1.2 mL/min
Time span of measurement: Twenty minutes after elution
of the peak of demethylbleomycin A 2 after the solvent peak.
System suitability—
System performance: When the procedure is run with
20 mL of the sample solution under the above operating conditions,
bleomycin A 2 and bleomycin B 2 areelutedinthis
order with the resolution between these peaks being not less
than 5.
System repeatability: When the test is repeated 6 times
with 20 mL of the sample solution under the above operating
conditions, the relative standard deviation of the peak area
of bleomycin A 2 is not more than 2.0z.
Purity (1) Clarity and color of solution—A solution
obtained by dissolving 0.08 g of Bleomycin Sulfate in 4 mL
of water is clear and colorless.
(2) Copper—Dissolve exactly 75 mg of Bleomycin Sulfatein10mLofdilutednitricacid(1in100),andusethis
solution as the sample solution. Separately, to exactly 15 mL
of Standard Copper Solution add diluted nitric acid (1 in
100) to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with the sample solution
and the standard solution as directed under the Atomic Absorption
Spectrophotometry according to the following conditions:
the absorbance of the sample solution is not more
than that of the standard solution (not more than 200 ppm).
Gas: Combustible gas—Acetylene
Supporting gas—Air
Lamp: Copper hollow-cathode lamp
Wavelength: 324.8 nm
Loss on drying Not more than 3.0z (60 mg, in vacuum,
phosphorus (V) oxide, 609C, 3 hours). Take the sample to be