Working document QAS/16.691

May 2018

Draft for comments

1 2

IVERMECTIN 3

(IVERMECTINUM) 4

DRAFT MONOGRAPH FOR INCLUSION IN 5

The International Pharmacopoeia 6

(May 2018) 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

The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 37 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 38 Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 39

All reasonable precautions have been taken by the World Health Organization to verify the information contained in 40 this draft. However, the printed material is being distributed without warranty of any kind, either expressed or 41 implied. The responsibility for the interpretation and use of the material lies with the reader. In no event, shall the 42 World Health Organization be liable for damages arising from its use. 43

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/16.691

page 2

SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/16.691: 46

Ivermectin 47

(Ivermectinum) 48

49

50

51

52

53

54

Date

Monograph drafted December 2016

Discussion at consultation on specifications

for medicines 2–4 May 2018

Laboratory investigations to develop,

optimize, verify, or validate the proposed

analytical tests and specifications

May 2017–March 2018

Draft revision sent out for public

consultation June–August 2018

Presentation to WHO Expert Committee on

Specifications for Pharmaceutical

Preparations

October 2018

Further follow-up action as required

Working document QAS/16.691

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IVERMECTIN 55

(IVERMECTINUM) 56

57

[Note from the Secretariat. The draft monograph is proposed for inclusion in The 58

International Pharmacopoeia. It was elaborated based on laboratory investigations 59

performed by a collaborating laboratory and on information provided by the 60

manufacturer and found in other pharmacopoeias.] 61

62

Molecular formula 63

64

Component R Molecular formula

H2B1a CH2-CH3 C48H74O14

H2B1b CH3 C47H72O14

65

Relative molecular mass. Component H2B1a 875.09; Component H2B1b 861.07. 66

Graphic formula 67

68

Chemical name. Mixture of 69

(2aE,4E,5′S,6S,6′R,7S,8E,11R,13R,15S,17aR,20R,20aR,20bS)-7-[[2,6-dideoxy-4-O-(2,70

6-dideoxy-3-O-methyl-α-L-arabino-hexopyranosyl)-3-O-methyl-α-L-arabino-hexopy71

ranosyl]oxy]-20,20b-dihydroxy-5′,6,8,19-tetramethyl-6′-[(1S)-1-methylpropyl]-3′,4′,5′72

,6,6′,7,10,11,14,15,17a,20,20a,20b-tetradecahydrospiro[11,15-methano-2H,13H,17H-f73

uro[4,3,2-pq][2,6]benzodioxacyclooctadecene-13,2′-[2H]pyran]-17-one (or 74

Working document QAS/16.691

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5-O-demethyl-22,23-dihydroavermectin A1a) (component H2B1a) and 75

(2aE,4E,5′S,6S,6′R,7S,8E,11R,13R,15S,17aR,20R,20aR,20bS)-7-[[2,6-dideoxy-4-O-(2,76

6-dideoxy-3-O-methyl-α-L-arabino-hexopyranosyl)-3-O-methyl-α-L-arabino-hexopy77

ranosyl]oxy]-20,20b-dihydroxy-5′,6,8,19-tetramethyl-6′-(1-methylethyl)-3′,4′,5′,6,6′,778

,10,11,14,15,17a,20,20a,20b-tetradecahydrospiro[11,15-methano-2H,13H,17H-furo[4,79

3,2-pq][2,6]benzodioxacyclooctadecene-13,2′-[2H]pyran]-17-one (or 80

5-O-demethyl-25-de(1-methylpropyl)-25-(1-methylethyl)-22,23-dihydroavermectin 81

A1a) (component H2B1b); CAS Reg. No. Ivermectin B1a: 71827-03-7; Ivermectin B1b: 82

70209-81-3. 83

84

Description. A white or yellowish-white, crystalline powder. 85

86

Solubility. Practically insoluble in water, freely soluble in dichloromethane R, soluble 87

in dehydrated ethanol R. 88

89

Category. Antifilarial. 90

91

Storage. Ivermectin should be kept in tightly closed containers, protected from light. 92

It should be stored between 2° and 8°. Where the use of an antioxidant is allowed, 93

store at 25°. Excursions are permitted between 15° and 30°. 94

95

Additional information. Ivermectin is slightly hygroscopic. It is a semisynthetic 96

product derived from a fermentation product. 97

98

Requirements 99

Definition. Ivermectin contains not less than 95.0% and not more than 102.0% of the 100

sum of the Ivermectin components H2B1a (C48H74O14) and H2B1b (C47H72O14), 101

calculated with reference to the anhydrous and solvent-free substance. The ratio of the 102

area percentages of component H2B1a / (H2B1a + H2B1b) is not less than 90.0%. 103

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104

Identity tests 105

Either test A alone or tests B or C together with test D may be applied. 106

107

A. Carry out the examination as described under 1.7 Spectrophotometry in the 108

infrared region. The infrared absorption spectrum is concordant with the 109

spectrum obtained from ivermectin RS or with the reference spectrum of 110

ivermectin. 111

112

B. Carry out the test as described under 1.14.4 High-performance liquid 113

chromatography using the conditions given under “Assay”. The retention time 114

of the principal peaks in the chromatogram obtained with solution (1) 115

corresponds to the retention time of the peaks due to ivermectin in the 116

chromatogram obtained with solution (2). 117

118

C. Carry out the test as described under 1.14.1 Thin-layer chromatography using 119

silica gel R4 or R2 as the coating substance and a mixture of 90 volumes of 120

dichloromethane R, 8 volumes of methanol R and 0.8 volume of ammonia 121

(~260 g/L) TS as the mobile phase. Apply separately to the plate 30 µL of each 122

of the following 2 solutions in methanol R containing (A) 7 mg of the test 123

substance per mL and (B) 7 mg of ivermectin RS per mL. Develop the plate. 124

After removing the plate from the chromatographic chamber allow it to dry in 125

air or in a current of air. Examine the chromatogram under ultraviolet light (254 126

nm). 127

128

The two partly separated spots in the chromatogram obtained with solution (A) 129

correspond in position, appearance, and intensity with the two partly separated 130

principal spots due to ivermectin in the chromatogram obtained with solution 131

(B). 132

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133

Specific optical rotation (1.4). Dissolve 0.250 g of the test substance in methanol R 134

and dilute to 10.0 mL with the same solvent; 20

Dα = −20 to –17 with reference to the 135

anhydrous and solvent-free substance. 136

137

Heavy metals. Use 1.0 g for the preparation of the test solution as described under 138

2.2.3 Limit test for heavy metals, Procedure 3; determine the heavy metals content 139

according to Method A; not more than 20 μg/g. 140

141

Sulfated ash (2.3). Not more than 1.0 mg/g, using about 1.0 g of the substance. 142

143

Clarity and colour of solution. Dissolve 1.0 g in 50 mL toluene R. This solution is 144

clear and not more intensely coloured than reference solution BY5, when compared as 145

described under 1.11.2 Degree of coloration of liquids, Method II. 146

147

Water. Determine as described under 2.8 Determination of water by the Karl Fischer 148

method, method A, using 0.500 g of the substance; the water content is not more than 149

10 mg/g. 150

151

Related substances Carry out the test as described under 1.14.4 High-performance 152

liquid chromatography, using the conditions given below under “Assay”. 153

154

Prepare the following solutions in methanol R. For solution (1) dissolve 40.0 mg of 155

the test substance and dilute to 50.0 mL. For solution (2) dilute 1.0 mL of solution (1) 156

to 100.0 mL. For solution (3) dilute 5.0 mL of solution (2) to 100.0 mL. 157

158

Inject alternately 20 μL each of solution (1), (2) and (3). Record the chromatograms 159

for about 2 times the retention time of the principal peak. 160

161

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The test is not valid unless in the chromatogram obtained with solution (1) the 162

resolution between the peak due to component H2B1b (with a relative retention time of 163

about 0.74 with reference to component H2B1a) and the peak due to component H2B1a 164

(retention time about 34 minutes) is at least 3.0, and the symmetry factor of the peak 165

due to component H2B1b is not greater than 2.5. In the chromatogram obtained with 166

solution (3) the signal-to-noise ratio of the principal peak is at least 10. 167

168

In the chromatogram obtained with solution (1): 169

the area of any impurity peak with a relative retention of 1.3 to 1.5 with 170

reference to the principal peak is not greater than 2.5 times the area of the 171

principal peak in the chromatogram obtained with solution (2) (2.5%); 172

the area of any other impurity peak is not greater than the area of the principal 173

peak in the chromatogram obtained with solution (2) (1 %); 174

the sum of the areas of all impurity peaks is not greater than 5 times the area 175

of the principal peak in the chromatogram obtained with solution (2) (5%). 176

Disregard any peak with an area less than the area of the principal peak in the 177

chromatogram obtained with solution (3) (0.05%). 178

179

Ethanol and formamide. Carry out the test as described under 1.14.5 Gas 180

chromatography, using a fused-silica capillary column 30 m long and 0.53 mm in 181

internal diameter coated with macrogol 20 000 R (film thickness: 1 µm). 182

183

As a detector use a flame ionization detector. Keep the detector at 250° C. Use 184

nitrogen for chromatography R as the carrier gas at a flow rate of 7.5 mL per minute. 185

Use as split injection ratio of 1:10. Keep the injection port at 200° C. 186

187

Use the gradient conditions described below. 188

189

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190

Time

(minutes) Temperature Comment

0–2 50° to 80° Linear gradient

2–8 80° to 240° Linear gradient

191

Prepare the following solutions: 192

193

Internal standard solution: Dilute 0.5 mL of propanol R to 100.0 mL with water R. 194

195

For solution (1) transfer 120 mg of the test substance to a centrifuge tube, dissolve in 196

2.0 mL of m-xylene R (if the solution is hazy, heat slightly to 30–40 °C while mixing), 197

add 2.0 mL of water R, mix thoroughly and centrifuge. Keeping the aqueous layer, 198

remove the upper m-xylene layer and extract it with an additional 2.0 mL of water. 199

Discard the m-xylene layer and combine the two aqueous layers. Add 1.0 mL of the 200

internal standard solution. Centrifuge and discard any remaining m-xylene. 201

202

For solution (2) dilute 3.0 g of dehydrated ethanol R to 100.0 mL with water R. 203

204

For solution (3) dilute 1.0 g of formamide R to 100.0 mL with water R. 205

206

For solution (4) dilute 5.0 mL of solution (2) and 5.0 mL of solution (3) to 50.0 mL 207

with water R. Transfer 2.0 mL of this solution to a centrifuge tube, add 2.0 mL of 208

m-xylene R, mix thoroughly and centrifuge. Remove the upper layer and extract it 209

with 2.0 mL of water R. Discard the upper layer and combine the aqueous layers. Add 210

1.0 mL of the internal standard solution. Centrifuge and discard any remaining 211

m-xylene. 212

213

For solution (5) dilute 10.0 mL of solution (2) and 10.0 mL of solution (3) to 50.0 mL 214

Working document QAS/16.691

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with water R. Transfer 2.0 mL of this solution to a centrifuge tube, add 2.0 mL of 215

m-xylene R, mix thoroughly and centrifuge. Remove the upper layer and extract it 216

with 2.0 mL of water R. Discard the upper layer and combine the aqueous layers. Add 217

1.0 mL of the internal standard solution. Centrifuge and discard any remaining 218

m-xylene. 219

220

Inject alternately 1 µL each of solutions (1), (4) and (5). Prepare calibration plots for 221

ethanol and formamide by plotting the ratios of peak responses of any peak due to 222

ethanol or formamide to the area of the corresponding peak due to the internal 223

standard versus the concentrations of solutions (4) and (5). Determine the 224

concentration of ethanol and formamide in solution (1), if present. In the event the 225

peak response is significantly outside the range found with solutions (4) and (5), 226

additional dilutions of solutions (2) and (3) should be prepared and injected to obtain 227

peak responses framing those found for solution (1); not more than 50 mg/g of ethanol 228

and not more than 30 mg/g of formamide. 229

230

Assay. Carry out the test as described under 1.14.4 High-performance liquid 231

chromatography, using a stainless-steel column (25 cm × 4.6 mm) packed with 232

particles of silica gel, the surface of which has been modified with chemically-bonded 233

octadecylsilyl groups (5 µm).1 234

235

As the mobile phase use a mixture of 13 volumes of water R, 35 volumes of methanol 236

R and 53 volumes of acetonitrile R. Operate with a flow rate of 1.5 mL per minute. As 237

a detector use an ultraviolet spectrophotometer set at a wavelength of 245 nm. 238

239

Prepare the following solutions in methanol R. For solution (1) dissolve 25.0 mg of 240

the test substance and dilute to 100.0 mL. For solution (2) dissolve 25.0 mg of 241

ivermectin RS and dilute to 100.0 mL. 242

1 A Restek or Agilent PoroShell column or a Zorbax SB C18 column were found suitable.

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243

Inject alternately 10 μL each of solution (1) and (2). Record the chromatograms for 244

about 2 times the retention time of the principal peak. 245

246

The test is not valid unless in the chromatogram obtained with solution (2) the 247

resolution between the peaks due to component H2B1b (with a relative retention of 248

about 0.74 with reference to component H2B1a) and due to component H2B1a 249

(retention time about 34 minutes) is at least 3.0 and the symmetry factor of the peak 250

due to component H2B1b is not greater than 2.5. 251

252

Measure the areas of the peaks corresponding to the components H2B1a and H2B1b 253

obtained in the chromatograms of solution (1) and (2) and calculate the percentage 254

content of ivermectin (component H2B1a and component H2B1b), considering the 255

assigned contents of component H2B1a and component H2B1b in ivermectin RS, and 256

the ratio H2B1a / (H2B1a + H2B1b). 257

258

Impurities 259

260

A. 5-O-demethylavermectin A1a (avermectin B1a), (synthesis-related impurity) 261

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262

B. 5-O-demethyl-25-de(1-methylpropyl)-25-(1-methylethyl) avermectin A1a 263

(avermectin B1b), (synthesis-related impurity) 264

265

C. (23S)-5-O-demethyl-23-hydroxy-22,23-dihydroavermectin A1a(avermectin B2a), 266

(synthesis-related impurity) 267

268

D. 5-O-demethyl-28-oxo-22,23-dihydroavermectin A1a (28-oxoH2B1a), (degradation 269

product) 270

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271

E. 5-O,12-didemethyl-12-ethyl-22,23-dihydroavermectin A1a 272

(12-demethyl-12-ethyl-H2B1a), (degradation product) 273

274

F. 275

5-O,12-didemethyl-25-de(1-methylpropyl)-12-ethyl-25-(1-methylethyl)-22,23-dihydr276

oavermectin A1a (12-demethyl-12-ethyl-H2B1b), (degradation product) 277

278

G. 279

(6R,13S,25R)-5-O-demethyl-28-deoxy-6,28-epoxy-13-hydroxy-25-[(1S)-1-methylpro280

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pyl]milbemycin B (H2B1a aglycone), (degradation product) 281

282

H. 283

4′-O-de(2,6-dideoxy-3-O-methyl-α-L-arabino-hexopyranosyl)-5-O-demethyl-22,23-d284

ihydroavermectin A1a, (degradation product) 285

286

I. 2,3-didehydro-5-O-demethyl-3,4,22,23-tetrahydroavermectin A1a (Δ2,3

H2B1a), 287

(degradation product) 288

289

J. 290

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2,3-didehydro-5-O-demethyl-25-de(1-methylpropyl)-25-(1-methylethyl)-3,4,22,23-tet291

rahydroavermectin A1a (Δ2,3

H2B1b), (degradation product) 292

293

K. (4R) and (4S)-5-O-demethyl-3,4,22,23-tetrahydroavermectin A1a (H4B1a isomers) 294

(synthesis-related impurity). 295

296

297

New reagents 298

m-xylene R 299

1,2-Dimethylbenzene; C8H10. 300

Description. Clear, colourless, flammable liquid, practically insoluble in water, 301

miscible with ethanol (~750 g/L) TS 302

Relative density = 0.881. 303

Refractive index =1.505. 304

Boiling point. About 144°C. 305

306

Reference substance to be established 307

Ivermectin RS 308

309

*** 310