methylthioninium chloride …...working document qas/16.675 page 3 57 methylthioninium chloride 58...
TRANSCRIPT
Working document QAS/16.675
July 2016
Draft for comments
1 2
METHYLTHIONINIUM CHLORIDE 3
(METHYLTHIONINII CHLORIDUM) 4
DRAFT MONOGRAPH FOR INCLUSION IN 5
The International Pharmacopoeia 6
(July 2016) 7
DRAFT FOR COMMENTS 8
9 Gg 10
DRAFT FOR COMMENTS 11
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18 19 20 21
© World Health Organization 2016 22
All rights reserved. 23
This draft is intended for a restricted audience only, i.e. the individuals and organizations having received this draft. 24 The draft may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in 25 part or in whole, in any form or by any means outside these individuals and organizations (including the 26 organizations' concerned staff and member organizations) without the permission of the World Health Organization. 27 The draft should not be displayed on any website. 28
Please send any request for permission to: 29 Dr Sabine Kopp, Group Lead, Medicines Quality Assurance Programme, Technologies Standards and Norms, 30 Department of Essential Medicines and Health Products, World Health Organization, CH-1211 Geneva 27, 31 Switzerland. Fax: (41-22) 791 4730; email: [email protected]. 32
The designations employed and the presentation of the material in this draft do not imply the expression of any 33 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, 34 territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines 35 on maps represent approximate border lines for which there may not yet be full agreement. 36
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This draft does not necessarily represent the decisions or the stated policy of the World Health Organization. 44
45
Should you have any comments on this draft, please send these to Dr Herbert Schmidt,
Medicines Quality Assurance Programme, Technologies Standards and Norms, Department of
Essential Medicines and Health Products, World Health Organization, 1211 Geneva 27,
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Working document QAS/16.675
page 2
SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/16.675: 46
METHYLTHIONINIUM CHLORIDE (METHYLTHIONINII CHLORIDUM) 47
48
49
Date
First draft received from a collaborating
laboratory
April 2016
Discussion at informal consultation on
quality control laboratory tools and
specifications for medicines
9–11 May 2016
Draft monograph sent out for public
consultation
July 2016
Presentation to WHO Expert Committee on
Specifications for Pharmaceutical
Preparations
October 2016
Further follow-up action as required
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Working document QAS/16.675
page 3
METHYLTHIONINIUM CHLORIDE 57
(METHYLTHIONINII CHLORIDUM) 58
59
60
[Note from the Secretariat. It is proposed to revise the monograph on 61
Methylthioninium chloride. 62
63
Changes from the current monograph are indicated in the text by insert or delete.] 64
65
66
Molecular formula. C16H18ClN3S (anhydrous); C16H18ClN3S∙H2O (monohydrate); 67
C16H18ClN3S∙3H2O (trihydrate); C16H18ClN3S∙5H2O (pentahydrate). 68
69
Relative molecular mass. 319.9 (anhydrous); 337.9 (monohydrate); 373.9 (trihydrate); 409.9 70
(pentahydrate). 71
72
Graphic formula 73
74
N
SNH3C
CH3
NCH3
CH3
Cl · n H2O
75
n=0 (anhydrous) 76
n=1 (monohydrate) 77
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n=3 (trihydrate) 78
n=5 (pentahydrate) 79
80
Chemical name. C.I. Basic Blue 9; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride; 81
CAS Reg. No. 61-73-4 (anhydrous). 82
C.I. Basic Blue 9 monohydrate; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride 83
monohydrate; CAS Reg. No. 122965-43-9 (monohydrate). 84
C.I. Basic Blue 9 trihydrate; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride trihydrate; 85
CAS Reg. No. 7220-79-3 (trihydrate). 86
C.I. Basic Blue 9 pentahydrate; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride 87
pentahydrate; CAS Reg. No. 32680-41-4 (penatahydrate). 88
89
Other name. Methylene blue 90
91
Description. Dark green crystals with a metallic lustre or a dark green, crystalline powder; 92
odourless or almost odourless. 93
94
Solubility. Sparingly soluble in water R; slightly soluble in ethanol (~750 g/L) TS; practically 95
insoluble in ether R. 96
97
Category. Antidote. 98
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99
Storage. Methylthioninium chloride should be kept in a tightly closed container, protected 100
from light, at a temperature not exceeding 30 °C. 101
102
Additional information. Methylthioninium chloride is hygroscopic. 103
104
Requirements 105
Definition. Methylthioninium chloride contains not less than 97.0% and not more than 106
101.0% not less than 93.0% and not more than 102.0% (“Assay”, method A) or not less than 107
98.0% and not more than 102.0% (“Assay”, method B) of C16H18ClN3S, calculated with 108
reference to the dried substance. 109
110
Identity tests 111
A. The absorption spectrum of a 5 μg/mL solution in hydrochloric acid (~70 g/l) TS, when 112
observed between 230 nm and 800 nm, exhibits 4 maxima at about 258 nm, 288 nm, 113
680 nm, and 745 nm. 114
B. Dissolve 1 mg in 10 mL of water; a deep blue colour is produced. Add 2.0 mL of 115
hydrochloric acid (~70 g/l) TS and 0.25 g of zinc R powder; the colour of the solution 116
is discharged; filter and expose the filtrate to the air; the blue colour of the solution 117
reappears. 118
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C. Mix 0.05 g with 0.5 g of anhydrous sodium carbonate R in a porcelain crucible. 119
Carefully heat the mixture to a red glow for 10 minutes. Cool, dissolve the residue in 120
10 mL of nitric acid (~130 g/l) TS and filter. The filtrate yields reaction A described 121
under 2.1 General identification tests as characteristic of chlorides. 122
Either tests A and F or any two of tests B, C, D or E together with test F may be applied. 123
124
A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared 125
region. The infrared absorption spectrum is concordant with the spectrum obtained from 126
methylthioninium chloride RS or with the reference spectrum of methylthioninium 127
chloride. 128
129
B. Carry out the test as described under 1.14.4 High-performance-liquid chromatography 130
using the conditions given under “Assay”, method A. The retention time of the principal 131
peak in the chromatogram obtained with solution (1) corresponds to the retention time of 132
the peak due to methylthioninium in the chromatogram obtained with solution (2). 133
134
C. The absorption spectrum (1.6) of a 5 μg/mL solution in hydrochloric acid (~70 g/L) TS, 135
when observed between 230 nm and 800 nm, exhibits 4 maxima at about 258 nm, 288 136
nm, 680 nm, and 745 nm. 137
138
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D. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel 139
R6 as the coating substance and a mixture of 3 volumes of acetic acid R, 3 volumes of 140
ethanol R and 4 volumes of water R as the mobile phase. Apply separately to the plate 2 141
µL of each of the following 2 solutions in methanol R containing (A) 0.1 mg of the test 142
substance per mL and (B) 0.1 mg of methylthioninium chloride RS per mL. After 143
removing the plate from the chromatographic chamber allow it to dry in air or in a 144
current of cool air. Examine the chromatogram in daylight. 145
146
The principal spot obtained with solution (A) corresponds in position, appearance and 147
intensity to that obtained with solution (B). 148
149
E. Dissolve 1 mg in 10 mL of water R; a deep blue color is produced. Add 2.0 mL of 150
hydrochloric acid (~70 g/L) TS and 0.25 g of zinc R powder; the color of the solution is 151
discharged. Filter and expose the filtrate to the air; the blue color of the solution 152
reappears. 153
154
F. Mix 0.05 g of the substance to be investigated with 0.5 g of anhydrous sodium carbonate 155
R in a porcelain crucible. Carefully heat the mixture to a red glow for 10 minutes. Cool, 156
dissolve the residue in 10 mL of nitric acid (~130 g/L) TS and filter. The filtrate yields 157
reaction A described under 2.1 General identification tests as characteristic of chlorides. 158
159
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Copper or zinc. Prepare the following solutions. For solution (1) Iignite 1.0 g in a porcelain 160
crucible using as low a temperature as practicable, until all of the carbon is oxidized. Cool the 161
residue, add 15 mL of nitric acid (~130 g/L) TS and boil for 5 minutes. For solution (2) 162
Separately prepare a reference solution by boiling boil a quantity of copper(II) sulfate R, 163
equivalent to 200 μg of Cu, with 15 mL of nitric acid (~130 g/L) TS for 5 minutes. Filter 164
separately the cooled test and reference solutions (1) and (2) and wash any residue with 10 165
mL of water. Combine the filtrate and washings of the test solution (1) and similarly combine 166
the filtrate and washings of the reference solution (2); add to each an excess of ammonia 167
(~100 g/L) TS and filter the solutions into 50 mL volumetric flasks. Wash the precipitates 168
with small portions of water, adding the washings to the filtrates; dilute the contents of each 169
flask with water to volume, mixing thoroughly. To 25 mL of each of the solutions add 10 mL 170
of hydrogen sulfide TS; no turbidity is produced within 5 minutes (absence of zinc) and any 171
dark colour produced in the test solution (1) is not more intense than that of the reference 172
solution (2) (the copper content is not more than 0.20 mg/g). 173
174
Iron. Mix 4 g with 200 mL of water R in a long-necked, round-bottomed flask, add 15 mL of 175
nitric acid (~1000 g/L) TS, heat carefully to boiling and continue boiling until the volume of 176
liquid is reduced to about 20 mL. Allow to cool, add 10 mL of sulfuric acid (~1760 g/L) TS 177
and mix. Heat to boiling and add small successive quantities of nitric acid (~1000 g/L) TS, 178
cooling before each addition, until a colourless liquid is obtained. Heat until white fumes are 179
evolved; if darkening occurs at this stage continue the treatment with nitric acid (~1000 g/L) 180
Working document QAS/16.675
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TS. Finally heat until white fumes are again evolved. Allow the colourless liquid to cool, add 181
25 mL of a saturated solution of ammonium oxalate R in water, and boil until the slight froth 182
completely subsides. Cool, dilute to 50 mL with water; 5 mL of the diluted solution complies 183
with the 2.2.4 Limit test for iron; not more than 0.10 mg/g. 184
185
Sulfated ash. Not more than 10 2.5 mg/g. 186
187
Loss on drying. Dry to constant weight at 105 °C for 5 hours; it loses not less than 80 mg/g 188
and not more than 220 240 mg/g. (The dried substance may be used to produce solution (4) of 189
the test “Related substances”). 190
191
Foreign dyes. Carry out the test as described under 1.14.1 Thin-layer chromatography, using 192
as the coating substance a slurry prepared from silica gel R1 and a mixture of equal volumes 193
of potassium dihydrogen phosphate (27.2 g/l) TS and disodium hydrogen phosphate (28.4 g/l) 194
TS. As the mobile phase, use a mixture of 20 volumes of 1-propanol R, 4 volumes of 195
anhydrous formic acid R, and 1 volume of water. Apply to the plate 2 μl of a solution 196
prepared by dissolving 25 mg of the test substance in sufficient methanol R to produce 10 197
mL. After removing the plate from the chromatographic chamber, allow it to dry in an oven at 198
105°C. At an Rf value of about 0.5, 3-4 spots appear, placed very close to each other, the 199
lowest spot being violet in colour and the others red, the intensity of the colour increasing in 200
ascending order of the spots. No other spot is detected. 201
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202
Related substances. 203
Carry out test as described under 1.14.4 High-performance liquid chromatography using the 204
chromatographic conditions as described under "Assay", method A. 205
206
Prepare the following solutions using as the diluent a mixture of 70 volumes of a 0.1% (v/v) 207
solution of trifluoroacetic acid R (mobile phase A) and 30 volumes of acetonitrile R (mobile 208
phase B). 209
210
For solution (1) dissolve about 50 mg of the substance to be examined and dilute to 50.0 mL. 211
Sonicate for 5 minutes. For solution (2) dilute 1.0 mL of solution (1) to 100.0 mL. For 212
solution (3) dilute 5.0 mL of solution (2) to 50.0 mL. For solution (4) dissolve 2.5 mg 213
methylthioninium chloride impurity A RS and dilute to 10.0 mL. Transfer 1.0 mL of this 214
solution to a 10 mL volumetric flask and make up to volume with solution (1). Alternatively, 215
dry the substance to be examined at 105°C for 5 h (the dried substance of the test “Loss on 216
drying” may be used), dissolve 100 mg of the dried substance and dilute to 100.0 mL. 217
Sonicate for 5 minutes. 218
219
Inject alternately 5 µL each of solutions (1), (2), (3), (4). 220
221
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Use the chromatograms obtained with solution (4) and solution (1) to identify the peak due to 222
impurity A. Impurity A is eluted at the relative retention of about 0.8 with reference to 223
methylthioninium (retention time about 11 minutes). The test is not valid unless the resolution 224
between the peaks corresponding to methylthioninium and impurity A is at least 3.5. 225
226
In the chromatogram obtained with solution (1): 227
the area of any peak corresponding to impurity A is not greater than 5 times the area 228
of the principal peak obtained with solution (2) (5.0%); 229
the area of any other impurity peak is not greater than the area of the principal peak 230
obtained with solution (3) (0.10%); 231
the sum of the areas of all impurity peaks, other than the peak corresponding to 232
impurity A, is not greater than 5 times the area of the principal peak obtained with 233
solution (3) (0.5 %). Disregard any peak with an area less than 0.5 times the area of 234
the principal peak obtained with solution (3) (0.05%). 235
236
Assay. Transfer about 0.3 g, accurately weighed, to a 100-mL volumetric flask, dissolve in 30 237
mL of water by warming on a water-bath, and allow the solution to cool. While shaking, add 238
50.0 mL of potassium dichromate (0.0167 mol/l) VS, dilute to volume with water, and mix. 239
Repeat the shaking intermittently for 10 minutes, and filter; discard the first 20 mL of the 240
filtrate. Transfer 50.0 mL of the filtrate to a glass-stoppered flask, add 40 mL of sulfuric acid 241
(~190 g/l) TS and 1 g of potassium iodide R, mix, and allow the closed flask to stand in the 242
Working document QAS/16.675
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dark for 5 minutes. Add 100 mL of water and titrate with sodium thiosulfate (0.1 mol/l) VS, 243
using starch TS as indicator, until a blue-green colour is obtained. Repeat the operation 244
without the substance being examined and make any necessary corrections. Each mL of 245
potassium dichromate (0.0167 mol/l) VS is equivalent to 10.66 mg of C16H18ClN3S. 246
247
Assay 248
• Either method A or B may be applied. 249
250
A. Carry out test as described under 1.14.4 High-performance liquid chromatography 251
using a stainless steel column (10 cm x 4.6 mm) packed with particles of silica gel, the 252
surface of which has been modified with chemically-bonded phenylsilyl groups (3.5 253
µm).1 254
255
Use the following conditions for gradient elution: 256
mobile phase A: 0.1 % (v/v) solution of trifluoroacetic acid R 257
mobile phase B: acetonitrile R. 258
259
Time
(minutes)
Mobile phase A
(% v/v)
Mobile phase B
(% v/v)
Comments
0–5 80 20 Isocratic
5–25 80 to 30 20 to 70 Linear gradient
1 An X-Bridge Phenyl column and a Phenomenex Luna 3 μm Phenyl-Hexyl column were found suitable.
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25–32 30 70 Isocratic
32–35 30 to 80 70 to 20 Return to initial
composition
35–40 80 20 Re-equilibration
260
Operate with a flow of 1.0 mL/min. As a detector us an ultraviolet spectrophotometer 261
set at a wavelength of 246 nm. Maintain the column temperature at 30 °C. 262
263
Prepare the following solutions using as diluent a mixture of 30 volumes of acetonitrile 264
R and 70 volumes of mobile phase A. For solution (1) dissolve about 50 mg of the 265
substance to be examined, accurately weighed, and dilute to 50.0 mL. Sonicate for 5 266
minutes. For solution (2) dissolve 50.0 mg of methylthioninium chloride RS and dilute 267
to 50.0 mL. Sonicate for 5 min. 268
269
Inject alternately 5 µL each of solutions (1) and (2). The test is not valid unless the 270
symmetry factor of methylthioninium is not more than 3.0. 271
272
Measure the areas of the peak responses obtained in the chromatograms from solutions 273
(1) and (2) and calculate the percentage content of methylthioninium chloride 274
(C16H18ClN3S), using the declared content of C16H18ClN3S in methylthioninium 275
chloride RS. 276
277
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B. Dissolve about 100 mg, accurately weighed, in sufficient ethanol (~457 g/L) TS to 278
produce 250.0 mL. Dilute 5.0 mL of this solution to 100.0 mL with ethanol (~457 g/L) 279
TS. Dilute 5.0 mL of this solution to 50.0 mL with ethanol (~457 g/L) TS. Measure the 280
absorbance (1.6) of a 1 cm layer of the diluted solution at the maximum at about 664 281
nm and calculate the percentage content of methylthioninium chloride (C16H18ClN3S) 282
using the absorptivity value of 2950 methylthioninium chloride. 283
[Note from the Secretariat. The absorptivity value is so far based on a single 284
determination. It is intended to perform further independent determinations to confirm 285
the value.] 286
287
288
Additional requirements for Methylthioninium chloride for parenteral use 289
290
Complies with the monograph for Parenteral preparations. 291
292
Bacterial endotoxins. If intended for use in the manufacture of a parenteral dosage forms 293
without a further appropriate procedure for the removal of bacterial endotoxins, carry out the 294
test as described under 3.4 Test for bacterial endotoxins; contains not more than 2.5 IU of 295
endotoxin RS per mg. 296
297
298
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Impurities 299
N
SNH
H3CN
CH3
CH3 300
A. 3-(Dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride (azure B). 301
302
N
SH2N NCH3
CH3 303
304
B. 3-Amino-7-(dimethylamino)phenothiazin-5-ium (azure A) 305
306
307
N
SH2N NH
CH3
308
309
C. 3-amino-7-(methylamino)phenotziazin-5-ium (azure C) 310
311
312
313
*** 314