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: kopps@who.int. 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

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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

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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