CIPROFLOXACIN HYDROCHLORIDE - WHO · 81 ciprofloxacin hydrochloride RS or with the reference...

Working document QAS/19.809

July 2019

Draft for comments

CIPROFLOXACIN HYDROCHLORIDE 1

(CIPROFLOXACINI HYDROCHLORIDUM) 2

Draft proposal for revision for The International Pharmacopoeia 3

(July 2019) 4

DRAFT FOR COMMENTS 5

6

7 © World Health Organization 2019 8 9 All rights reserved. 10 11 This draft is intended for a restricted audience only, i.e. the individuals and organizations having received this draft. The draft 12 may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in part or in whole, in any 13 form or by any means outside these individuals and organizations (including the organizations' concerned staff and member 14 organizations) without the permission of the World Health Organization. The draft should not be displayed on any website. 15 16 Please send any request for permission to: 17 18 Dr Sabine Kopp, Group Lead, Medicines Quality Assurance, Technologies Standards and Norms, Department of Essential 19 Medicines and Health Products, World Health Organization, CH-1211 Geneva 27, Switzerland, e-mail: [email protected]. 20 21 The designations employed and the presentation of the material in this draft do not imply the expression of any opinion 22 whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or 23 of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate 24 border lines for which there may not yet be full agreement. 25 26 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 27 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and 28 omissions excepted, the names of proprietary products are distinguished by initial capital letters. 29 30 All reasonable precautions have been taken by the World Health Organization to verify the information contained in this draft. 31 However, the printed material is being distributed without warranty of any kind, either expressed or implied. The responsibility 32 for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for 33 damages arising from its use. 34 35 This draft does not necessarily represent the decisions or the stated policy of the World Health Organization. 36

Please send any comments you may have on this draft working document to Dr Herbert Schmidt,

Technical Officer, Medicines Quality Assurance, Technologies Standards and Norms (email:

[email protected]) by 15 September 2019.

Working documents are sent out electronically and they will also be placed on the WHO Medicines website

(http://www.who.int/medicines/areas/quality_safety/quality_assurance/guidelines/en/) for comments under

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http://www.who.int/medicines/areas/quality_safety/quality_assurance/guidelines/en/



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SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/19.809: 37

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41

42

43

[Note from the Secretariat. It is proposed to revise the monograph on Ciprofloxacin 44

hydrochloride in The International Pharmacopoeia. The monograph on Ciprofloxacin is 45

proposed for omission. 46

The revision is based on information found in other pharmacopoeias, in the scientific literature 47

and on laboratory investigations performed by a collaborating laboratory. 48

Changes from the current monograph are indicated in the text by insert or delete.] 49

50

51

52

53

54

Description Date

Monograph drafted. April 2019

Discussion at the informal Consultation on Screening

Technologies and Pharmacopoeial Specifications for

Medicines.

2–3 May 2019

Draft revision sent out for public consultation. July-September 2019

Presentation to the Fifty-fourth WHO Expert Committee on

Specifications for Pharmaceutical Preparations. October 2019

Further follow-up action as required.


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57

Graphic formula 58

59

Molecular formula. C17H18FN3O3,HCl,xH2O 60

Relative molecular mass. 367.8 (anhydrous) 61

Chemical name. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-62

quinolinecarboxylic acid hydrochloride; CAS Reg. No. 86483-48-9 (anhydrous) . 63

Description. A pale yellow, crystalline powder. 64

Solubility. Soluble in water R, slightly soluble in methanol R, very slightly soluble in 65

dehydrated ethanol R, practically insoluble in acetone R, in ethyl acetate R and in 66

dichloromethane R. 67

Category. Antibacterial, antituberculosis. 68

Storage. Ciprofloxacin hydrochloride should be kept in a tightly closed container, protected 69

from light. 70

Additional information. Ciprofloxacin hydrochloride is slightly hygroscopic. 71

Requirements 72

Definition. Ciprofloxacin hydrochloride contains not less than 98.0% and not more than 102.0% 73

(“Assay”, Method A) and not less than 99.0% and not more than 101.0% (“Assay”, Method B) 74


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of ciprofloxacin hydrochloride (C17H19ClFN3O3) calculated with reference to the anhydrous 75

substance. 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 spectrum obtained from 80

ciprofloxacin hydrochloride RS or with the reference spectrum of ciprofloxacin 81

hydrochloride. 82

B. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel 83

R6 as the coating substance and a mixture of 1 volume of acetonitrile R, 2 volumes of 84

ammonia (~260 g/l) TS, 4 volumes of methanol R, 4 volumes of dichloromethane R and 85

as the mobile phase. Apply separately to the plate as 1 cm bands, 5 μl of each of 2 86

solutions containing (A) 10 mg of the test substance per mL and (B) 10 mg of 87

ciprofloxacin hydrochloride RS per mL. Place an evaporating-dish containing 50 mL of 88

ammonia (~260 g/L) TS in the chromatographic chamber. Expose the plate to the 89

ammonia vapour in the closed chamber for 15 minutes. Withdraw the plate and transfer 90

to another chromatographic chamber containing the mobile phase to develop. After 91

removing the plate from the chromatographic chamber, allow it to dry in air for about 15 92

minutes, and examine the chromatogram in ultraviolet light (254 nm and 365 nm). 93

The principal spot obtained with solution (A) corresponds in position, appearance and 94

intensity to that obtained with solution (B). 95

C. Carry out the test as described under 1.14.4 High-performance liquid chromatography 96

using the conditions given under “Test for related substances”. Prepare the following 97

solutions in a mixture of 87 volumes of buffer solution and 13 volumes of acetonitrile. 98

For solution (1), dissolve 10.0 mg of the test substance in 100.0 mL. For solution (2), 99

dissolve 10.0 mg of ciprofloxacin hydrochloride RS in 100.0 mL. Inject 20 µL of 100

solutions (1) and (2). The retention time of the principle peak in the chromatogram 101

obtained with solution (1) correspond to the retention time of the peak due to 102

ciprofloxacin in the chromatogram obtained with solution (2). 103


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D. A 0.1 g/mL solution yields reaction B described under 2.1 General identification tests as 104

characteristic of chlorides. 105

pH value. pH of a 25 mg/mL solution of the test substance in carbon-dioxide-free water R, 106

3.5-4.5. 107

Clarity and colour of solution. A solution, containing 12.5 g of the test substance in carbon-108

dioxide-free water R, is clear and not more intensely coloured than reference solution GY5, 109

when compared as described under 1.11.2 Degree of coloration of liquids, Method II. 110

Heavy metals. For the preparation of the test solution, dissolve 0.25 g in water R and dilute 111

to 30 mL with the same solvent. Carry out a prefiltration. Determine the heavy metals content 112

in the filtrate as described under 2.2.3 Limit test for heavy metals, Procedure 1; Method B; not 113

more than 20 μg/g. 114

Sulfated ash (2.3). Not more than 1.0 mg/g. 115

Water. Determine, as described under 2.8 Determination of water by Karl Fischer Method, 116

Method A. Use 0.200 g of the test substance. The water content is not more than 67.0 mg/g. 117

Related substances. Prepare fresh solutions protected from light and perform the test without 118

delay. 119

Carry out the test as described under 1.14.4 High-performance liquid chromatography using a 120

stainless steel column (25 cm x 4.6 mm), packed with end-capped and base-deactivated 121

particles of silica gel, the surface of which has been modified with chemically-bonded 122

octadecylsilyl groups (5 μm).1 123

Prepare the following buffer solution. Carefully add 3.4 mL of phosphoric acid (~1440 g/L) 124

TS to 1600 ml of water R and mix. Adjust the pH with triethylamine R to a pH of 3.0 (+/- 0.1) 125

and dilute to 2000 mL with water R. 126

Use the following conditions for gradient elution: 127

1A Prodigy ODS 3 column was found suitable.


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• mobile phase A: buffer solution; and 128

• mobile phase B: acetonitrile R. 129

Time

(minutes)

Mobile phase A

(% v/v)

Mobile phase B

(% v/v)

Comments

0–10 87 13 Isocratic

10–11 87 to 50 13 to 50 Linear gradient

11–16 50 50 Isocratic

16–16.1 50 to 87 50 to 13 Return to initial composition

16.1–20 87 13 Re-equilibration

Operate with a flow rate of 1.5 mL per minute. As a detector, use an ultraviolet 130

spectrophotometer set at a wavelength of 278 nm and, for impurity A, at 263 nm. Maintain the 131

column temperature at 40 °C. 132

Prepare the solutions (1) to (3) in a mixture of 87 volumes of buffer solution and 13 volumes 133

of acetonitrile R. For solution (1), dissolve 25.0 mg of the test substance in 50.0 mL. For 134

solution (2), dilute 1.0 mL of solution (1) to 100.0 mL. Dilute 1.0 mL of this solution to 10.0 135

mL. For solution (3), dissolve 2.5 mg of ciprofloxacin hydrochloride for peak identification 136

RS (containing ciprofloxacin and the impurities B, C, D and E) and dilute to 5.0 mL. For 137

solution (4), dissolve 5.0 mg of ciprofloxacin impurity A RS in 5 mL ammonia (~17 g/L) TS 138

and dilute to 50.0 mL with water R. Dilute 2.0 mL to 100.0 mL with water R. 139

Inject alternately 50 µL each of solutions (1), (2), (3) and (4). 140

Use the chromatogram obtained with solution (3) to identify the peaks due to the impurities B, 141

C, D and E. Use the chromatogram obtained with solution (4) to identify the peak due to the 142

impurity A. The impurities are eluted at the following relative retention with reference to 143

ciprofloxacin (retention time about 9 minutes); impurity E about 0.4; impurity B about 0.6; 144

impurity C about 0.7; impurity D about 1.2; impurity A about 1.89. 145


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The test is not valid unless, in the chromatogram obtained with solution (3), the resolution 146

between the peaks due to impurity B and the peak due to impurity C is at least 1.3. Also, in 147

the chromatogram obtained with solution (2), the signal-to-noise ratio of the peak due to 148

ciprofloxacin is at least 20 and, in the chromatogram obtained with solution (4), the signal-to-149

noise ratio of the peak due to impurity A is at least 10. 150

In the chromatogram obtained with solution (1): 151

• the area of any peak corresponding to impurity E, when multiplied by a correction factor 152

of 6.7, is not greater than three times the area of the peak due to ciprofloxacin in the 153

chromatogram obtained with solution (2) (0.3 %) ; 154

• the area of any peak corresponding to impurity B, when multiplied by a correction factor 155

of 0.7, is not greater than two times the area of the peak due to ciprofloxacin in the 156

chromatogram obtained with solution (2) (0.2 %); 157

• the area of any peak corresponding to impurity C, when multiplied by a correction factor 158



• the area of any peak corresponding to impurity D, when multiplied by a correction factor 161



• the area of any peak corresponding to impurity A, recorded at 263 nm, is not greater than 164

two times the area of the peak due to impurity A, recorded at 263 nm, in the 165

chromatogram obtained with solution (4) (0.2%); 166

• the area of any other impurity peak is not greater than the area of the peak due to 167

ciprofloxacin in the chromatogram obtained with solution (2) (0.10 %); 168

• the sum of the corrected areas of any peak corresponding to impurities E, B, C or D and 169

the areas of all other impurity peaks, other than any peak corresponding to impurity A, 170

is not greater than five times the area of the peak due to ciprofloxacin in the 171

chromatogram obtained with solution (2) (0.5 %). Disregard any peak with an area less 172

than 0.5 times the area of the peak due to ciprofloxacin in the chromatogram in the 173

chromatogram obtained with solution (2) (0.05%). 174

Assay 175


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• Either test A or test B may be applied. 176

A. Carry out the test as described under 1.14.4 High-performance liquid chromatography, 177

using the conditions given below under “Related substances” with the following 178

modifications. 179

Prepare the following solutions in a mixture of 87 volumes of buffer solution and 13 180

volumes of acetonitrile R. For solution (1), dissolve 50.0 mg of the test substance in 181

100.0 mL. For solution (2), dissolve 50.0 mg of Ciprofloxacin hydrochloride RS in 100.0 182

mL. 183

Inject alternately 10 μL each of solutions (1) and (2). 184

Measure the areas of the peaks corresponding to ciprofloxacin obtained in the 185

chromatograms of solutions (1) and (2) and calculate the percentage content of 186

ciprofloxacin hydrochloride (C17H19ClFN3O3) using the declared content of 187

ciprofloxacin hydrochloride (C17H19ClFN3O3) in ciprofloxacin hydrochloride RS. 188

B. Prepare fresh solutions protected from light and perform the test without delay. 189

Dissolve 0.300 g in 80 mL of glacial acetic acid R and titrate with perchloric acid (0.1 190

mol/l) VS as described under 2.6. Non-aqueous titrations, determining the end point 191

potentiometrically. Each mL of perchloric acid (0.1 mol/l) VS is equivalent to 36.78 mg 192

of C17H19ClFN3O3. 193

194

195

Impurities 196

197


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A. 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 198

(fluoroquinolonic acid) (synthesis related impurity). 199

200

B. 1-cyclopropyl-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid 201

(desfluoro compound) (synthesis related impurity). 202

203

C. 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-204

carboxylic acid (ethylenediamine compound) (degradation product). 205

206

207

208

D. 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic 209

acid (7-Chloro-6-piperazinyl analog) (synthesis related impurity). 210


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211

E. 1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (decarboxylated 212

compound) (degradation product, synthesis related impurity). 213

214

F. 1-cyclopropyl-6-hydroxy-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic 215

acid (synthesis related impurity). 216

Reference substances evoked 217

Ciprofloxacin hydrochloride RS 218

ICRS under establishment. 219

220

Ciprofloxacin impurity A RS 221

ICRS to be established. 222

Ciprofloxacin hydrochloride for peak identification RS (containing ciprofloxacin and the 223

impurities B, C, D and E) 224

It is intended to refer to the corresponing reference substance established for the European 225

Pharmacopoeia. 226

C17H18FN3O3,HCl,H2O 227


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Relative molecular mass. 385.8 228

Chemical name. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-229

quinolinecarboxylic acid monohydrochloride monohydrate; CAS Reg. No. 86393-32-0. 230

Description. A pale yellow, crystalline powder. 231

Solubility. Soluble in water; slightly soluble in methanol R; very slightly soluble in ethanol 232

(~750 g/l) TS; practically insoluble in acetone R and dichloromethane R. 233

Category. Antibacterial. 234

Storage. Ciprofloxacin hydrochloride should be kept in a tightly closed container, protected 235

from light. 236

Requirements 237

Ciprofloxacin hydrochloride contains not less than 98.0% and not more than 102.0% of 238

C17H18FN3O3,HCl, calculated with reference to the anhydrous substance. 239

Identity tests 240

A. Carry out the examination as described under 1.7 Spectrophotometry in the infrared region. 241

The infrared absorption spectrum is concordant with the spectrum obtained from ciprofloxacin 242

hydrochloride RS or with the reference spectrum of ciprofloxacin hydrochloride. 243

B. Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R4 244

as the coating substance and a mixture of 4 volumes of methanol R, 4 volumes of 245

dichloromethane R, 2 volumes of ammonia (~260 g/l) TS, and 1 volume of acetonitrile R as 246

the mobile phase. Apply separately to the plate as 1-cm bands, 5 μl of each of 2 solutions 247

containing (A) 10mg of Ciprofloxacin hydrochloride per mL, and (B) 10 mg of ciprofloxacin 248

hydrochloride RS per mL. Place an evaporating-dish containing 50ml of ammonia (~260g/l) 249

TS in the chromatographic chamber. Expose the plate to the ammonia vapour in the closed 250

chamber for 15 minutes. Withdraw the plate and transfer to another chromatographic chamber 251

containing the mobile phase to develop. After removing the plate from the chromatographic 252

http://apps.who.int/phint/en/d/Jb.7.1.7/#toc_1_7

http://apps.who.int/phint/en/d/Jb.7.1.14.1/#toc_1_14_1


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chamber, allow it to dry in air for about 15 minutes, and examine the chromatogram in 253

ultraviolet light (254nm and 365nm). 254

The principal spot obtained with solution A corresponds in position, appearance, and intensity 255

with that obtained with solution B. 256

C. A 0.1 g/mL solution yields reaction B described under 2.1 General identification tests as 257

characteristic of chlorides. 258

Heavy metals. For the preparation of the test solution dissolve 0.25 g in water and dilute to 30 259

mL with the same solvent. Carry out the prefiltration. Determine the heavy metals content in 260

the filtrate as described under 2.2.3 Limit test for heavy metals, Method B; not more than 261

20μg/g. 262

Clarity and colour of solution. A solution of 0.25 g in 10 mL of carbondioxide- free water R 263

is clear and not more intensely coloured than standard colour solution Gn4 when compared as 264

described under 1.11.1 Colour of liquids. 265

Sulfated ash. Not more than 1.0 mg/g. 266

Water. Determine as described under 2.8 Determination of water by the Karl Fischer method, 267

Method B, using about 0.2 g of the substance; the water content is between 0.047 g/g and 0.067 268

g/g. 269

pH value. pH of a 25 mg/mL solution in carbon-dioxide-free water R, 3.0-4.5. 270

Fluoroquinolonic acid. Carry out the test as described under 1.14.1 Thin-layer 271

chromatography, using silica gel R4 as the coating substance and a mixture of 4 volumes of 272

methanol R, 4 volumes of dichloromethane R, 2 volumes of ammonia (~260 g/l) TS, and 1 273

volume of acetonitrile R as the mobile phase. Apply separately to the plate 5μl of each of 2 274

solutions containing (A) 10mg of Ciprofloxacin hydrochloride per mL, and for solution (B) 275

dissolve 10mg of fluoroquinolonic acid RS in a mixture of 0.10ml of ammonia (~100 g/l) TS 276

and 90 mL of water, and dilute to 100 mL with water. Dilute 2.0ml of this solution to 10 mL 277

with water. Place an evaporating-dish containing 50ml of ammonia (~260 g/l) TS in the 278

chromatographic chamber. Expose the plate to the ammonia vapour in the closed chamber for 279








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15 minutes. Withdraw the plate and transfer to another chromatographic chamber containing 280

the mobile phase to develop. After removing the plate from the chromatographic chamber, 281

allow it to dry in air for about 15 minutes, and examine the chromatogram in ultraviolet light 282

(254 nm). 283

The spot corresponding to fluoroquinolonic acid in the chromatogram obtained with solution 284

A is not more intense than that obtained with solution B (0.2%). 285

Related substances. Carry out the test as described below under "Assay". 286

Inject 50μl each of solutions A and F. Record the chromatogram for twice the retention time 287

of ciprofloxacin. 288

Measure the areas of the peak responses obtained in the chromatograms from solutions A and 289

F, and calculate the content of the related substances as a percentage. In the chromatogram 290

obtained with solution A, the areas of the peaks corresponding to the ethylenediamine 291

compound and the by-compound A are not greater than the corresponding peaks in the 292

chromatogram obtained with solution F (0.2%); the area of any other peak is not greater than 293

the area of the peak corresponding to the ethylenediamine compound in the chromatogram 294

obtained with solution F (0.2%); the sum of the areas of all the peaks, other than the principal 295

peak, is not greater than 2.5 times the area of the peak corresponding to the ethylenediamine 296

compound in the chromatogram obtained with solution F (0.5%). Disregard any peak with an 297

area less than 0.25 times the area of the peak corresponding to the ethylenediamine compound 298

in the chromatogram obtained with solution F (0.05%). 299

Assay. Determine as described under 1.14.4 High-performance liquid chromatography, using 300

a stainless steel column (25cm × 4.6mm) packed with particles of silica gel, the surface of 301

which has been modified with chemically bonded octadecylsilyl groups (5μm). As the mobile 302

phase, use a mixture of 87 volumes of phosphoric acid (~2.45 g/l) TS, adjusted to a pH of 3.0 303

with triethylamine R and 13 volumes of acetonitrile R. 304

Prepare the following solutions in the mobile phase to produce 50 mL: solution (A) contains 305

0.50 mg of Ciprofloxacin hydrochloride per mL; solution (B) contains 0.50 mg of ciprofloxacin 306

hydrochloride RS per mL; solution (C) contains 0.050 mg of 1-cyclopropyl-1,4-dihydro-4-oxo-307

7-(1-piperazin-1-yl)quinoline-3-carboxylic acid RS per mL (desfluoro compound); solution 308



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(D) contains 0.050 mg of 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-309

quinoline-3-carboxylic acid RS (ethylenediamine compound) per mL; solution (E) contains 310

0.050 mg of 7-chloro-1-cyclopropyl-1,4-dihydro-4-oxo-6-(piperazin-1-yl)quinoline-3-311

carboxylic acid RS (by-compound A) per mL. For solution (F) mix 0.1 mL of solution A with 312

1.0 mL of solution C, 1.0 mL of solution D, 1.0 mL of solution E, and dilute to 50 mL with the 313

mobile phase. 314

Operate with a flow rate of 1.5 mL per minute. As a detector use an ultraviolet 315

spectrophotometer set at a wavelength of about 278 nm. Maintain the temperature of the 316

column at 40 °C. 317

Inject 50μl of solution F. The following order of elution is obtained: desfluoro compound, 318

ethylenediamine compound, ciprofloxacin and by-compound A. The retention time of 319

ciprofloxacin is about 9 minutes. Adjust the sensitivity of the system so that the height of the 320

peak due to the ethylenediamine compound is at least 40% of the full scale of the recorder. The 321

assay is not valid unless the resolution between the peaks corresponding to the desfluoro 322

compound and the ethylenediamine compound is at least 1.3, and the resolution between the 323

peaks corresponding to ciprofloxacin and the by-compound A is at least 3.0. Inject 10μl of 324

solution B. The assay is not valid unless the relative standard deviation of the peak area of 325

ciprofloxacin is at most 1.0%. 326

Inject alternately 10μl each of solutions A and B. 327

Measure the areas of the peak responses obtained in the chromatograms from solutions A and 328

B, and calculate the percentage content of C17H18FN3O3,HCl. 329

330

*** 331

CIPROFLOXACIN HYDROCHLORIDE - WHO · 81 ciprofloxacin hydrochloride RS or with the reference...

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