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SEPARATION AND CHARACTERIZATIONOF THE IMPURITIES AND ISOMERS INCEFMENOXIME HYDROCHLORIDE BYHPLC-UV-MSn

Jian Wang a , Dan Ruan b & Weiguang Shan ba Zhejiang Institute for Food and Drug Control , Hangzhou , P. R.Chinab College of Pharmacy, Zhejiang University of Technology ,Hangzhou , P. R. ChinaPublished online: 09 Jun 2013.

To cite this article: Jian Wang , Dan Ruan & Weiguang Shan (2013): SEPARATION ANDCHARACTERIZATION OF THE IMPURITIES AND ISOMERS IN CEFMENOXIME HYDROCHLORIDE BY HPLC-UV-MSn , Journal of Liquid Chromatography & Related Technologies, 36:15, 2125-2141

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SEPARATION AND CHARACTERIZATION OF THE IMPURITIES ANDISOMERS IN CEFMENOXIME HYDROCHLORIDE BY HPLC-UV-MSn

Jian Wang,1 Dan Ruan,2 and Weiguang Shan2

1Zhejiang Institute for Food and Drug Control, Hangzhou, P. R. China2College of Pharmacy, Zhejiang University of Technology, Hangzhou, P. R. China

& The twelve impurities and isomers in cefmenoxime hydrochloride were separated and identifiedby high-performance liquid chromatography-electrospray ionization tandem mass spectrometry(HPLC-ESI-MSn). A discipline of mass fragmentation pattern and structure for the E-isomer andD3-isomer of oxime cephalosporin antibiotics was presented to distinguish their structures. The col-umn was Alltima C18(250� 4.6mm, 5lm). The mobile phase was water-acetic acid-acetonitrile(85:1:15). In positive mode, full scan LC-MS was first performed in order to obtain the m=z valueof the protonated molecules of all detected peaks. LC-MS-MS and LC-MS-MS-MS were then carriedout on the compounds of interest. The complete fragmentation patterns of twelve impurities were stud-ied and used to obtain information about the structure of these impurities. The relationship of massfragmentation pattern and structure for cefmenoxime, E-isomer of cefmenoxime and D3-isomer of cef-menoxime was studied. The structures of the twelve impurities and isomers in cefmenoxime hydro-chloride were deduced based on the HPLC-MSn data, assisted by the UV spectra and stress testing.

Keywords cefmenoxime hydrochloride, HPLC-ESI-MSn, identification, impurity,isomer, structure

INTRODUCTION

Cefmenoxime Hydrochloride is a third generation cephalosporinantibiotics. It is widely used because of high activity against a large numberof both gram-positive and gram-negative micro-organisms and its resistanceto b-lactamases. Cefmenoxime hydrochloride is unstable. Some degradationproducts are produced readily during the production of cefmenoximehydrochloride and during storage. The clinical effect will be impactedbecause of the reduction of these impurities’ antibacterial activities andthe increase of these impurities’ toxicities. Therefore, it is necessary tocharacterize and control these impurities.

Address correspondence to Weiguang Shan, College of Pharmacy, Zhejiang University ofTechnology, Hangzhou 310014, P. R. China. E-mail: swg@zjut.edu.cn

Journal of Liquid Chromatography & Related Technologies, 36:2125–2141, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.712935

Journal of Liquid Chromatography & Related Technologies, 36:2125–2141, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.712935

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The identifications of impurities could be performed by preparativeliquid chromatography followed by spectra. It is evident that such a pro-cedure is labor intensive and requires a large amount of degradationmaterial. Moreover, this indirect technique can induce degradation of thecompound of interest. Consequently, it is difficult to know whether the ident-ified compound was really a impurity or simply an artifact. This is especiallytrue for b-lactams which have limited stability in organic solvents likemethanol or acetonitrile. A direct coupling method is thus more suitable.

The on-line combination of liquid chromatography and massspectrometry (HPLC-MSn) has developed quickly as an identification tool.HPLC-MSn combines chromatographic strong separation with mass spectro-metric strong qualitative advantages. Much structure information can beobtained through LC-MS-MS and LC-MS-MS-MS analysis, which shows greatsuperiority to identify the impurities. Structural identifications of the impu-rities in cephalosporin antibiotics have been reported,[1–12] but structuralidentification of the impurities in cefmenoxime hydrochloride has not beenreported. The aim of this study was to separate and characterize the impu-rities in Cefmenoxime Hydrochloride by HPLC-ESI-MSn, assisted by theUV spectra and stress testing. Twelve impurities and isomers in cefmenox-ime hydrochloride drug substance were separated and characterized, anda complete structure was proposed for twelve of these.

The relationship of mass fragmentation pattern and structure for cefme-noxime, E-isomer of cefmenoxime, and D3-isomer of cefmenoxime wasstudied. The relationship of mass fragmentation pattern and structurefor deacetylcefotaxime (a impurity), E-isomer of deacetylcefotaxime, andD3-isomer of deacetylcefotaxime was studied. A discipline of mass fragmen-tation pattern and structure for the E-isomer and D3-isomer of oximecephalosporin antibiotics was presented to distinguish their structures.

EXPERIMENTAL

Chemicals and Reagents

Cefmenoxime hydrochloride reference substance and drug substance(batch numbers: 101217) and intermediates were provided by ZhejiangJinhua Kangenbei Pharmaceutical Co. Ltd. (Jinhua, China). Acetic acidand acetonitrile were chromatographic grade.

Instrumentation

LC ApparatusAn Agilent 1260 series liquid chromatography (LC) system equipped

with a binary pump and a UV detector was connected to an Agilent

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G1313A autosampler. Chromatographic separation was carried out at roomtemperature using an Alltima C18 analytical column (250� 4.6mm, 5mm).The mobile phase consisted of water-acetic acid-acetonitrile (85:1:15). Theflow rate was 0.8mL=min. Column oven temperature was maintained at30�C and injection volume was 20 mL.

Mass Spectrometry

LC-MS experiment was carried out on an AB SCIEX 4000 Q TRAP com-posite triple quadrupole=linear ion trap tandem mass spectrometer. Thecolumn effluent was split using a zero-dead-volume ‘‘T’’ connector, withapproximately one quarter of the flow being fed to the mass spectrometer.The MSD was equipped with an ESI source. The ionization mode was posi-tive. The interface and MSD parameters were as the follows: nebulizerpressure (40 p.s.i.), dry gas pressure (40 p.s.i.), curtain gas pressure (10p.s.i.), dry gas temperature (650�C), spray capillary voltage(5500v), CAD:high, DP: 80V, EP: 20V, CE: 45 V.

All data were acquired and processed by Analyst software.

Sample Solution Preparation

Dissolve adequate cefmenoxime hydrochloride drug substance withwater-acetic acid-acetonitrile (70:1:30) to make the solution of 1.0mg=mL�1.

RESULTS AND DISCUSSION

Selection of Chromatographic Conditions

In order to permit the use of liquid chromatography-mass spectrometryanalysis, acid in the mobile phase must be a volatile acid and its concen-tration should be as low as possible. By testing, we found that the retentiontime of cefmenoxime was appropriate, and cefmenoxime could be com-pletely separated from intermediates, excipient, and degradation productswhen using water-acetic acid-acetonitrile (85:1:15) as the mobile phase. Thechromatogram of cefmenoxime hydrochloride drug substance is shown inFigure 1.

Stress Testing and Results

Samples were stored under relevant stress conditions (light, heat,acid=base hydrolysis, and oxidation, respectively). The results are shownin Table 1.

Impurities and Isomers in Cefmenoxime Hydrochloride 2127

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Acid DegradationThe 20mg of cefmenoxime hydrochloride was introduced into a test

tube. Then, 0.5mL of 1M HCl was added and heated for 30min at 60�C.After 30min, the drug treated with 1M HCl was neutralized with 1M NaOHand diluted with water-acetic acid-acetonitrile (70:1:30) to 20mL.

Basic DegradationThe 20mg of cefmenoxime hydrochloride was introduced into a test

tube. Then, 0.5mL of 1M NaOH was added and heated for 30min at

TABLE 1 Change of the Impurity Content Under the Stress Condition and Natural Degradation

No.

Stress TestingNatural

Degradation

BasicDamage

AcidDamage

HighTemperature

Damage

UltravioletLight

DamageOxidativeDamage

HighTemperature

Damageof Solution

UltravioletLight

DamageofSolution

NaturalDegradation

of OneWeek

I – – – – þ þ þ þ þII þ þ – – – þ þ þþ þ þIII – – – – – þ þ þþ þ þIV – – – þ þ þ þ þ þ þ þ þV – – – – – þ þ þVI – – – – þ – – þVII – – þ þ – – þ þ þþ þ þVIII – þ þ þ þ – – þ þ – þ þIX þ þ – þ þ – – – – –X – – – – – – – þXI þ – – – – – – –XII – – – þ þ – – þ þ þ þ

Note: –, meant that the impurity content had no change.þ, meant that the impurity content increased.þ þ, meant that the impurity content increased obviously.

FIGURE 1 Chromatogram of cefmenoxime hydrochloride drug substance. 1) impurity; 2) Impurity; 3)impurity III; 4) impurity IV; 5) impurity V; 6) impurity VI; 7) impurity VII; 8) impurity VIII; 9) impurityIX; 10) impurity X; 11) cefmenoxime hydrochloride; 12) impurity XI; 13) impurity XII. (Color figureavailable online.)

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100�C. After 30min, the drug treated with 1M NaOH was neutralized with1M HCl and diluted with water-acetic acid-acetonitrile (70:1:30) to 20mL.

Oxidative DegradationThe 20mg of cefmenoxime hydrochloride was introduced into a test

tube. Then, 0.2mL of 3% H2O2 was added and heated for 30min at60�C. After 30min, the drug treated with 3% H2O2 was diluted with water--acetic acid-acetonitrile (70:1:30) to 20mL.

Heat DegradationThe 20mg of cefmenoxime hydrochloride was heated for 3 hr at 105�C.

After 3 hr, the drug was diluted with water-acetic acid-acetonitrile (70:1:30)to 20mL.

Photolytic DegradationThe 20mg of cefmenoxime hydrochloride was introduced into a Petri

dish which was exposed to ultraviolet light at 254 nm for 24hr. After 24 hr,the drug was diluted with water-acetic acid-acetonitrile (70:1:30) to 20mL.

Solution Heat DegradationDissolve adequate cefmenoxime hydrochloride drug substance with

water-acetonitrile (70:30) to make the solution of 1.0mg=mL�1. This sol-ution was heated for 1 hr at 60�C.

Solution Photolytic DegradationDissolve adequate cefmenoxime hydrochloride drug substance with

water-acetonitrile (70:30) to make the solution of 1.0mg=mL�1. Thissolution is exposed to ultraviolet light at 254 nm for 24hr.

Selection of MS Conditions

APCI and ESI are two common used LC=MS ion source, APCI is moresuitable to the analysis of neutral or small polar compounds, while ESI ismore suitable to the analysis of polar, thermo-labile alkaline, or acidic com-pounds. According to the structural characteristic of cefmenoxime hydro-chloride and its impurities, ESI was selected to analyze the impurities incefmenoxime hydrochloride. Compared with negative mode, the impuritiesin cefmenoxime hydrochloride had higher MS response and sensitivity inpositive mode. In order to obtain the proper fragment ions, various inter-face and MSD parameters were tested. The results showed that the interfaceand MSD parameters in Mass Spectrometry were optimal. Full scan LC-MS

Impurities and Isomers in Cefmenoxime Hydrochloride 2129

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was first performed in order to obtain the m=z value of the protonatedmolecules of all detected peaks. In order to obtain as much structural infor-mation as possible, LC-MS-MS and LC-MS-MS-MS were then carried out onthe compounds of interest. The complete fragmentation patterns of twelveimpurities were studied by MSn and used to obtain information about thestructure of these impurities. Mass spectra results of twelve impurities incefmenoxime hydrochloride are shown in Table 2.

Structure Elucidation

Impurity IX, Impurity XI, and Impurity XIIImpurity IX, impurity XI, and impurity XII exhibited the same molecu-

lar species [M þH]þ at m=z 512, [M þNa]þ at m=z 534 and [M þK]þ at m=z550 in LC-MS analysis, indicating the same molecular mass of the threeimpurities as 511, which is equivalent to the mass of cefmenoxime. There-fore, they were suggested as isomers of cefmenoxime. The MS-MS spectraand MS-MS-MS spectra is presented in Figure 2.

Figure 3 presents the following fragmentation sequence of cefmenox-ime: a loss of a 1-methyl-1H-tetrazole-5-thiol with the formation of theion at m=z 396; a loss of a neutral of 44 Da (CO2) conducting to the frag-ment at m=z 352; a loss of 28 Da (CO) leading to the ion at m=z 324. Theloss of CO before CO2 also happened from the ion at m=z 396 to givethe ions at m=z 368 and 324, respectively. The subsequent fragmentationinvolved the loss of 157 leading to the ion at m=z 167. Another fragmen-tation pathway concerned the cleavage of the b-lactam ring from the ionat m=z 396 conducting to the fragmentation at m=z 241.

The content of impurity XII increased obviously after cefmenoximehydrochloride was introduced into a Petri dish which was exposed to ultra-violet light at 254 nm for 24hr. That E-isomers of several oxime cephalos-porin antibiotics formed by cis-trans isomerization of the oxime functionwhen exposed to ultraviolet light have been reported,[3] suggesting theimpurity XII as E-isomer of cefmenoxime. It was supported further bythe MS-MS spectrum and MS-MS-MS spectrum of the literature [2] and theimpurity XII. It concerned the loss of CH3O- from the ion atm=z 324 leadingto the fragment at m=z 293. The fragmentation at m=z 293 in cefmenoxime(Z) had low abundance because the methoxy group of the oxime withamine close to it formed N-O hydrogen bonding. The formed structurewas stable, and the methoxy group of the oxime was not easily lost. However,the fragmentation at m=z 293 in the impurity XII (E-isomer of cefmenox-ime) was abundant because the methoxy group of the oxime function withamine away from it did not easily form N-O hydrogen bonding and themethoxy group of the oxime function was easily lost. Figure 4 presentsthe fragmentation sequence of the impurity XII.

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TABLE

2MassSp

ectraResultsofTwelve

Impurities

inCefmen

oximeHydroch

loride

Impurity

t R(m

in)

[MþH]þ

(m=z)

MajorMS=

MS

Fragm

entation

Ions(m

=z)

MajorMS=

MS=

MSF

ragm

entation

Ions(m

=z)

ProposedStructure

oftheIm

purity

I3.1

396

352

321

247

230

352!

321

230

II3.5

243

126

III

3.7

414

241

197

126

241!

197

197!

166

IV4.3

414

285

241

227

200

285!

254

257

225

(Continued

)

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TABLE2

Continued

Impurity

t R(m

in)

[MþH]þ

(m=z)

MajorMS=

MS

Fragm

entation

Ions(m

=z)

MajorMS=

MS=

MSF

ragm

entation

Ions(m

=z)

ProposedStructure

oftheIm

purity

V4.9

414

285

241

227

285!

257

225

VI

5.3

528

412

394

366

412!

394

350

394!

366

VII

7.2

117

9974

VII

9.3

396

241

227

200

227!

200

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IX11

.451

239

635

224

139

6!24

119

720

119

716

615

2

X12

.239

828

524

122

724

1!16

7

14.6

512

396

368

352

396!

324

277

241

324

277

241

211

167

117

XI

23.2

512

396

368

352

396!

324

277

241

324

277

241

211

167

XII

27.2

512

396

324

293

396!

352

324

293

276

182

126

241

112

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The ultraviolet absorption of the impurity IX shifted toward short wave-length compared with cefmenoxime (D2), which indicated its conjugatedsystem was reduced. This suggested the impurity IX as D3-isomer of cefme-noxime. It was supported further by the MS-MS spectrum and MS-MS-MSspectrum of the literature[1] and the impurity IX. The loss of CO fromthe ion at m=z 396 provided the ions at m=z 368. However, the absence ofthe fragment ion at m=z 368 in the impurity IX, attributed for cefmenox-ime, was not detected in this case. The subsequent fragmentation ions atm=z 277 and at m=z 167 in the impurity IX attributed to cefmenoximewas also not detected. This was because different positions of double bondsin D2=D3-cefmenoxime caused electron density different in the ring.Figure 5 presents the fragmentation sequence of the impurity IX.

FIGURE 2 The MS=MS spectra of Cefmenoxime (a), impurity IX (b1), impurity XI (c1), impurity XII(d1), and MS=MS=MS spectra of impurity IX (b2), impurity XI (c2), and impurity XII (d2). (Colorfigure available online.)

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The fragmentation ion patterns of the impurity XI and cefmenoximewere very similar suggesting that it was a structural analogue of the maindrug. The content of impurity XI increased obviously in the basic condition.The 7-epimer of several cephalosporin antibiotics produced in basiccondition have been reported,[3] suggesting the impurity XI as 7-epimerof cefmenoxime.

Impurity III, Impurity IV, and Impurity VThe impurity III, impurity IV, and impurity V exhibited the same proto-

nated molecule [M þH]þ at m=z 414 in LC-MS analysis, indicating the samemolecular mass of the three impurities as 413. The molecular masses ofthem were equivalent to the mass of deacetylcefotaxime, an impurity ofcefotaxime listed in the British Pharmacopoeia, which were, therefore,suggested as deacetylcefotaxime and isomers of deacetylcefotaxime. TheMS-MS spectra and MS-MS-MS spectra of the impurity III, impurity IV,

FIGURE 3 Proposed fragmentation pathways of cefmenxime.

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and impurity V verified that their structures were in agreement with deace-tylcefotaxime. Figure 6 presents the proposed fragmentation patterns ofimpurity IV, impurity IV, and impurity V.

The fragmentation pathways of impurity IV and impurity V concernedthe cleavage of the b-lactam ring conducting to the fragmentation at m=z285 and at m=z 241. The content of impurity IV increased, but the contentof impurity V did not change after cefmenoxime hydrochloride was intro-duced into a Petri dish that was exposed to ultraviolet light at 254 nm for24 hr,[3] suggesting the impurity IV as E-isomer, and the impurity V asZ-isomer. It was supported further by the MS-MS spectra and MS-MS-MSspectra of the literature,[2] the impurity IV and the impurity V. It concernedthe loss of CH3O- from the ion at m=z 285 leading to the fragmentation atm=z 254. The fragmentation at m=z 254 in the impurity V (Z-isomer) hadlow abundance because the methoxy group of the oxime with amine closeto it formed N-O hydrogen bonding. The formed structure was stable,and the methoxy group of the oxime was not easily lost. On the contrary,the fragmentation at m=z 254 in the impurity IV (E-isomer) was abundantbecause the methoxy group of the oxime function with amine away fromit did not easily formed N-O hydrogen bonding and the methoxy groupof the oxime function was easily lost.

The ultraviolet absorption of the impurity III shifted toward short wave-length compared with impurity IV and impurity V(D2), which indicated itsconjugated system reduced. This suggested the impurity III as D3-isomer.It was supported further by the MS-MS spectrum and MS-MS-MS spectrum

FIGURE 4 Proposed fragmentation pathways of the impurity XII (E Cefmenxime).

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FIGURE 5 Proposed fragmentation pathways of the impurity IX(D3 Cefmenxime).

FIGURE 6 Proposed fragmentation patterns of impurity III (a), impurity IV(b), and impurity V(c).

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of the impurity III. The fragmentation pattern of impurity III was differentwith the fragmentation patterns of impurity IV and impurity V. The absenceof the fragmentation at m=z 285 in the impurity III, attributed for impurityIV and impurity V, was not detected in this case. This was because differentpositions of double bond in D2=D3- isomers. The main MS-MS fragmen-tation of impurity III was at m=z 241. The subsequent fragmentationinvolved the loss of COOH from the ion at m=z 241 leading to the ion atm=z 197.

Impurity XThe impurity X exhibited the protonated molecule [M þH]þ at m=z

398 in LC-MS analysis, indicating the molecular weight of the impurity as397. Its structure is in agreement with an impurity of cefotaxime listed inBritish Pharmacopoeia, which was named as deacetoxycefotaxime. TheHPLC retention time and MS-MS spectra of impurity X and deacetoxycefo-taxime were identical to verify an identity between them. Figure 7 presentsthe proposed fragmentation patterns of impurity X.

Impurity I and Impurity VIIIThe impurity I and impurity VIII exhibited the same protonated mol-

ecule [MþH]þ at m=z 396 in LC-MS analysis, indicating the same molecularmass of the two impurities as 395. Therefirem they were suggested asisomers. The proposed fragmentation sequences of the impurity I andimpurity VIII are presented in Figure 8 and Figure 9.

The content of impurity I increased, but the content of impurity VIII didnot change after cefmenoxime hydrochloride solution is exposed to ultra-violet light at 254 nm for 24hr,[3] suggesting the impurity I as E-isomer,and the impurity VIII as Z-isomer. It was supported further by the MS-MSspectra and MS-MS-MS spectra of the literature,[2] the impurity I and theimpurity VIII. It concerned the loss of CH3O- from the ion at m=z 352 lead-ing to the fragmentation at m=z 321. The fragmentation at m=z 321 in theimpurity VIII (Z-isomer) had low abundance because the methoxy groupof the oxime with amine close to it formed N-O hydrogen bonding. Theformed structure was stable, and the methoxy group of the oxime function

FIGURE 7 Proposed fragmentation pattern of the impurity X (deacetoxycefotaxime).

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was not easily lost. On the contrary, the fragmentation at m=z 321 in theimpurity I (E-isomer) was abundant because the methoxy group of theoxime function with amine away from it did not easily formed N-O hydrogenbonding and the methoxy group of the oxime function was easily lost.

Impurity IIThe impurity II exhibited themolecular species [MþH]þ atm=z 243 and

[MþNa]þ atm=z 265 in LC-MS analysis, indicating themolecular mass of theimpurity as 242. Its structure was deduced based on the HPLC-MSn data.Figure 10 presents the proposed fragmentation sequence of impurity II.

Impurity VIIThe impurity VII exhibited the protonated molecule [M þH]þ at m=z

117 in LC-MS analysis, indicating the molecular mass of the impurity as116. Its structure is in agreement with a intermediate, which was named

FIGURE 9 Proposed fragmentation pathways of the impurity VIII.

FIGURE 8 Proposed fragmentation pathways of the impurity I.

Impurities and Isomers in Cefmenoxime Hydrochloride 2139

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as 1-methyl-1H-tetrazole-5-thiol (MMTZ). The HPLC retention time andMS-MS spectra of impurity VII andMMTZ were identical to verify an identitybetween them.

Impurity VIThe impurity VI exhibited the molecular species [M þH]þ at m=z 528,

[M þNa]þ at m=z 550 and [M þK]þ at m=z 566 in LC-MS analysis, indicatingthe molecular mass of the impurity as 527, which is 16 Da more than that ofcefmenoxime. The content of impurity VI increased obviously after cefme-noxime hydrochloride was damaged by oxidation, suggesting the impurityVI as 5S-oxide of cefmenoxime. Figure 11 presents the proposed fragmen-tation patterns of impurity VI.

CONCLUSIONS

This study was to separate and characterize the impurities in Cefmenox-ime Hydrochloride by HPLC-ESI-MSn, assisted by the UV spectra and stresstesting. Twelve impurities and isomers in cefmenoxime hydrochloride drugsubstance were separated and characterized, and their mass spectrometrysplitting rules were studied. A discipline of mass fragmentation patternand structure for the E-isomer and D3-isomer of oxime cephalosporin anti-biotics was presented to distinguish their structures.

Cefmenoxime hydrochloride is unstable. Some degradation productsare produced readily during the production of cefmenoxime hydrochloride

FIGURE 10 Proposed fragmentation pathways of [MþH]þ of the impurity II.

FIGURE 11 Proposed fragmentation pathways of impurity VI.

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and during storage. These were found to be structural analogues of themain compound as suggested by the close m=z values of the protonatedmolecules [MþH]þ and the similar main fragmentation patterns. Further-more, the results suggested that some stereoisomers could be producedby degradation.

REFERENCES

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