1402 Naproxen / Official Monographs
hydroxide (7 in 10) and 5 mL of pyridine, and heat at 1009C
for 5 minutes: a red color is produced (chlorobutanol).
(2) Place 10 mL of Naphazoline and Chlorpheniramine
Solution in a glass-stoppered test tube, add 10 mL of ethanol
(95), 2 mL of sodium hydroxide TS and 1 mL of a solution
of copper (II) chloride dihydrate in ethanol (95) (1 in 10),
and shake: a blue color is produced (glycerin).
(3) To 20 mL of Naphazoline and Chlorpheniramine
Solution add 5 mL of sodium hydroxide TS, extract with 10
mL of diethyl ether, and separate the diethyl ether layer.
Take 5 mL of this solution, distil off the solvent, dissolve the
residue in 5 mL of methanol, and use this solution as the
sample solution. Separately, dissolve 0.01 g each of napha-
zoline nitrate and Chlorpheniramine Maleate RS in 10 mL
and 5 mL of methanol, respectively, and use these solutions
as standard solutions (1) and (2). Perform the test with these
solutions as directed under Thin-layer Chromatography
<2.03>. Spot 5 mL each of the sample solution and standard
solutions on a plate of silica gel with fluorescent indicator
for thin-layer chromatography. Develop the plate with a
mixture of chloroform, methanol, acetone and ammonia so-
lution (28) (73:15:10:2) to a distance of about 10 cm, and air-
dry the plate. Examine under ultraviolet light (main wave-
length: 254 nm): two spots obtained from the sample solu-
tion exhibit the same R f values as the spots from standard
solutions (1) and (2). Spray evenly Dragendorff's TS on the
plate: the spots from standard solutions (1) and (2) and the
corresponding spot from the sample solutions reveal an
orange color.
Assay Pipet 4 mL of Naphazoline and Chlorpheniramine
Solution, add exactly 4 mL of the internal standard solution,
then add water to make 10 mL, and use this solution as the
sample solution. Weigh accurately about 50 mg of naphazo-
line nitrate for assay, dried at 1059C for 2 hours, and about
0.1 g of Chlorpheniramine Maleate RS, dried at 1059C for 3
hours, dissolve in water to make exactly 100 mL. Pipet 4 mL
of this solution, add exactly 4 mL of the internal standard
solution, then add water to make 10 mL, and use this solu-
tion as the standard solution. Perform the test with 10 mL
each of the sample solution and standard solutions as di-
rected under Liquid Chromatography <2.01> according to
the following conditions, and calculate the ratios, QTa and
QTb, of the peak height of naphazoline and chlorphenira-
mine to that of the internal standard of the sample solution,
and the ratios, QSa and QSb, of the peak height of naphazo-
line and chlorpheniramine to that of the internal standard of
the standard solution.
Amount (mg) of naphazoline nitrate (C14H14N2.HNO3)
＝ MSa × QTa/QSa × 1/25
Amount (mg) of chlorpheniramine maleate
(C16H19ClN2.C4H4O4)
＝ MSb × QTb/QSb × 1/25
MSa: Amount (mg) of naphazoline nitrate for assay taken
MSb: Amount (mg) of Chlorpheniramine Maleate RS
taken
Internal standard solution—A solution of ethenzamide in
methanol (1 in 1000).
Operating conditions—
Detector: An ultraviolet absorption photometer (wave-
length: 254 nm).
Column: A stainless steel column, about 4 mm in inside
diameter and 25 to 30 cm in length, packed with octadecyl-
silanized silica gel for liquid chromatography (5 mm in parti-
cle diameter).
The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
JP XVIII
Column temperature: Room temperature.
Mobile phase: A mixture of acetonitrile and a solution of
sodium laurylsulfate (1 in 500) in diluted phosphoric acid
(1 in 1000) (1:1).
Flow rate: Adjust so that the retention time of chlor-
pheniramine is about 10 minutes.
Selection of column: Proceed with 10 mL of the standard
solution under the above operating conditions. Use a column
giving well-resolved peaks of the internal standard, naphazo-
line and chlorpheniramine in this order.
Containers and storage Containers—Tight containers.
Storage—Light-resistant.
Naproxen
ナプロキセン
C14H14O3: 230.26
(2S )-2-(6-Methoxynaphthalen-2-yl)propanoic acid
[22204-53-1]
Naproxen, when dried, contains not less than 98.5z
of naproxen (C14H14O3).
Description Naproxen occurs as white, crystals or crystal-
line powder. It is odorless.
It is freely soluble in acetone, soluble in methanol, in
ethanol (99.5) and in chloroform, sparingly soluble in diethyl
ether, and practically insoluble in water.
It dissolves in sodium hydroxide TS.
Identification (1) Dissolve 0.01 g of Naproxen in 5 mL of
methanol, add 5 mL of water, then add 2 mL of potassium
iodide TS and 5 mL of a solution of potassium iodate (1 in
100), and shake: a yellow to yellow-brown color develops.
To this solution add 5 mL of chloroform, and shake: a light
red-purple color develops in the chloroform layer.
(2) To 1 mL of a solution of Naproxen in ethanol (99.5)
(1 in 300) add 4 mL of hydroxylamine perchlorate-ethanol
TS and 1 mL of N, N?-dicyclohexylcarbodiimide-ethanol TS,
shake well, and allow to stand in lukewarm water for 20
minutes. After cooling, add 1 mL of iron (III) perchlorate-
ethanol TS, and shake: a red-purple color develops.
(3) Determine the absorption spectrum of a solution of
Naproxen in ethanol (99.5) (1 in 50,000) as directed under
Ultraviolet-visible Spectrophotometry <2.24>, and compare
the spectrum with the Reference Spectrum: both spectra
exhibit similar intensities of absorption at the same wave-
lengths.
(4) Determine the infrared absorption spectrum of
Naproxen, previously dried, as directed in the potassium
bromide disk method under Infrared Spectrophotometry
<2.25>, and compare the spectrum with the Reference Spec-
trum: both spectra exhibit similar intensities of absorption at
the same wave numbers.
Optical rotation <2.49> [a]25
D : ＋63.0 – ＋68.59(after dry-
ing, 0.1 g, chloroform, 10 mL, 100 mm).
Melting point <2.60> 154 – 1589
C
Purity (1) Clarity and color of solution—Dissolve 2.0 g
of Naproxen in 20 mL of acetone: the solution is clear. Per-
JP XVIII Official Monographs / Nartograstim (Genetical Recombination)
form the test with this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>: the absorbance at 400 nm
is not more than 0.070.
(2) Heavy metals <1.07>—Proceed with 2.0 g of Napro-
xen according to Method 2, and perform the test. Prepare
the control solution with 2.0 mL of Standard Lead Solution
(not more than 10 ppm).
(3) Arsenic <1.11>—Prepare the test solution with 2.0 g
of Naproxen according to Method 3, and perform the test
(not more than 1 ppm).
(4) Related substances—Conduct this procedure without
exposure to light, using light-resistant vessels. Dissolve
0.10 g of Naproxen in 10 mL of a mixture of ethanol (99.5)
and chloroform (1:1), and use this solution as the sample so-
lution. Pipet 2 mL of the sample solution, and add a mixture
of ethanol (99.5) and chloroform (1:1) to make exactly 100
mL. Pipet 5 mL of this solution, add a mixture of ethanol
(99.5) and chloroform (1:1) to make exactly 50 mL, and use
this solution as the standard solution. Perform the test with
these solutions as directed under Thin-layer Chromatogra-
phy <2.03>. Spot 10 mL each of the sample solution and
standard solution on a plate of silica gel with fluorescent in-
dicator for thin-layer chromatography. Develop the plate
with a mixture of hexane, dichloromethane, tetrahydrofuran
and acetic acid (100) (50:30:17:3) to a distance of about 12
cm, and air-dry the plate. Examine under ultraviolet light
(main wavelength: 254 nm): the spots other than the princi-
pal spot and the spot of the starting point obtained from the
sample solution are not more intense than the spot from the
standard solution.
Loss on drying <2.41> Not more than 0.5z (1 g, 1059C,
3 hours).
Residue on ignition <2.44> Not more than 0.1z (1 g).
Assay Weigh accurately about 0.5g of Naproxen, previ-
ously dried, add 100 mL of diluted methanol (4 in 5), dis-
solve by gentle warming if necessary, and titrate <2.50> with
0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phe-
nolphthalein TS). Perform a blank determination in the
same manner, and make any necessary correction.
Each mL of 0.1 mol/L sodium hydroxide VS
＝ 23.03 mg of C14H14O3
Containers and storage Containers—Well-closed contain-
ers.
Storage—Light-resistant.
Nartograstim (Genetical
Recombination)
ナルトグラスチム（遺伝子組換え）
C850H1344N226O245S8: 18905.65
[134088-74-7]
Nartograstim (Genetical Recombination) is an
aqueous solution in which a desired product is a
recombinant human granulocyte colony-stimulating
factor (G-CSF) analog. It is N-methionylated, and
threonine, leucine, glycine, proline and cysteine
The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
1403
residues at the positions, 1, 3, 4, 5 and 17 of G-CSF
are substituted by alanine, threonine, tyrosine, argi-
nine and serine, respectively. It is a glycoprotein con-
sisting of 175 amino acid residues.
It contains not less than 0.9 mg and not more than
2.1 mg of protein per mL, and not less than 4.0 × 108
units per mg of protein.
Description Nartograstim (Genetical Recombination) oc-
curs as a clear and colorless, liquid.
Identification (1) To a suitable amount of Nartograstim
(Genetical Recombination) add tris-sodium chloride buffer
solution (pH 8.0) so that each mL contains 1 mg of protein,
and use this solution as the sample solution. Put 0.1 mL of
the sample solution in the well of a 96-well microplate, allow
to stand at 59 C for not less than 10 hours, then remove the
liquid, and wash the well. Then to the well add 0.25 mL of
blocking TS for nartograstim test, and allow to stand at
room temperature for 1 hour. Remove the blocking TS, add
0.1 mL of rabbit anti-nartograstim antibody TS to the well,
and shake gently at room temperature for 3 hours. Remove
the rabbit anti-nartograstim antibody TS, and wash the well.
Then, add 0.1 mL of peroxidase labeled anti-rabbit antibody
TS, shake gently at room temperature for 2 hours, remove
the TS, and wash the well. Then, add 0.1 mL of 2,2?-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammoni-
um salt TS, allow to stand at room temperature for 10
minutes, add 0.1 mL of a solution of oxalic acid dihydrate (1
in 50), and name this well as the sample well. Separately,
proceed with 0.1 mL of tris-sodium chloride buffer solution
(pH 8.0) in the same manner as for the sample solution, and
name the well so obtained as the control well. When com-
pare the sample well and the control well, the sample well re-
veals a green color, while the control well reveals no color.
Washing procedure of well: To the well add 0.25 mL of
washing fluid for nartograstim test, allow to stand for 3
minutes, and remove the washing fluid. Repeat this proce-
dure 2 times more.
(2) To a suitable amount of Nartograstim (Genetical
Recombination) add water so that each mL contains 1 mg of
protein. Replace the solvent of 2 mL of this solution with
tris-calcium chloride buffer solution (pH 6.5). To 0.5 mL of
the solution so obtained add 0.5 mL of tris-calcium chloride
buffer solution (pH 6.5) and 5 mL of thermolysin solution (1
in 1000), allow to stand at 379C for 21 hours, and use this
solution as the sample solution. Separately, proceed with 2
mL of Nartograstim RS in the same manner as for the sam-
ple solution, and use the solution so obtained as the standard
solution. Perform the test with 20 mL each of the sample so-
lution and standard solution as directed under Liquid Chro-
matography <2.01> according to the following conditions,
and compare these chromatograms: the similar peaks appear
at the same retention times.
Operating conditions—
Detector: An ultraviolet absorption photometer (wave-
length: 220 nm).
Column: A stainless steel column 6 mm in inside diameter
and 15 cm in length, packed with octadecylsilanized silica gel
for liquid chromatography (5 mm in particle diameter).
Column temperature: A constant temperature of about
359C.
Mobile phase A: A mixture of water and trifluoroacetic
acid (1000:1).
Mobile phase B: A mixture of acetonitrile, water and
trifluoroacetic acid (900:100:1).
Flowing of mobile phase: Control the gradient by mixing
the mobile phases A and B as directed in the following table.