Study of the Fragmentation Patterns of Nine

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Study of the Fragmentation Patterns of Nine Fluoroquinolones by Tandem Mass Spectrometry a

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Qingfa Tang , Feilong Chen & Xuefeng Xin

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Key Laboratory of Research of Traditional Chinese Medicine and New Drug, Southern Medical University, Guangzhou, P. R. China Version of record first published: 14 Dec 2011

To cite this article: Qingfa Tang, Feilong Chen & Xuefeng Xin (2012): Study of the Fragmentation Patterns of Nine Fluoroquinolones by Tandem Mass Spectrometry, Analytical Letters, 45:1, 43-50 To link to this article: http://dx.doi.org/10.1080/00032719.2011.565448

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Analytical Letters, 45: 43–50, 2012 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print=1532-236X online DOI: 10.1080/00032719.2011.565448

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Original Research Paper STUDY OF THE FRAGMENTATION PATTERNS OF NINE FLUOROQUINOLONES BY TANDEM MASS SPECTROMETRY Qingfa Tang, Feilong Chen, and Xuefeng Xin Key Laboratory of Research of Traditional Chinese Medicine and New Drug, Southern Medical University, Guangzhou, P. R. China Fluoroquinolones (FQs) are a group of structurally related antibacterial agents, the fragmentation patterns of which are poorly investigated. This work reports a systematic study on the fragmentation pattern of a group of related FQs by electrospray ionization with multistage mass spectrometry (ESI-MSn) in positive mode. Potential dissociation pathways for the seven 7-piperazidine FQs and two 7-piperidine FQs are proposed and discussed. First, the loss of the peripheral groups was observed. Second, further steps involve the rearrangement of heterocyclic ring. In addition, the loss of CO2 or H2O in different pathways was observed. These fragmentation patterns provide important information for the determination of these compounds by liquid chromatography (LC)-MS. Keywords: Collision-induced dissociation; Fluoroquinolones; Fragmentation patterns

INTRODUCTION Fluoroquinolones (FQ) antibiotics are synthetic antibacterial compounds widely used in human as well as in veterinary medicine for the treatment of digestive, urinary, and pulmonary infections. The widespread use of FQs in agriculture has resulted in the potential residues in foodstuffs from animal origin, and in parallel, to an upsetting increase of resistant human pathogens. The great chemical variety of FQs and the possibility of trace level residues made it necessary to develop sensitive screening methods. Most reports have described current methods of FQs analysis are based on liquid chromatography, mainly with fluorescence (McMullen, Schenck, and Vega 2009; Can˜ada-Can˜ada et al. 2009; Lee, Peart, and Svoboda 2007), ultraviolet (UV) or MS detection (Lillenberg et al. 2009; Medvedovici et al. 2009; Samanidou et al. 2008). Several papers reported the analytical methods of FQs by capillary electrophoresis with MS (Yang and Wang 2009; Lara et al. 2008).

Received 5 July 2010; accepted 5 January 2011. Address correspondence to Dr. Qingfa Tang, Key Laboratory of Research of Traditional Chinese Medicine and New Drug, Southern Medical University, Guangzhou, 510515, P. R. China. E-mail: tqf18@ live.cn 43

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The ESI-MS is the most sensitive and specific method for the analysis of FQs. Because of the soft ionization characteristics of the Electrospray Ionization Mass Spectrometry (ESI-MS) technique, it usually shows only protonated molecules ions and=or molecules adduct ions. Additional techniques such as in-source collision induced dissociation or tandem mass spectrometry (MS=MS) have been applied to produce the necessary structurally significant fragment ions for confirmation of identity (Vanessa et al. 2008). To our knowledge, only a few studies have reported on the fragmentation patterns of FQs. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was applied to study the negative ion mass spectra of seven quinolone drugs (Guo et al. 1995). Time of flight mass spectrometry (TOF-MS), with different electron energies for electron ionization (EI) and different gas pressure for chemical ionization (CI), of levofloxacin lactate was studied (Li and Yin 2003). The FQs were analyzed using electrospray ion trap mass spectrometry in a multistage MS full scan mode (Ma et al. 2006). Fragmentations observed by ESI-MSn may be different from those observed with other mass spectrometric methods. This work characterizes fragmentation patterns of FQs by ESI-MSn. These results have potential application for residue analysis of FQs by LC-MSn. EXPERIMENTAL Materials Norfloxacin, enrofloxacin, and ciprofloxacin reference standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Ofloxacin and fleroxacin reference standards were supplied by Chengdu Tianyin Pharmaceutical Co. Ltd (Chengdu, China). Nadifloxacin and rufloxacin reference standards were supplied by Spring Pharmaceutical Co. Ltd (Beijing, China). Balofloxacin and lomefloxacin hydrochloride reference standards were supplied by Huanghe Medical (Yancheng, China), purity >98%, checked by LC-MS. All of the other chemicals were of analytical grade. Norfloxacin, ciprofloxacin, enrofloxacin, and balofloxacin were dissolved in 0.1 M hydrochloric acid. Ofloxacin was dissolved in 30% methanol. Lomefloxacin hydrochloride and nadifloxacin were dissolved in 0.1 M sodium hydrate. Rufloxacin hydrochloride was dissolved in water. Fleroxacin was dissolved in 0.1 M hydrochloric acid. All prepared at a concentration of 0.1 mg=ml standard stock solution. Mass Spectrometry Quasi-MS3 analysis was performed using a Finnigan TSQ Quantum Discovery MAX system consisting of a triple quadrupole TSQ Quantum (Q1q2Q3) mass spectrometer, a syringe pump, and an ESI source in the positive ion mode, run by Xcalibur 2.0 software (Thermo Electron Corporation). The spray needle voltage was set at 5.0 kV and the spray was stabilized with a nitrogen sheath gas (30 psi). The capillary temperature was 350
C. A syringe pump delivering 5 ml=min was used for the direct loop injections of pure compounds. Mass spectra were acquired using dwell times of 0.1 s per 0.7 Da step in full mass scan mode and recorded with a limited mass range of m=z 10–500.

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In MS2 experiments, Q1 was scanned at a rate of 0.1 s=scan over the range m=z 10–500 for conventional full-scan experiments. According to different compounds and their detected ions, in-source CID was optimized by varying the cone voltage between 0 and 40 V. MS=MS in the product ion scan, precursor ion scan, and multiple reaction monitoring (MRM) modes was performed using argon as collision gas at a pressure of p(Ar) ¼ 1.50 mTorr in the collision quadrupole, q2. The collision offset voltage was changed during analysis (collision offset voltage optimizations and mixed-mode experiments). Product ion spectra were obtained by scanning Q3 over the range m=z 10–500. RESULTS AND DISCUSSION ESI-MS of Nine FQs A comparative analysis of the ESI spectra of nine FQs (see Figure 1) was performed on the triple quadrupole TSQ quantum (Q1q2Q3) mass spectrometer. The detected ions and their corresponding relative abundances are summarized in Table 1. Under the ESI conditions used, the positive-ion mode ESI mass spectrum of FQs displayed [M þ H]þ as the protonated molecule base peak. CID-MS/MS of the Protonated Molecule of 7-piperazidine FQs Ciprofloxacin, enrofloxacin, feroxacin, lomefloxacin, norfloxacin, rufloxacin, and ofloxacin are FQs with 7-piperazidine substituent. To get a comprehensive view of the fragmentations of FQs with 7-piperazidine, low-energy CID-MS=MS processes were conducted on the molecular species arising from FQs. The CID

Figure 1. Chemical structures of fluoroquinolones.

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Q. TANG ET AL. Table 1. Mass spectral data of fluoroquinolones in the MS2 analysis with the relative abundance (%) in parentheses Compounds [MW]

MS1

Ciprofloxacin [331]

332 (40) 314 (25) 288 (25) 268 (25) 360 (5) 342 (100) 316 (100) 296 (100) 370(18) 352 (57) 326 (10) 306 (20) 352 (36) 334 (17) 308 (15) 288 (17) 320 (100) 302 (26) 276 (46) 256 (9) 362 (23) 344 (53) 318 (20) 298 (40) 364 (45) 346 (13) 320 (10) 300 (15) 390 (9) 359 (100) 315 (37) 283 (13) 271 (100) 361 (13) 343 (35) 283 (25) 257 (38)

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Enrofloxacin [359]

Fleroxacin [369]

Lomefloxacin [351]

Norfloxacin [319]

Ofloxacin [361]

Rufloxacin [363]

Balofloxacin [389]

Nadifloxacin [360]

MS2 314 294 268 240 342 322 296 268 352 332 306 278 334 314 288 260 302 282 256 228 344 324 298 270 346 326 300 272 359 315 283 239 243 343 283 200 241

(90) 288 (100) 268 (22) 245 (65) (14) 286 (7) 243 (100) (17) 245 (100) 231 (92) (80) 225 (100) (65) 316 (95) 296 (15) 245 (100) (8) 314 (46) 243 (80) (11) 245(73) 231 (85) (57) 225 (50) (18) 326 (89) 306 (10) 269 (100) (6) 224 (65) 267 (100) (73) 269 (100) (100) 249 (72) (15) 308 (65) 288 (8) 265 (100) (34) 306 (12) 263 (78) (13) 265 (100) 237 (85) (100) 245 (53) (50) 276 (45) 256 (10) 219 (28) (29) 274 (17) 231 (100) (56) 233 (98) 219 (48) (100) 213 (78) (7) 318 (60) 298 (5) 261 (25) (11) 316 (65) 259 (100) (9) 261 (100) (90) 241 (100) (8) 320 (100) 300 (18) 264 (40) (23) 318 (10) 269 (57) (12) 263 (87) 235 (100) (100) 243 (73) (100) 315 (42) 283 (47) 271 (57) (77) 283 (29) 271 (37) (43) 271 (76) (96) 227 (100) (76) 229 (57) 215 (80) (100) 283 (57) 257 (40) (100) 257 (65) (90) 186 (100) (80) 189 (100) 161 (45)

experiments were carried out on the FQs. The major product ions of the CID spectra were formed from the precursor ions [M þ H]þ, together with other weaker product ions. Taking into account all of these results, we propose a series of stepwise decomposition pathways of FQs, considering that several peaks are related to direct cleavages of the precursor ion. In the first step, the losses of CO2 and H2O from the 3-carboxy group are observed. Further step display the loss of other peripheral groups, one spectra show losses of CH2CHNH2 or CH3CH2NCH2 from the 7-piperazidine substituent followed by losses of CO2, other spectra show losses of HF and CO followed by losses of H2O. An example of a compound containing 7-piperazidine is norfloxacin (see Figure 2), which showed that the precursor ion

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Figure 2. (a) In-sources ESI-CID-MS1 spectra of norfloxacin; (b) ESI-CID-MS2 spectra of the ion at m=z 276; (c) ESI-CID-MS2 spectra of the ion at m=z 302; (d) Scheme of stepwise dissociation pathway of norfloxacin.

[M þ H]þ at m=z 320. The produced ions were obtained by two different pathways, which define three stepwise dissociation pathways noted in a and b: 1. In the a pathway, the precursor ion at m=z 320 may decompose, via the a1 process, into the product ion at m=z 276 (A1). This described the intramolecular rearrangement loss of CO2 from the 3-carboxy group. After a1 dissociation, the 7-piperazidine cyclic structure cleavage step occurred by a nucleophilic attack of the N group, and then proton was transfered to give rise to the product ions at m=z 233 (A2-1) and m=z 219 (A2-2) (a2 step). The ion at m=z 233 was the main fragment of the m=z 276 product ion, which was a loss of 43 Da from the rearrangement of 7-piperazidine. This process began with 40 -N with a protonated, bond cleavage at 40 -50 (C-N). The proton then migrated from the 30 -C to 10 -N atom and bond cleavage at 10 -20 (C-N), leading to the loss of CH2CHNH2. The formation of the ion at m=z 219 was produced from the product ion at m=z 276 by the loss of 57 Da; this process began with the protonation of 10 -N. At the same time, bond cleavage at 10 -20 (C-N) and 50 -60 (C-C), lead to the loss of CH3CH2NCH2. The product ion at m=z 276 also can lose HF from 6-fluorine to yield the second generation ion at m=z 256. The product ion at m=z 256 underwent consecutive releases of CO and CH2CHNH2 to yield the product ion at m=z 228 and m=z 213.

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Figure 3. (a) In-sources ESI-CID-MS1 spectra of balofloxacin; (b) ESI-CID-MS2 spectra of the ion at m=z 359; (c) ESI-CID-MS2 spectra of the ion at m=z 315; (d) ESI-CID-MS2 spectra of the ion at m=z 283; (e) ESI-CID-MS2 spectra of the ion at m=z 271; (f) Scheme of stepwise dissociation pathway of balofloxacin.

2. In the b pathway, the step b1 initially described the positive charge induced carboxyl group single bond removed H2O to yield the product ion at m=z 302 (B1), which was observed as a pathway competing with the loss of CO2. The second step b2 consisted of charge parting, yielding the product ion at m=z 282 (B2-1) and m=z 231 (B2-2). The former was due to the loss of HF from 6-fluorine. The latter was observed with a very high abundance, which was due to the loss of CO from the 3-carboxy group and successive loss of CH2CHNH2 from the 7-piperazidine cyclic structure.

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CID-MS/MS of the Protonated Molecule of 7-piperidine FQs Balofloxacin and nadifloxacin are FQs with 7-piperidine substituent. The CID experiments of FQs with 7-piperidine were conducted on the molecular [M þ H]þ species. It was observed that their characteristic cleavage differs from those with 7-piperazidine. The fragmentation pattern was rationalized by taking into account the formation of complexes, as previously done for the positive-ion mode. The first fragment ion was observed resulting from the loss of the side chain of 7-piperidine using low-energy collision. The further characterized fragment ions indicate rearrangement of the 7-piperidine substituent and then loss of the peripheral groups. The important fragment ion was always observed at [M þ H – 44]þ depending on the substituent of the 3-carboxy group. Balofloxacin with 7-piperidine substituent groups as an example, for which a fragmentation mechanism is proposed in Figure 3. The CID spectrum of [M þ H]þ displayed the product ion at m=z 359 (C1) as the base peak; the product ion at m=z 359 is resulted from the loss of the side chain of 7-piperidine. This occurred from CH3NH cleavage assisted by a nucleophilic attack of the piperidine side chain CH3NH2 group followed by removal of CH3NH2 to yield the ion at m=z 359. Then, through intramolecular rearrangement loss of CH3CH2CH3 from piperidine ring yields the product ion at m=z 315. The two common product ions at m=z 271 and m=z 283 were decomposed by the product ion at m=z 315; the product ion at m=z 271 loss of CO2 from the 3-COOH is observed as a main pathway competing with the product ion at m=z 283 loss of CH3OH from the 8-OCH3. CONCLUSIONS Mass spectrometry is an excellent tool for the detection of FQs and its analogues. Here, we show that CID experiments constitute a method of choice to investigate the fragmentation of FQs. In this study, ESI always produces [M þ H]þ as the main peak. It is observed that FQs gave characteristic fragments such as the neutral loss of CO2, H2O, HF, and CO. These correspond to the carboxy, fluorine, and 4-carbonyl groups in their structures. The decarboxylation products and heterocyclic rearrangement products are their main fragmentations. The results show that the stepwise fragmentation pathways are different between FQs with piperazidine and those with piperidine. This work proposes general rules for the fragmentation of related FQs as a basis for the determination by LC-ESI-MSn. REFERENCES Can˜ada-Can˜ada, F., J. A. Arancibia, G. M. Escandar, G. A. Iban˜ez, A. Espinosa-Mansilla, A. Mun˜oz de la Pen˜a, and A. C. Olivieri. 2009. Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones. J. Chromatogr. A 1216 (24): 4868–4876. Guo, Y. L., K. Y. Yu, B. R. Xiang, and D. K. An. 1995. Negative ion mass spectra of quinolone drugs. J. Chin. Pharm. Univer. 26: 149–151. Lara, F. J., A. M. Garcı´a-Campan˜a, F. Ale´s-Barrero, and J. M. Bosque-Sendra. 2008. In-line solid-phase extraction preconcentration in capillary electrophoresis-tandem mass spectrometry

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