Development of high performance liquid chromatography methods with charged aerosol detection for the determination of lincomycin, spectinomycin and its impurities in pharmaceutical products

Accepted Manuscript Title: Development of high performance liquid chromatography methods with charged aerosol detection for the determination of lincomycin, spectinomycin and its impurities in pharmaceutical products Author: K. Stypulkowska A. Blazewicz A. Brudzikowska M. Warowna – Grzeskiewicz K. Sarna Z. Fijalek PII: DOI: Reference:

S0731-7085(15)00213-7 http://dx.doi.org/doi:10.1016/j.jpba.2015.03.036 PBA 10029

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

16-1-2015 27-3-2015 29-3-2015

Please cite this article as: K. Stypulkowska, A. Blazewicz, A. Brudzikowska, M.W. Grzeskiewicz, K. Sarna, Z. Fijalek, Development of high performance liquid chromatography methods with charged aerosol detection for the determination of lincomycin, spectinomycin and its impurities in pharmaceutical products, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.03.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

 Simple and repeatable LC-CAD methods have been developed and tested.

2

 A parallel connection of HPLC system with MS and CAD was used.

3

 All peaks were identified by HPLC-MS/MS-TOF.

4

 Methods could be used as routine assay and purity test methods of pharmaceuticals.

ip t

1

5

cr

6 87

Ac ce p

te

d

M

an

us

9

1

Page 1 of 26

9

Development of high performance liquid chromatography methods

10

with charged aerosol detection for the determination of

11

lincomycin, spectinomycin and its impurities in pharmaceutical products

ip t

12 K. Stypulkowskaa*, A. Blazewicza, A. Brudzikowskaa,b ,

14

M. Warowna – Grzeskiewicza,c , K. Sarnaa, Z. Fijaleka,b a

National Medicines Institute, Pharmaceutical Chemistry Department, 30/34 Chelmska Str.,

16

00-725 Warsaw, Poland

17

b

18

Medical University of Warsaw, Department of Bioanalysis and Drug Analysis,

c

20

an

1 Banacha Str., 02-097 Warsaw, Poland

19

Medical University of Warsaw, Department of Inorganic and Analytical Chemistry, 1 Banacha Str., 02-097 Warsaw, Poland

M

21

Abstract

d

22 23

us

15

cr

13

Novel and simple liquid chromatography methods with charged aerosol detection (LC-

25

CAD) for simultaneous quantitation of lincomycin and spectinomycin and its related

26

substances have been developed and tested. This type of analysis is complicated due to the

27

different chromatographic behavior of these two agents and the lack of chromophores in

28

spectinomycin complex. CAD seems to be a promising alternative to overcome these

29

difficulties. It shows a consistent inter-analyte response, independent of chemical structure of

30

an analyte. It also enables the direct quantification of related substances for which no

31

reference standards were available, with good accuracy and precision. Chromatographic

32

separations were achieved using a C18 Hypersil® Gold column, with mobile phases consisting

33

of water, acetonitrile and trifluoroacetic acid. All impurities were identified using time-of-

34

flight mass spectrometry with electrospray ionization. The developed methods have been

35

successfully used in the routine quality control analysis of pharmaceutical preparations.

Ac ce p

te

24

2

Page 2 of 26

36 37 38 39

* Corresponding author, Tel.: +48 228412121, Fax: +48 223311563 e-mail address: [email protected] (K. Stypulkowska) Keywords: charged aerosol detector; spectinomycin; lincomycin; related substances.

41

ip t

40 1. Introduction

Spectinomycin produced by Streptomyces spectabilis belongs to aminoglycoside

43

antibiotics and lincomycin produced by Streptomyces lincolnensis is a lincosamide antibiotic.

44

Pharmaceuticals being a combination of these two antibiotics (in a 2:1 ratio) are widely used

45

in veterinary medicine for the treatment of bacterial gastrointestinal and respiratory infections

46

[11,22]. In human medicine, spectinomycin is used principally against Neisseria gonorrhoeae

47

[[33]]. Due to their natural synthesis, both of them remain a mixture of similar compounds.

48

Spectinomycin is the principal component of its complex. In solution, it undergoes a ring

49

opening and closure of the hemiketal function. This results in an equilibrium mixture of four

50

possible anomers, which may, or may not, co-elute in a chromatographic analysis. The

51

chemical structures of both mentioned antibiotic and their related substances are presented in

52

Fig. 1 and Fig. 2.

us

an

M

d

te

Ac ce p

53

cr

42

Few chromatographic methods combined with electrochemical detection (ED) [[44]]

54

and mass spectrometry (MS) [[55],[66]] have been reported in literature for the determination

55

of lincomycin. The European Pharmacopoeia (Ph. Eur.) defines a HPLC-UV method for

56

lincomycin assay and related substances test [[77]]. This method is rather simple to apply, it

57

uses a very popular UV detector and a C8 column and produces repeatable results, so it may

58

be used in a routine analysis successfully.

59

For spectinomycin, the current Ph. Eur. monographs define a reversed phase HPLC-

60

ED method after post-column derivatization [[88],[99]], whereas the United States

61

Pharmacopoeia (US pharmacopoeia ) prescribes gas chromatography with flame ionization

3

Page 3 of 26

detection (GC-FID) [[1010],[1111]] for the assay determination, without defining the related

63

substances test. Pharmacopoeial methods are hardly reproducible, so other methods have been

64

developed. Some are performed using pre-column or post-column derivatization procedures

65

[[1212],[1313],[1414]], which are typically cumbersome, difficult to reproduce and may

66

result in introduction of non-controlled impurities. Therefore, non-derivatizing HPLC

67

methods with ED [[1515],[1616],[1717]], MS [[1818],[1919]] and evaporative light scattering

68

detection (ELSD) [[2020],[2121],[2222]] have been developed.

cr

ip t

62

As far as we are aware, there are only two chromatographic methods with pulsed

70

amperometric detector (PAD) [[2323]] or MS [[2424]] describing the simultaneous

71

determination of both compounds in pharmaceuticals in one run. This type of analysis is

72

complicated due to the difference in the chromatographic behavior of these two agents.

M

an

us

69

Corona Charged Aerosol Detection (CAD) seems to be a promising alternative to

74

overcome the difficulties mentioned above. It shows consistent inter-analyte response,

75

independent of chemical structures of an analyte, under identical mobile phase and CAD

76

conditions.Its features have been described previously [[2525]] and An extensive overview of

77

CAD’s applications has been reported by Almeling et al. [[2626]]. Also, its usefulness in the

78

quantitative analysis of aminoglycosides has been reported [25,[2727],[2828],[2929]].

te

Ac ce p

79

d

73

This manuscript describes the development and validation of simple, repeatable and

80

accurate HPLC-CAD methods to determine the assay of lincomycin and spectinomycin with

81

its related substances in 18 minutes.

82 83

2. Materials and methods

84

2.1. Chemicals

85

All reagents were LC grade: methanol (MeOH) and acetonitrile (ACN) (Rathburn

86

Chemicals Ltd., Walkerburn, Scotland), trifluoroacetic acid (TFA) (AppliChem GmbH,

4

Page 4 of 26

Darmstadt, Denmark). Doubly distilled water additionally purified in the Nanopure

88

DiamondTM UV Deionization System from Barnstead (a part of Thermo Scientific, Marietta,

89

United States) was used throughout. Reference standards: Lincomycin hydrochloride CRS,

90

Spectinomycini dihydrochloridum CRS and Spectinomycin hydrochloride for system

91

suitability CRS containing impurities A, C, D, E, F and G were purchased from EDQM

92

(Strasbourg, France).

cr

ip t

87

The methods were tested on two products: soluble powder containing lincomycin

94

hydrochloride and spectinomycin sulphate at the concentration of 222 g + 444 g/1000g,

95

respectively (Sample A), and solution for injection containing 50 mg/ml of lincomycin

96

hydrochloride hydrate and 100 mg/ml of spectinomycin sulphate tetrahydrate (Sample B).

97

The interesting fact is that both products had the same trade name, but one was a registered

98

and commercially available pharmaceutical product. The other one was not registered in

99

Poland (the illegal product), but it was provided from the legal Polish market.

an

M

2.2. Equipment and conditions

d

100

us

93

An LC UltiMate® 3000 system consisted of a pump, a degasser, an autosampler, a

102

column heater and a pulse damper (DionexTM Softron GmbH, a part of Thermo Scientific,

103

Germering, Germany) was used. LC system was coupled simultaneously to a CoronaTM ultra

104

RSTM Charged Aerosol Detector (Thermo Scientific TM DionexTM) and a maXis 4G mass

105

spectrometer (Bruker Daltonik GmbH, Bremen, Germany) by a 1/10 T-split. Nitrogen

106

generator LCMS30-0 (Parker Donick Hunter, Cleveland, OH, USA) provided nitrogen gas to

107

the both aerosol detectors. Additional nitrogen of high purity >99 % was introduced to the

108

collision cell from a nitrogen cylinder. The CAD detection was performed using a medium

109

filter and the 100 pA detection range. LC-CAD data were processed with ChromeleonTM 7

110

and ChromeleonTM Validation ICH softwares (Thermo ScientificTM DionexTM).

Ac ce p

te

101

5

Page 5 of 26

A time-of-flight mass spectrometer with electrospray ionization (ESI-MS-TOF) maXis

112

4G from Bruker Daltonik was used for the peaks’ identification. The MS settings were: ESI

113

in the positive ion mode, dry gas (nitrogen) flow rate 8.0 l/min, the dry heater 180°C, the

114

capillary voltage 4500 V and end plate offset 500 V. MS data were recorded in the full scan

115

mode (from 50 to 1000 m/z). MS data analysis was evaluated with Hystar 3.2., ESI Compass

116

1.3 for micrOTOF/maXis and Data Analysis® Version 4.0 SP 4 softwares from Bruker

117

Daltonik.

cr

ip t

111

The chromatographic separations by LC-CAD were performed on a C18 Hypersil®

119

Gold analytical column (150 x 4.6 mm; 3 μm particle size, Thermo Scientific). The mobile

120

phases consisted of a mixture of 0.3 % TFA and acetonitrile in a ratio 98:2 (v/v) for

121

spectinomycin analysis and 80:20 (v/v) for lincomycin analysis, respectively. A flow rate was

122

0.6 ml/min, a column temperature was set at 25oC. Samples were maintained at 8oC in

123

autosampler and the injection volume was 10 µl in partial loop injection mode.

d

M

an

us

118

125

te

124

2.3. Standard Solutions Preparation

The standards of the analyzed substances were accurately weighed into volumetric

Ac ce p

126 127

flasks and dissolved with water to produce stock solutions, which were successively diluted

128

with water to obtain the required concentrations.

129

2.4. Sample Preparation

130

Samples were dissolved and diluted with water. For the assay determination samples

131

were prepared and left for 24 h so that equilibrium between spectinomycin and its anomers

132

was reached. For related substances tests, the solutions were prepared immediately before use

133

[8].

134

6

Page 6 of 26

135

3. Results and Discussion

136

3.1. LC methods development The hydrophilic nature of both compounds demanded the use of a rather highly

138

aqueous mobile phase with addition of an ion-pair agent. Different water-MeOH and water-

139

ACN mobile phases with addition of TFA or HFBA in isocratic and gradient elution modes

140

were investigated. A Hypersil® Gold C18 column was used, because it was suitable for the

141

analysis of other aminoglycosides previously [25,27]. The noise level when using HFBA was

142

too high, so TFA was chosen for further investigations. Mixtures of water-ACN showed a

143

more powerful separation. The effect of ACN content (0, 5, 10, 20, 40 %), TFA content (0.1,

144

0.2, 0.3, 0.4 %), flow rate (0.5, 0.6, 0.7 ml/min) and column temperature (20, 25, 35ºC) on the

145

peaks’ resolution were studied.

M

an

us

cr

ip t

137

For the spectinomycin analysis, the best mobile phase composition was 0.3 % TFA-

147

ACN in a 98:2 (v/v) ratio, whereas for lincomycin assay a higher content of acetonitrile (20

148

%) was demanded. To circumvent this problem, a one-step gradient was introduced in the 12

149

minute after the spectinomycin analysis. With such gradient each compound was eluted on a

150

defined plateau with a constant mobile phase composition, that is crucial for the usefulness of

151

CAD’s universal response. What is more, depending on the type of the analyzed product, each

152

analysis part may be used separately. The final elution program used is described in Table 1.

153

After each run a four-minute step in the initial chromatographic conditions, for the system

154

equilibration, was used.

te

Ac ce p

155

d

146

The other chromatographic parameters were as follow: a flow rate 0.6 ml/min, a

156

temperature column set at 25oC, a sampler temperature set at 8oC and the injection volume 10

157

µl. These conditions were optimal for the separation of ten related substances from the

158

spectinomycin in 12 minutes and for the separation lincomycin from lincomycin B and other

7

Page 7 of 26

159

impurities in 6 minutes. The representative LC-CAD chromatogram obtained under these

160

conditions is presented in Fig. 3. Also, different C18 columns were investigated. The best resolution was obtained with

162

the initial Hypersil® Gold column, however, similar results were also obtained when using a

163

TSKgel® ODS-100V column (150 x 4.6 mm; 3 μm, Tosoh Bioscience) and an Ascentis®

164

Express C18 column (150 x 4.6 mm; 5 μm, Sigma-Aldrich).

cr

165 3.2. Peaks assignment

us

166

ip t

161

Spectinomycin and lincomycin were identified as the major peaks in standard

168

solutions at 6.6 min and 15.3 min, respectively. The standards of (4R)-dihydrospectinomycin

169

and lincomycin B were not available, so they were identified as the minor peaks in standard

170

solutions and confirmed by LC-ESI-MS/MS-TOF at 8.6 min (4R)-dihydrospectinomycin) and

171

14.0 min (lincomycin B). Other impurities of spectinomycin were identified by injecting the

172

Spectinomycin for system suitability CRS solution (500 µg/ml) in the LC-MS full scan,

173

positive ion mode. Chromatograms were obtained by plotting the total ion current (TIC) as a

174

time function for [M+H]+ ions. For every peak MS and MS/MS spectra were recorded. The

175

MS spectra showed ions that were consistent with the expected protonated molecular ions

176

[M+H]+. However, for spectinomycin and impurities D and E the dominant ions were

177

[M+H2O+H]+. Figure 4 presents typical MS and MS/MS spectra and fragmentation pathways

178

of spectinomycin. The fragmentation scheme presented in this figure is also applicable to the

179

related substances.

Ac ce p

te

d

M

an

167

180

During the LC-CAD analysis a peak at the retention time of 9.7 min was found. The

181

LC-MS analysis identified its molecular ion mass as m/z [M+H]+ 305.2, which is 28 u less

182

than m/z [M+H]+ =333.2 of spectinomycin. This corresponds to a loss of carbon monoxide

183

from spectinomycin. Thus, the proposed molecular formula is C13H24N2O6.

8

Page 8 of 26

Degradation studies (see Section 3.3.7.1.) showed that during basic hydrolysis a big

185

peak at 7.8 min has appeared. On the basis of LC-ESI-MS/MS-TOF analysis this peak was

186

identified as the actinospectinoic acid (impurity B). The full peak assignment on the basis of

187

LC-ESI-MS/MS-TOF results is presented in Table 2.

ip t

184

188 3.3. Method validation

cr

189

The quantitative aspects of the proposed methods were examined according to the ICH

191

guideline [[3030]]. Validation parameters were evaluated using standard solutions of

192

spectinomycin dihydrochloride and lincomycin hydrochloride. Peak areas were evaluated in

193

the whole validation, except the LOD and LOQ determination.

194

3.3.1. Specificity

M

an

us

190

The specificity of the proposed method was determined by comparing any interfering

196

peaks from the blank injection and by checking peak purity of each peak via LC-ESI-MS-

197

TOF. The LC-MS analysis demonstrated no co-elution of any peak of interest. Also, two

198

peaks from the mobile phase at retention time of 3.5 and 3.8 min were observed, but no

199

interferences with the other peaks occurred (Fig. 3).

200

3.3.2. Linearity

te

Ac ce p

201

d

195

For both active substances the linearity was tested in two concentration ranges: the

202

first one for the assay and the second one for the related substances quantitation. Six

203

concentration levels were included. in the investigating ranges, in triplicate injections at each

204

level.

205

The obtained results were plotted as a linear and power function. The results are

206

presented in Table 3. There were no differences in the investigated ranges in the coefficient of

207

determination values (R2) between the linear and power functions. The linearity was found

9

Page 9 of 26

with very good R2 values, for both compounds, within a concentration range of one-order of

209

magnitude. The obtained results confirmed that, although the CAD response is nonlinear at a

210

range of four orders of magnitude, the signal is nearly linear in the smaller ranges

211

[[3131],[3232]].

212

3.3.3. Limits of detection and quantitation

ip t

208

The detection limit (LOD) and quantitation limit (LOQ) were defined as a signal-to-

214

noise ratio of 3:1 and 10:1, respectively. Peaks heights were evaluated for this validation

215

parameter. For lincomycin the LOD and LOQ values were 0.3 µg/ml and 1.0 µg/ml,

216

respectively. For spectinomycin the LOD and LOQ values were 0.5 µg/ml and 2.0 µg/ml,

217

respectively.

an

us

cr

213

The LOD and LOQ values for lincomycin and spectinomycin are different, because

219

the mobile phase composition is different. CAD is an aerosol-based detector and the intensity

220

of the response depends on the composition of a mobile phase – the higher organic content is,

221

the higher response is observed.

222

3.3.4. Accuracy

d

te

Ac ce p

223

M

218

The accuracy of the methods was evaluated by injecting in triplicate standard solutions

224

covering the linearity range. It was assessed by calculating the difference between the

225

measured concentrations of standard solutions and their known concentrations, and expressed

226

as percentage. The concentrations of the active substances in standard solutions were

227

measured by putting the peak area into the calibration curve created during the linearity study.

228

The accuracy results are shown in Table 4 at the α = 0.05 and they are satisfactory.

229

3.3.5. Precision

230

The precision of the methods was determined by intra-day precision (repeatability) and

231

inter-day (intermediate) precision experiments. The relative standard deviations concern the 10

Page 10 of 26

calculated concentrations of spectinomycin and lincomycin. The repeatability was calculated

233

on the basis of three consecutive injections at each concentration level. The inter-day

234

precision was calculated on the basis of the results from two days in two weeks’ period. All

235

data is summarized in Table 4.

ip t

232

Also, the precision of the methods in sample analysis was evaluated using 6 solutions

237

of Sample A at the test concentrations (see Section 3.4). Samples were tested due to

238

determination of the percentage contents of spectinomycin dihydrochloride, lincomycin

239

hydrochloride, (4R)-dihydrospectinomycin dihydrochloride and lincomycin B hydrochloride.

240

The RSD values for the assay of spectinomycin and lincomycin were 0.3 % and 0.2 %,

241

respectively. The RSD values for the content determination of (4R)-dihydrospectinomycin

242

and lincomycin B were 0.8 % and 0.5 %, respectively.

243

3.3.6. Robustness

M

an

us

cr

236

The methods’ robustness was evaluated during the method development. It was tested

245

by the variation of the acetonitrile content (0 and 5 %), TFA content (0.2 and 0.4 %), column

246

temperature (23 and 27 ºC) and flow rate (0.5 and 0.7 ml/min). The resolution (Rs) between

247

the peaks was used as the robustness index. The method is robust for the column temperature

248

± 2ºC and flow rate ± 0.1 ml/min. The acetonitrile and TFA content were demonstrated to be

249

critical parameters for the peaks resolution. Also, different C18 columns were investigated.

250

Similar results were obtained when using TSKgel® ODS-100V column (150 x 4.6 mm; 3 μm,

251

Tosoh Bioscience) and Ascentis® Express C18 column (150 x 4.6 mm; 5 μm, Sigma-Aldrich).

252

3.3.7. Stability

253

3.3.7.1. Stability of the methods

Ac ce p

te

d

244

254

The stability of the methods was investigated by conducting stress degradation studies

255

under following conditions: heat (dry heat 80 ºC for 72 h), acidic hydrolysis (1M HCl, dry

11

Page 11 of 26

heat 80ºC for 2 h), basic hydrolysis (1M NaOH, dry heat 80 ºC for 2 h) and oxidation (H2O2

257

3%, v/v, dry heat 80ºC for 0.5 h). The sample solutions at concentration of 500 µg/ml

258

spectinomycin and 250 µg/ml lincomycin were analyzed. Then, peak’s purity was conducting

259

by LC-ESI-MS-TOF. In all samples all peaks of interest had good peak purity.

ip t

256

The stability studies showed that analyzed substances are sensitive to stress conditions.

261

In heat conditions only small amount of both active substances decomposed, but during the

262

both type of the hydrolysis and oxidation spectinomycin and lincomycin decomposed almost

263

completely and a big amount of related compounds appeared.

264

3.3.7.2. Stability of the solutions

an

us

cr

260

The stability of the standard and sample solutions was evaluated by repeated injections

266

of the same samples during a period of 24 hours, one week and one month. Peak areas were

267

compared. Lincomycin solutions were stable during the stability test. Spectinomycin solutions

268

were unstable. After 24 hours, three other peaks appeared at the retention time of 5.6 min, 7.4

269

min

270

dihydrospectinomycin anomers (retention time 5.6 min and 7.4 min) and impurity B

271

(retention time 7.8 min.). After one month, the peak at retention time 7.8 min. increased

272

significantly and the peak area of spectinomycin decreased slightly. Therefore, spectinomycin

273

solutions for the related substances test were prepared immediately before use.

275

min.

These

d

7.8

peaks

te

and

were

identified

by

LC-ESI/MS/MS-TOF

as

Ac ce p

274

M

265

3.4. Application to pharmaceutical preparations

276

The developed and validated LC-CAD methods were tested on the commercially

277

available products. Peak areas were used for the calculations. The assay of spectinomycin was

278

calculated according to Ph. Eur. Thus, the factor of 1.062 was applied for the calculation of

279

spectinomycin

280

dihydrochloride [9]. The assay of the actives was determined in diluted samples (50 µg/ml of

sulphate

occurring

in

veterinary

12

preparations

from

spectinomycin

Page 12 of 26

lincomycin hydrochloride and 100 µg/ml of spectinomycin dihydrochloride after calculations)

282

with a reference to standard solutions. The quantity of related substances was evaluated in

283

samples at the concentration of 300 µg/ml of lincomycin hydrochloride and 500 µg/ml of

284

spectinomycin dihydrochloride. Standard solutions at concentration 5 µg/ml of each active

285

substance were used.

ip t

281

The assay of both actives was in agreement with the label. The content of

287

spectinomycin impurities A, C, D, E and F (according to the Ph. Eur.) was less than LOQ (2

288

µg/ml). The quantity of other related substances and lincomycin B complied with

289

pharmacopoeial requirements, so both analyzed preparations were good quality products. All

290

results are presented in Table 5.

an

us

cr

286

292

M

291 4. Conclusions

The elaborate LC-CAD methods provide a rapid, simple and robust analysis for

294

lincomycin and lincomycin B, spectinomycin and its related substances, due to a simple

295

composition of the mobile phases and a relatively short analysis time. The proposed

296

methodology may be applied successfully for the routine analysis of samples that contain only

297

one active agent or a mixture of such agents. In this paper we have demonstrated a Corona®

298

CAD to be an ideal detector for the analysis of different non-chromophore compounds

299

without any derivatization. It enables the direct quantitation of related substances for which

300

no reference standards are available, with good accuracy and precision. Due to unique

301

synergistic approach with MS and ELSD to the mobile phase composition (only volatile

302

components), the developed chromatographic conditions may be transferred directly from an

303

LC-CAD analysis to an LC-MS or LC-ELSD analysis. However, unlike these detectors, CAD

304

is more reproducible and robust. To the best of our knowledge, no analytical method has been

Ac ce p

te

d

293

13

Page 13 of 26

305

proposed for the simultaneous quantitation of lincomycin and spectinomycin and its

306

impurities in pharmaceuticals by LC-CAD so far.

307 Acknowledgements

309

Authors thank Sigma-Aldrich Poland for providing an Ascentis® Express column for this

310

project.

cr

ip t

308

311 Figure Legends

us

312 313

Fig. 1. Chemical structures of lincomycin and its related substances.

an

314

316

M

315

Fig. 2. Chemical structures of spectinomycin and its related substances.

d

317

Fig. 3. LC-CAD chromatogram acquired for a mixture of Spectinomycin dihydrochloride

319

CRS

µg/ml)

and

Lincomycin

hydrochloride

CRS

(250

µg/ml)

solutions.

Ac ce p

320

(500

te

318

321

Fig. 4. MS and MS/MS spectra acquired for Spectinomycin dihydrochloride CRS solution

322

(500 µg/ml) and ESI-MS2 fragmentation pathways of spectinomycin.

323 324

Referencesp 1

A. Hamdy, D. Kratze, L. Paxton, B. Roberts, Effect of a single injection of lincomycin, spectinomycin, and linco-spectin on early chick mortality caused by Escherichia coli and Staphyllococcus aureus, Avian Dis. 23 (1979) 164-173. 2 D. Jordan, C. Venning, Treatment of ovine dermatophilosis with long-acting oxytetracycline or a lincomycin-spectinomycin combination, Aust. Vet. J. 72 (1995) 234-236. 3 J. Sherrard, Gonorrhoea, Medicine 38 (2010) 245-248. 4 M. Chiu, H. Yang , C. Liu, J. Zen, Determination of lincomycin in urine and some foodstuffs by flow injection analysis coupled with liquid chromatography and electrochemical

14

Page 14 of 26

us

cr

ip t

detection with a preanodized screen-printed carbon electrode, J. Chromatogr. B 877 (2009) 991-994. 5 D. Sin, Y. Wong, A. Ip, Quantitative analysis of lincomycin in animal tissues and bovine milk by liquid chromatography electrospray ionization tandem mass spectrometry, J. Pharm. Biomed. Anal. 34 (2004) 651–659. 6 M. Dousa, Z. Sikac , M. Halama, K. Lemr, HPLC determination of lincomycin in premixes and feedstuffs with solid-phase extraction on HLB OASIS and LC–MS/MS confirmation, J. Pharm. Biomed. Anal. 40 (2006) 981-986. 7 European Pharmacopoeia ver. 8.5, Monograph 01/2012:0583, Council of Europe, Strasbourg, France (2015). 8 European Pharmacopoeia ver. 8.5, Monograph 01/2008:1152, Council of Europe, Strasbourg, France (2015). 9 European Pharmacopoeia ver. 8.5, Monograph 01/2008:1658, Council of Europe, Strasbourg, France (2015). 10 USP 35, monograph: Spectinomycin dihydrochloride,4679, USA, 2012. 11

USP 35, monograph: Spectinomycin for injectable suspension,4680, USA, 2012. H. Yan, P. Xu, H. Huang, J. Qiu, Analysis of spectinomycin in fermentation broth by reversed-phase chromatography, Chem. Pap. 63 (2009) 635-640. 13 N. Haagsma, P. Scherpenisse, R.J. Simmonds, S.A. Wood, S.A Rees, High-performance liquid chromatographic determination of spectinomycin in swine, calf and chicken plasma using post-column derivatization, J. Chromatogr. B 672 (1995) 165-171. 14 A. Bergwerff, P. Scherpenisse, N. Haagsma, HPLC determination of residues of spectinomycin in various tissue types from husbandry animals, Analyst 123 (1998) 21392144. 15 D. Debremaeker, E. Adams, E. Nadal, B. van Hove, E. Roets, J. Hoogmartens, Analysis of spectinomycin by liquid chromatography with pulsed electrochemical detection, J. Chromatogr. A 953 (2002) 123-132. 16 L. Elrod, J.F. Brauer, S.L. Messner, Determination of spectinomycin dihydrochloride by liquid chromatography with electrochemical detection, Pharm. Res. 5 (1988) 664-667. 17 J.G. Phillips, C. Simmonds, Determination of spectinomycin using cation-exchange chromatography with pulsed amperometric detection, J. Chromatogr. A 675 (1994) 123-128. 18 M.C. Carson, D.N. Heller, Confirmation of spectinomycin in milk using ion-pair solidphase extraction and liquid chromatography–electrospray ion trap mass spectrometry, J. Chromatogr. B 718 (1998) 95-102. 19 H. Berrada, J.C. Moltó, J. Mañes, G. Font, Determination of aminoglycoside and macrolide antibiotics in meat by pressurized liquid extraction and LC-ESI-MS, J. Sep. Sci. 33 (2010) 522–529. 20 M.J Wang, Ch.Q Hu, Analysis of Spectinomycin by HPLC with Evaporative LightScattering Detection, Chromatographia 63, (2006) 255-260. 21 J. Wang , X. Hu, Y. Tu, K.Ni, Determination of spectinomycin hydrochloride and its related substances by HPLC–ELSD and HPLC–MSn, J. Chromatogr. B 834 (2006) 178-182.

Ac ce p

te

d

M

an

12

22

J. Zhou, L. Zhang, Y. Wang, Ch. Yan, HPLC-ELSD analysis of spectinomycin dihydrochloride and its impurities, J. Sep. Sci. 34 (2011) 1811-1819. 23 J. Szu´nyog, E. Adams, K. Liekens, E. Roets, J. Hoogmartens, Analysis of a formulation containing lincomycin and spectinomycin by liquid chromatography with pulsed electrochemical detection, J. Pharm. Biomed. Anal. 29 (2002) 213-220. 24 K. Vucicevic-Prcetic, R. Cservenak, N. Radulovic, Development and validation of liquid chromatography tandem mass spectrometry methods for the determination of gentamicin,

15

Page 15 of 26

Ac ce p

te

d

M

an

us

cr

ip t

lincomycin, and spectinomycin in the presence of their impurities in pharmaceutical formulations, J. Pharm. Biomed. Anal. 56 (2011) 736-742. 25 K. Stypulkowska, A. Blazewicz, Z. Fijalek, M. Warowna-Grzeskiewicz, K. Srebrzynska, Determination of neomycin and related substances in pharmaceutical preparations by reversed-phase high performance liquid chromatography with mass spectrometry and charged aerosol detection, J. Pharm. Biomed. Anal. 76 (2013) 207-214. 26 S. Almeling, D. Ilko, U. Holzgrabe, Charged aerosol detection in pharmaceutical analysis, J. Pharm. Biomed. Anal. 69 (2012) 50-63. 27 K. Stypulkowska, A. Blazewicz, Z. Fijalek, K. Sarna, Determination of Gentamicin Sulphate Composition and Related Substances in Pharmaceutical Preparations by LC with Charged Aerosol Detection, Chromatographia 72 (2010) 1225-1229. 28 U. Holzgrabe, C.J. Nap, N. Kunz, S. Almeling, Identification and control of impurities in streptomycin sulfate by high-performance liquid chromatography coupled with mass detection and corona charged aerosol detection, J. Pharm. Biomed. Anal. 56 (2011) 271279. 29 A. Joseph, S. Patel, A. Rustum, Development and validation of a RP-HPLC method for the estimation of netilmicin sulfate and its related substances using charged aerosol detection, J. Chromatogr. Sci. 48 (2010) 607-612. 30 International Conference on Harmonization (ICH), Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, Geneva, 2005, www.emea.europa.eu/pdfs/human/ich/038195en.pdf 31 R.W. Dixon, D.S. Peterson, Development and testing of a new detector for liquid chromatography based on aerosol charging, Anal. Chem. 74 (2002) 2930-2937. 32 T. Vehovec, A. Obreza, Review of operating principle and applications of the charged aerosol detector, J. Chromatogr. A 1217 (2010) 1549-1556.

16

Page 16 of 26

cr

i

*Graphical Abstract

Intens.

+MS, 6.8 min.

351.18

us

x105

M an

1.0

333.18 0.0 100

150

ce pt

ed

Intens.

HPLC

250

300

350

5

6

400

450

7

8

500 m/z

(TIC)

1.0

1 : 10

Ac

T-split

MS

[x1 MS 04] 2.0

200

0.0

CAD [mAU] 10.0 5.0 0.0

1

2

3

4

9 Time [min]

CAD Page 17 of 26

cr

ip t

Table(s)

B (%, v/v)

0.0

98

2

12.0

98

2

12.1

80

18.0

80

18.1

98

2

us

A (%, v/v)

an

Time (min)

M

Table 1. Elution program used in the final chromatographic conditions.

20

Ac c

ep te

d

20

Page 18 of 26

cr

ip t

Table2

us

Table 2. Peak assignment of spectinomycin related substances in LC-CAD on the basis of LC-MS/MS results. Compound

Chemical formula

m/z [M+H]+

m/z [M+H2O+H]+

0.63

Impurity A

C8H18N2O4

207.14

n.a.*

0.66

Impurity F

C14H26N2O7

335.18

353.19

0.76

Impurity D

C14H26N2O8

351.18

369.19

C14H24N2O7

333.17

351.18

C14H26N2O7

335.18

n.a.

C13H22N2O7

337.16

M

Impurity E

319.15

1.00

Spectinomycin

C14H24N2O7

333.18

351.19

1.11

Dihydrospectinomycin anomer

C14H26N2O7

335.18

n.a.

1.20

Impurity B

C14H26N2O8

351.18

n.a.

1.31

4R-dihydrospectinomycin

C14H26N2O7

335.37

n.a.

1.45

Unknown impurity

C13H24N2O6

305.18

n.a.

1.61

Impurity C

C14H26N2O7

335.18

n.a.

Ac c

0.93

ep te

0.87

Spectinomycin anomer Dihydrospectinomycin anomer

d

0.80

an

RRT*

* RRT = relative retention time to retention time of spectinomycin, n.a. = not applicable

Page 19 of 26

cr

ip t

Table3

LOQ (μg/ml)

Range (μg/ml)

R2

Equation

R2

y = 0.0101x + 0.0007

0.9998

y = 0.0093x1.0287

0.9998

y = 0.0069x + 0.2116

0.9990

y = 0.0212x0.8148

0.9990

1 - 25

y = 0.0259x + 0.0268

0.9968

y = 0.0346x0.9366

0.9968

40 - 120

y = 0.0190x + 0.3152

0.9991

y = 0.0506x0.8216

0.9991

M

1.0

80 - 200

ep te

d

0.3

2.0

Ac c

Lincomycin

0.5

Power function (y = bxa)

Equation

2 - 45 Spectinomycin

Linear function (y = ax+b)

an

Compound

LOD (μg/ml)

us

Table 3. LOD, LOQ and regression data of spectinomycin and lincomycin determined by LC-CAD.

Page 20 of 26

cr

ip t

Table4

Spectinomycin 80 - 180

Lincomycin

1.65 ± 0.07 10.09 ± 0.09 25.07 ± 0.04 44.51 ± 0.44

84.2 103.2 102.5 101.1

79.81 ± 0.97 100.78 ± 1.72 123.14 ± 2.53 151.41 ± 1.63 180.73 ± 3.95 1.03 ± 0.00 2.51 ± 0.04 5.05 ± 0.06 10.19 ± 0.03

97.7 98.7 100.5 98.9 98.3 100.4 98.5 99.0 99.8

0.43 0.69 1.02 0.65 1.59 0.00 0.02 0.02 0.01

0.5 0.7 0.8 0.4 0.9 0.1 0.6 0.5 0.1

0.40 0.81 1.41 0.87 1.92 0.00 0.01 0.02 0.11

0.5 0.8 1.2 0.6 1.1 0.4 0.6 0.4 1.0

39.64 ± 0.41 51.63 ± 0.19 63.24 ± 0.17 84.12 ± 0.33 103.82 ± 0.54 122.51 ± 1.09

97.0 101.1 103.2 103.0 101.7 100.0

0.17 0.07 0.06 0.13 0.22 0.44

0.4 0.1 0.1 0.2 0.2 0.4

0.37 0.18 0.51 0.40 0.78 0.53

1.0 0.3 0.8 0.5 0.8 0.4

81.68 102.10 122.52 153.15 183.78 1.02 2.55 5.11 10.21

40 - 120

an

1.96 9.78 24.45 44.01

Ac c

1 - 10

Accuracy (%)

M

2 - 45

Inter-day precision (n=6) SD RSD (μg/ml) (%) 0.04 2.2 0.26 2.5 0.37 1.5 0.96 2.2

Concentration found mean ± t S x , p = 0.05 (μg/ml)

d

Range

Intra-day precision (n=3) SD RSD (μg/ml) (%) 0.03 1.8 0.04 0.3 0.02 0.9 0.18 0.1

Concentration added (μg/ml)

ep te

Compound

us

Table 4. Precision and accuracy data of spectinomycin and lincomycin determined by LC-CAD.

40.85 51.05 61.27 81.69 102.12 122.54

Page 21 of 26

cr

ip t

Table5

Table 5.

Linc. B

Spect.+ 4R-DHS

4RDHS

A (legal)

100.1

2.6

101.3

1.2

B (illegal)

103.3

0.3

100.9

0.6

Imp.A

Imp.C

Imp.D

an

Linc.+ Linc. B

< LOQ (~0.01) < LOQ (~0.01)

< LOQ (~0.01) < LOQ (~0.01)

Imp.E

0.1

0.2

< LOQ (~0.05)

0.2

Imp.F < LOQ (~0.01) < LOQ (~0.01)

Other

Sum of imp.

0.2

0.5

0.1

0.3

Ac c

ep te

d

M

Sample

us

Determination of active substances and impurities of spectinomycin in commercial samples by LC-CAD (%, m/m).

Page 22 of 26

N H

N H

O

R2

R1

R2

Lincomycin

CH3

C3H7

OH

Lincomycin B

CH3

C2H5

OH

Impurity A (α-amide epimer)

CH3

C3H7

Impurity C

H

C3H7

CH3

C3H7

S

Impurity D (7-epi-lincomycin)

H 3C N

H

N H

CH 3 OH H O

OH

O

N H

HO OH

OH S

Ac c

H 3C H 3C

H3C

ep te

HO O H

d

M

H3C

Compound

us

R1

CH3 OH H

an

HO O H

Impurity B (propylidene analogues)

cr

ip t

Figure1

H

CH3 OH H

H2N O

OH

"

H3C Impurity E (4-propyl hygric acid)

H3C

OH

S

Impurity F (methyl-1-thiolincosaminide)

Page 23 of 26

HO

R1

O O

H

O

Spectinomycin

CH3

OH R3 R2

R1

CH3

R4

R2

R3

R4

R2+R3=O

H

(4R-dihydrospectinomycin)

CH3

OH

H

H

Impurity C (4S-dihydrospectinomycin)

CH3

H

OH

H

Impurity D (dihydroxyspectinomycin)

CH3

H

OH

OH

Impurity E (N-desmethylspectinomycin)

H

R2+R3=O

H

Impurity G (tetrahydroxyspectinomycin)

CH3

R2+R3=O

OH

OH OH

HO H3C

Ac c

H3C

H N

ep te

d

M

HN

H

Compound

us

HN

OH H

an

CH3

cr

ip t

Figure2

OH

NH

Impurity A (Actinamine)

H3C

H N

CH3 OH H

COOH OH

HO H3C

O

O

H3C

H N

OH H

Impurity B (Actinospectinoic acid)

O

OH

H

H OH

NH

O

O OH

HO H3C

NH

CH3

Impurity F (Triol spectinomycin)

Page 24 of 26

Ac

ce pt

ed

M an

us

cr

i

Figure3

Page 25 of 26

i

Figure4

cr

Intens. +MS, 6.8min x105

us

351.177

1.0

100

150

M an

0.0

200

Intens. x105 +MS2 (351.177), 24eV

300

ed

1.2 m/z 207

350

400

450

500

m/z

C14H27N2O8 351.177

m/z 126

OH

1.0

ce pt

CH3NH

0.8

HO

O

O

0.6

CH3

OH O m/z 305

m/z 153

Ac

+ H2O

m/z 315

O

NHCH3

0.4

333.167

247.132 250

m/z 351 m/z 333

m/z 189

- H2O C14H25N2O7 333.167 - H2O C14H23N2O6 - CO 315.157 C13H24N2O6 305.157

0.2 - H2O C H N O C8H13N2O C8H17N2O3 8 19 2 4 207.135 153.103 189.125 100

150

200

250

300

350

m/z

Page 26 of 26