Working document QAS/16.

681
February 2018
Document for comment

1 CLAVULANATE POTASSIUM
2 (KALII CLAVULANAS)
3 Revised draft proposed monograph for The International
4 Pharmacopoeia
5 (February 2018)
6 DRAFT FOR COMMENT
7
Should you have any comments on this draft, please send these to Dr Herbert Schmidt,
8 Medicines Quality Assurance Programme,
DRAFT FORTechnologies Standards and Norms, Department of
COMMENTS
Essential Medicines and Health Products, World Health Organization, 1211 Geneva 27,
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23 Essential Medicines and Health Products, World Health Organization, CH-1211 Geneva 27, Switzerland. Fax: (41-22) 791
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37
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38 SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/16.681:

39 Clavulanate potassium
40 (Kalii clavulanas)
41

42
Date 43
Revision drafted taking into consideration July 2016
44
specifications and tests published other
pharmacopoeias and the scientific literature 45
Presentation to WHO Expert Committee on October 2016
46
Specifications for Pharmaceutical
Preparations

Laboratory investigations to validate and July 2016 to September 2017

verify the laboratory investigations
Presentation to WHO Expert Committee on October 2017
Specifications for Pharmaceutical
Preparations

Draft revision sent out for public February to April 2018

consultation

Further follow-up action as required

 Working document QAS/16.681
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47 Clavulanate potassium

48 (Kalii clavulanas)

49 [Note from the Secretariat. It is proposed to include the monograph on Clavulanate potassium in The
50 International Pharmacopoeia. The monograph is based on laboratory investigations and on
51 information found in the Chinese Pharmacopoeia, the European Pharmacopoeia and the United
52 States Pharmacopeia.

53 Comments are in particular sought regarding the nature of the impurities listed on the transparency
54 list, i.e. whether they are synthesis-related impurities, degradation products or both.]

55

56 Molecular formula. C8H8KNO5.

57 Relative molecular mass. 237.3.

58 Graphic formula

59

60 Chemical name. Potassium (2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-

61 azabicyclo[3.2.0]heptane-2-carboxylate, CAS Reg. No.61177-45-5.

62 Description. A white or almost white, crystalline powder.

63 Solubility. Freely soluble in water R, slightly soluble in ethanol (~710 g/L) TS, very slightly
64 soluble in acetone R.

65 Category. β-Lactamase inhibitor.

66 Storage. Potassium clavulanate should be kept in tightly closed containers, protected from
67 light, at a temperature of 2 °C to 8 °C.

68 Additional information. Potassium clavulanate is hygroscopic.

69 Requirements
70 Definition. Potassium clavulanate contains not less than 96.5% and not more than 102.0% of
71 C8H8KNO5, calculated with reference to the anhydrous substance.

72 Manufacture. The method of production is validated to demonstrate that the substance, if

73 tested, would comply with the limit of not more than 0.01% for clavam-2-carboxylate using a
74 suitable method.

75

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76 Identity tests

77  Either tests A and D or tests B, C and D may be applied.

78 A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared
79 region. The infrared absorption spectrum is concordant with the reference spectrum of
80 potassium clavulanate.

81 B. Carry out the test as described under 1.14.4 High-performance liquid chromatography
82 using the conditions given under “Assay”. The retention time of the principal peak in the
83 chromatogram obtained from solution (1) is similar to that obtained from solution (2).

84 C. [Note from the Secretariat. It is intended to add a TLC test specific for clavulanic acid
85 and amoxicilline.]

86 D. Ignite a small quantity, dissolve the residue in water and filter. Add 2 mL of sodium
87 hydroxide (~80 g/L) TS to the filtrate. It yields the reaction described under 2.1 General
88 identification tests as characteristic of potassium.

89 Solution S. Dissolve 0.400 g of the test substance in carbon-dioxide-free water R and dilute
90 to 20.0 mL with the same solvent.

91 pH value (1.13). Dilute 5 mL of solution S to 10 mL with carbon dioxide-free water R; the

92 value lies between 5.5 to 8.0.

Specific optical rotation (1.4). Use solution S; α D = + 53 to + 63 with reference to the

20
93
94 anhydrous substance.

95 Polymeric impurities and other impurities absorbing at 278 nm

96 Prepare fresh solutions and perform the test without delay.

97 Dissolve 50.0 mg of the test substance in phosphate buffer, pH 7.0 (0.1 mol/L) TS and dilute
98 to 50.0 mL with the same buffer solution. Measure the absorbance immediately. The
99 absorbance (1.6) of the solution determined at 278 nm is not greater than 0.40.

100 Water. Determine as described under 2.8 Determination of water by the Karl Fischer
101 method, method A, using about 0.50 g of the substance; the water content is not more than 5
102 mg/g.

103 Related substances. Carry out the test as described under 1.14.4 High-performance liquid
104 chromatography using a stainless steel column (10 cm × 4.6 mm) packed with particles of
105 silica gel, the surface of which has been modified with chemically-bonded octadecylsilyl
106 groups (5 µm).1

1
A Waters Atlantis T3 column was found suitable.
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107 Prepare the following phosphate buffer, pH 4.0. Dissolve 7.8 g of sodium dihydrogen
108 phosphate R in about 800 mL of water R, adjust to pH 4.0 with phosphoric acid (~105 g/L)
109 TS and dilute to 1000.0 mL with the same solvent.

110 Use the following conditions for gradient elution:

111 mobile phase A: phosphate buffer, pH 4.0;

112 mobile phase B: a mixture of equal volumes of methanol R and mobile phase A.

113

Time Mobile phase Mobile phase Comments

A B
(minutes)
(% v/v) (% v/v)

0–4 100 0 Isocratic

4–15 100 to 50 0 to 50 Linear gradient

15–18 50 50 Isocratic

18–19 50 to 100 50 to 0 Return to initial

composition

19–30 100 0 Re-equilibration

114

115 Operate with a flow rate of 1 mL per minute. As a detector use an ultraviolet
116 spectrophotometer set at a wavelength of 230 nm. Maintain the column temperature at 40 °C.

117 Prepare the following solutions immediately before use in mobile phase A. For solution (1)
118 dissolve about 25 mg of the test substance and dilute to 25.0 mL. For solution (2) dilute 1.0
119 mL of solution (1) to 100.0 mL. For solution (3) dissolve 10 mg of lithium clavulanate R and
120 10 mg of amoxicillin trihydrate R and dilute to 100 mL.

121 Inject 20 µL of solution (3). The test is not valid unless in the chromatogram obtained the
122 resolution between the peaks due to clavulanate (retention time about 3 minutes) and the peak
123 due to amoxicillin (with a relative retention of about [value to be determined]) is at least 13.

124 Inject alternately 20 μL each of solution (1) and (2).

125 In the chromatogram obtained with solution (1) the following impurities, if present, are eluted
126 at the following relative retention with reference to clavulanate (retention time about 3
127 minutes): impurity E about 2.3; impurity G about 3.6.

128 In the chromatogram obtained with solution (1):

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129  the area of any peak corresponding to either impurity E or impurity G is not greater
130 than the area of the peak due to clavulanic acid in the chromatogram obtained with
131 solution (2) (1.0%);
132  the area of any other impurity peak is not greater than 0.2 times the area of the peak
133 due to clavulanic acid in the chromatogram obtained with solution (2) (0.2%);
134  the sum of the areas of all impurity peaks is not greater than 2 times the area of the
135 peak due to clavulanic acid in the chromatogram obtained with solution (2) (2.0%).
136 Disregard any peak with an area less than 0.05 times the area of the peak due to
137 clavulanic acid in the chromatogram obtained with solution (2) (0.05%).

138 Aliphatic amines. The method can be used to determine the following aliphatic amines: 1,1-
139 dimethylethylamine (impurity H); N,N,N’,N’-tetramethylethylenediamine (impurity J);
140 1,1,3,3-tetramethylbutylamine (impurity K); N,N’-diisopropylethane-1,2-diamine (impurity
141 L); 2,2’-oxybis(N,N-)dimethylethylamine (impurity M).

142 Carry out the test as described under 1.14.5 Gas chromatography. Use a fused-silica capillary
143 column, 50 m long and 0.53 mm in internal diameter, coated with poly(dimethyl)(diphenyl)
144 siloxane R (film thickness: 5 µm).

145 As an internal standard use a solution containing 0.5 µL of 3-methylpentan-2-one R per mL

146 of water R. For solution (1) transfer 1.00 g of the test substance to a centrifuge tube. Add 5.0
147 mL of the internal standard solution, 5.0 mL of sodium hydroxide (~8.5 g/L) TS, 10.0 mL of
148 water R, 5.0 mL of 2-methylpropanol R and 5 g of sodium chloride R. Shake vigorously for 1
149 minute. Centrifuge to separate the layers and use the upper layer. For solution (2) dissolve
150 80.0 mg of each of the following amines: 1,1-dimethylethylamine R; tetramethylethylene
151 diamine R; 1,1,3,3-tetramethylbutylamine R; N,N’-diisopropylethylenediamine R and 2,2’-
152 oxybis(N,N-dimethylethylamine) R in hydrochloric acid (~70 g/L) TS and dilute to 200.0 mL
153 with the same acid. Transfer 5.0 mL of this solution into a centrifuge tube. Add 5.0 mL of the
154 internal standard solution, 10.0 mL of sodium hydroxide (~8.5 g/L) TS, 5.0 mL of 2-
155 methylpropanol R and 5 g of sodium chloride R. Shake vigorously for 1 minute. Centrifuge
156 to separate the layers and use the upper layer.

157 As a detector use a flame ionization detector.

158 Use nitrogen R as the carrier gas at an appropriate pressure and a split ratio 1:10 with a flow
159 rate of about 6 mL/min.

160 Maintain the temperature of the column at 35 °C for 7 minutes, then raise the temperature at a
161 rate of 30 °C per minutes to 150 °C and maintain for 15 minutes. Keep the temperature of the
162 injection port at 200 °C and that of the flame ionization detector at 250 °C.

163 Inject alternately 1 µL of solution (1) and solution (2).

164 In the chromatogram obtained with solution (1) the following impurities, if present, are eluted
165 at the following relative retention with reference to 3-methylpentan-2-one (internal standard,
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166 retention time about 11.4 minutes): impurity H about 0.55; impurity J about 1.07; impurity K
167 about 1.13; impurity L about 1.33; impurity M about 1.57.

168 Measure the peak responses of the aliphatic amines and of the internal standard. Calculate the
169 percentage content of each impurity using the ratios of the responses of the each aliphatic
170 amine to the responses of the internal standard. Use the ratios of the peak responses of the
171 corresponding reagents as a reference. The sum of the percentage contents of all aliphatic
172 amines is less than 0.2%.

173 2-Ethylhexanoic acid. Carry out the test as described under 1.14.5 Gas chromatography.
174 Use a fused-silica capillary column 10 m long and 0.53 mm in internal diameter coated with
175 macrogol 20000 2-nitroterephthalate R (film thickness: 1.0 µm).

176 As an internal standard use a solution containing 1.0 mg 3-cyclohexylpropionic acid R per
177 mL of cyclohexane R. For solution (1) transfer 1.00 g of the test substance to a centrifuge
178 tube. Add 4.0 mL of a 33% (V/V) solution of hydrochloric acid R. Shake vigorously for 1
179 minute with 1.0 mL of the internal standard solution. Allow the phases to separate (if
180 necessary, centrifuge for a better separation). Use the upper layer. For solution (2) dissolve
181 75.0 mg of 2-ethylhexanoic acid R in the internal standard solution and dilute to 50.0 mL
182 with the same solution. To 1.0 mL of the solution add 4.0 mL of a 33% (V/V) solution of
183 hydrochloric acid R. Shake vigorously for 1 minute. Allow the phases to separate (if
184 necessary, centrifuge for a better separation). Use the upper layer.

185 As a detector use a flame ionization detector.

186 Use nitrogen as the carrier gas at an appropriate pressure with a flow rate of about 6
187 mL/minute.

188 Maintain the temperature of the column at 40 °C for 2 minutes, then raise the temperature at a
189 rate of 30 °C per minutes to 200 °C and maintain for 3 minutes. Keep the temperature of the
190 injection port at 200 °C and that of the flame ionization detector at 300 °C.

191 Inject alternately 1 µL of solution (1) and solution (2).

192 The test is not valid unless the resolution between the peaks due to 2-ethylhexanoic acid (first
193 peak) and due to the internal standard is at least 2.0.

194 Measure the peak responses of 2-ethylhexanoic acid and of the internal standard. Calculate
195 the percentage content of 2-ethylhexanoic acid in the test substance using the ratios of the
196 responses of 2-ethylhexanoic acid to the responses of the internal standard; the content is not
197 more than 0.8%.

198 Assay. Carry out the test as described under 1.14.4 High-performance liquid chromatography
199 using a stainless steel column (40 cm x 4.6 mm) packed with particles of silica gel, the
200 surface of which has been modified with chemically-bonded octadecylsilyl groups (10 µm).2

2
A Zorbax eclipse XDB-C18 column was found suitable.

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201 Prepare the following phosphate buffer, pH 4.0. Dissolve 15 g of sodium dihydrogen
202 phosphate R in about 800 mL of water R, adjust to pH 4.0 with phosphoric acid (~105 g/L)
203 TS and dilute to 1000.0 mL with the same solvent.

204 As the mobile phase use a mixture of 5 volumes of methanol R and 95 volumes of phosphat
205 buffer, pH 4.0.

206 Operate with a flow rate of 1 mL per minute. As a detector use an ultraviolet
207 spectrophotometer set at a wavelength of 230 nm.

208 Prepare the following acetate buffer, pH 6.0. Dissolve 4.1 g of sodium acetate R in about 800
209 mL of water R, adjust to pH 6.0 with glacial acetic acid R and dilute to 1000.0 mL with the
210 same solvent.

211 Prepare the following solutions immediately before use, using acetate buffer, pH 6.0 as the
212 solvent. For solution (1) dissolve 50.0 mg of the test substance and dilute to 50.0 mL. For
213 solution (2) dissolve 50.0 mg of lithium clavulanate RS and dilute to 50.0 mL. For solution
214 (3) dissolve 10 mg of amoxicillin trihydrate R in 10 mL of solution (2).

215 Inject 10 μL of solution (3). The assay is not valid unless the resolution between the peaks
216 due to clavulanate (retention time about 5 minutes) and the peak due to amoxicillin (with a
217 relative retention of about [value to be determined]) is at least 3.5.

218 Measure the areas of the peaks corresponding to clavulanate obtained in the chromatograms
219 of solution (1) and (2) and calculate the percentage content of C8H8KNO5, using the declared
220 content of clavulanic acid (C8H9NO5) in lithium clavulanate RS. 1 mg of clavulanic acid
221 (C8H9NO5) is equivalent to 1.191 mg of potassium clavulanate C8H8KNO5.

222

223 Impurities

224

225

226 A. 2,2’-(pyrazine-2,5-diyl)diethanol

227

228

229 B. 3-[3,6-bis(2-hydroxyethyl)pyrazin-2-yl]propanoic acid

230
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231

232 C. 2,2’-(3-ethylpyrazine-2,5-diyl)diethanol

233

234

235 D. 4-(2-hydroxyethyl)-1H-pyrrole-3-carboxylic acid

236

237

238 E. (2R,4R,5Z)-2-(carboxymethyl)-5-(2-hydroxyethylidene)-3-[[(2R,3Z,5R)-3-(2-
239 hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]oxazolidine-4-
240 carboxylic acid

241

242

243 F. 4-[[[[4-(2-hydroxyethyl)-1H-pyrrol-3-yl]carbonyl]oxy]methyl]-1H-pyrrole-3-carboxylic
244 acid

245

246

247 G. 4-[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-4-oxobutanoic acid (N-

248 (hydrogensuccinyl)tyrosine)

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249

250

251 H. 2-methylpropan-2-amine (2-amino-2-methylpropane, tert-butylamine, 1,1-

252 dimethylethylamine)

253

254

255 J. N,N,N’,N’-tetramethylethane-1,2-diamine (1,2-bis(dimethylamino)ethane, N,N,N’,N’-

256 tetramethylethylenediamine)

257

258

259 K. 2,4,4-trimethylpentan-2-amine (2-amino-2,4,4-trimethylpentane, 1,1,3,3-

260 tetramethylbutylamine)

261

262

263 L. N,N’-diisopropylethane-1,2-diamine (N,N’-bis(1-methylethyl)ethane-1,2-diamine)

264

265

266 M. 2,2’-oxybis(N,N-dimethylethanamine), bis[2-(dimethylamino)ethyl] ether, N,N,N’,N’-

267 tetramethyl(oxydiethylene)diamine)

268

269
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270 Reagents to be established

271 Amoxicillin trihydrate R

272 Amoxicillin trihydrate of a suitable quality should be used.

273 3-Cyclohexylpropionic acid R

274 C9H16O2

275 Molecular weight. 156.2.

276 Description. Clear liquid.

277 Relative density . About 0.998.

278 Boiling point. About 130 °C.

279 N,N’-Diisopropylethylenediamine R

280 C8H20N2

281 Molecular weight. 144.3.

282 Other name. N,N’-Bis(1-methylethyl)-1,2-ethanediamine.

283 Description. Colourless or yellowish, hygroscopic liquid, corrosive, flammable.

284 Relative density . About 0.798.

285 Boiling point. About 170 °C.

286 1,1-Dimethylethylamine R

287 C4H11N

288 Molecular weight. 73.1.

289 Other names. 2-Amino-2-methylpropane, tert-Butylamine.

290 Description. liquid,miscible with ethanol (~710 g/L) TS.

291 Relative density . About 0.694.

292 Boiling point. About 46 °C.

293 2-Ethylhexanoic acid R

294 C8H16O2

295 Molecular weight. 144.2.

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296 Description. Colourless liquid.

297 Relative density . About 0.91.

298 Related substances. Carry out the test as described under 1.14.5 Gas chromatography using
299 the conditions given in the test for 2-ethylhexanoic acid in the monograph on Potassium
300 clavulanate. Prepare the following solution: suspend 0.2 g of 2-ethylhexanoic acid in 5 mL of
301 water R, add 3 mL of 33% (V/V) solution of hydrochloric acid R and 5 mL of hexane R,
302 shake for 1 minute, allow the layers to separate and use the upper layer. Inject 1 µL of this
303 solution. The sum of the area of any peaks, other than the principal peak and the peak due to
304 the solvent, is not greater than 2.5% of the area of the principal peak.

305 Lithium clavulanate R

306 Lithium clavulanate of a suitable quality should be used.

307 Macrogol 20000 R

308 Description. White or almost white solid with a waxy or paraffin-like appearance.

309 Solubility. Very soluble in water, soluble in methylene chloride, practically insoluble in
310 alcohol, in fatty oils and in mineral oils.

311 Macrogol 20000 2-nitroterephthalate R

312 Macrogol 20000 R modified by treating with 2-nitroterephthalate acid.

313 Description. A hard, white or almost white, waxy solid.

314 Solubility. Soluble in acetone R.

315 3-Methylpentan-2-one R

316 C6H12O

317 Molecular weight. 100.2.

318 Description. Colourless, flammable liquid.

319 Relative density . About 0.815.

320 Boiling point. About 118 °C.

321 2-Methylpropanol R

322 C4H10O

323 Molecular weight. 74.1.

324 Other names. Isobutyl alcohol, 2-Methylpropan-1-ol.

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325 Description. Clear colourless liquid.

326 Solubility. Soluble in water, miscible with ethanol (~710 g/L) TS.

327 Relative density . About 0.80.

328 Boing point. About 107 °C.

329 2,2’-Oxybis(N,N-dimethylethylamine) R

330 C8H20N2O

331 Molecular weight. 160.3.

332 Other name. Bis(2-dimethylaminoethyl) ether.

333 Description. Colourless, corrosive liquid.

334 Relative density . About 0.85.

335 Phosphate buffer, pH 7.0 (0.1 mol/L) TS

336 Procedure. Dissolve 1.361 g of potassium dihydrogen phosphate R in 100.0 mL of water.

337 Adjust the pH using a 14,20 g/L solution of anhydrous disodium hydrogen phosphate R.

338 Poly(dimethyl)(diphenyl)siloxane R

339 Stationary phase for gas chromatography. Contains 95% of methyl groups and 5% of phenyl
340 groups.

341 Sodium hydroxide (~8.5 g/L) TS

342 A solution of sodium hydroxide R containing about 8.5 g/L of NaOH.

343 1,1,3,3-Tetramethylbutylamine R

344 C8H19N

345 Molecular weight. 129.3.

346 Other name. 2-Amino-2,4,4-trimethylpentane.

347 Description. Clear, colourless liquid.

348 Relative density . About 0.805.

349 Boiling point. About 140 °C.

350 Tetramethylethylenediamine R

351 C6H16N2

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352 Molecular weight. 116.2.

353 Other name. N,N,N’,N’-Tetramethylethylenediamine.

354 Description. Colourless liquid, miscible with water and with ethanol (~710 g/L) TS.

355 Relative density . About 0.78.

356 Boiling point. About 121 °C.

357 ***