Research article Received: 26 January 2015,
Revised: 17 May 2015,
Accepted: 26 May 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3526
A validated HPLC-MS/MS method for the quantification of caderofloxacin in human plasma and its application to a clinical pharmacokinetic study Xianhua Zhang, Suodi Zhai, Jingli Duan and Li. Yang* ABSTRACT: A simple, selective and rapid HPLC-MS/MS method was developed and validated for the determination of caderofloxacin in human plasma. Sparfloxacin was used as the internal standard (IS). After precipitation with methanol and dilution with the mobile phase, the samples were injected into the HPLC-MS/MS system. The chromatographic separation was performed on a Zorbax XDB Eclipse C18 column (150 × 4.6 mm, 5 μm) with a mobile phase of ammonium acetate buffer (20 mm, pH 3.0)–methanol, 45:55 (v/v). The MS/MS analysis was done in positive mode. The multiple reaction monitoring transitions monitored were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The calibration curve was linear over the range of 50.0–8000 ng/mL with an aliquot of 100 μL plasma. The precision of the assay was 2.0–9.4 and 6.6–11.5% for the intra- and inter-run variability, respectively. The intra- and inter-run accuracy (relative error) was 4.4–10.0 and 1.2–4.0%. The total run time was 3.5 min. The assay was fully validated in accordance with the US Food and Drug Administration guidance. It was successfully applied to a pharmacokinetic study of caderofloxacin in healthy Chinese volunteers. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Caderofloxacin; HPLC-MS/MS; Pharmacokinetics; Human plasma
Introduction
126
Caderofloxacin, a newly developed active quinolone carboxylic acid (see the chemical structure in Fig. 1A), is a broad-spectrum antibacterial agent. As a potential member of the quinolone class of antimicrobial agents, it demonstrated activity against Gramnegative and Gram-positive organisms (Hooper and Wolfson, 1991; Biedenbach et al., 1995; Masuda et al., 1996). It is effective for the treatment of acute exacerbation of chronic bronchitis caused by Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae or Staphylococcus aureus bacteria. It is also used for the therapy of infectious pneumonia, acute sinusitis, urinary tract infections, glomerulonephritis and rectal infection (Masuda et al., 1996). Many methods, including HPLC/UV, HPLC/FLD and HPLC/MS/ MS have been reported for the determination of quinolones in biofluids (Huang et al., 2014; Raju et al., 2012; Roy et al., 2011; Jin et al., 2011; Fang et al., 2010; Pranger et al., 2010; Lin et al., 2009; Bian et al., 2007; Conte et al., 2006; Al-Dgither et al., 2006; Zoutendam et al., 2003; Vishwanathan et al., 2001; Zheng et al., 2006; Bai et al., 2009; Ma et al., 2003; Liu et al., 2007; Wang et al., 2009, 2011). However, only a few methods published were to determine caderofloxacin (Bai et al., 2009; Liu et al., 2007; Wang et al., 2011). Using an HPLC/UV method to determine caderofloxacin (Bai et al., 2009), the sensitivity was low and the lower limit of quantitation (LLOQ) was 100 ng/mL. Moreover, a large amount of sample volume (0.5 mL) was used to increase sensitivity. An HPLC/MS method was reported to determine caderofloxacin concentration in rat plasma (Liu et al., 2007); laborious liquid liquid extraction procedures were used to obtain
Biomed. Chromatogr. 2016; 30: 126–130
higher sensitivity and ‘cleaner’ extracts. Furthermore, a relatively long run time was used (10 min) to minimize the interferences. Thus far, there has been no literature about the clinical study of caderofloxacin using an HPLC-MS/MS method that meets the high throughput needs of modern drug development and gives rapid feedback of analytical information for pharmacokinetic studies. Here we developed and validated a simple, rapid and specific method for the determination of caderofloxacin in human plasma with an LLOQ of 50.0 ng/mL. Only an aliquot of 100 μL human plasma was used. Simple protein precipitation was used to prepare samples. Sparfloxacin was used as the internal standard (see the chemical structure in Fig. 1B.). The validated method was successfully applied to a clinical pharmacokinetic study of caderofloxacin in healthy volunteers.
Experimental Chemicals and reagents Caderofloxacin (lot number 130456–200502, purity 82%) and sparfloxacin (IS, lot number 130461–200501, purity 99.64%) were purchased from the National Institute for the Control of Biological Products (Beijing, China).
* Correspondence to: L. Yang, Department of Pharmacy, Peking University Third Hospital, Beijing 100191, People’s Republic of China. Email: [email protected] Department of Pharmacy, Peking University Third Hospital, Beijing100191, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of caderofloxacin in human plasma with HPLC/MS/MS Validation procedures
Figure 1. Chemical structures of (A) caderofloxacin and (B) IS.
HPLC-grade methanol was purchased from Fisher Chemical (So. Norwalk, CT, USA) and ammonium acetate was from Dikma Technologies (Lake Forest, CA, USA). Purified water was prepared using a Milli-Q purification system from Millipore Company (Bedford, MA, USA). Blank human plasma was provided by the hospital blood bank.
Preparation of calibration and quality control standards Two separate caderofloxacin stock solutions and the IS stock solution were prepared with methanol. Calibration and quality control (QC) working solutions were prepared by diluting the two caderofloxacin stock solutions with mobile phase. The IS working solution was obtained by diluting the IS stock solution with mobile phase and the final concentration was 8 μg/mL. All the solutions were stored at 4°C. Calibration and QC standards were prepared by adding 5 μL of the working solutions to 95 μL of blank plasma and mixed. There were eight calibrators ranged from 50.0 to 8000 ng/mL. Concentrations of the QC standards were 100, 500 and 5000 ng/mL.
Sample preparation A 10 μL aliquot of the IS working solution and a 400 μL aliquot of methanol was added to 100 μL of the plasma samples and votexed for 30 s. The mixture was centrifuged for 5 min at 10,000 rpm. Then 100 μL of the upper layer solution was transferred to another tube and diluted with 200 μL of the mobile phase. The mixture was transferred to the sample vials for injection.
Chromatographic and MS/MS conditions
Biomed. Chromatogr. 2016; 30: 126–130
Application to pharmacokinetic study The validated method was applied to a clinical pharmacokinetic study of caderofloxacin. The study was approved by the Institutional Review Board of Peking University Third Hospital. All the volunteers signed the informed consents to participate in the study according to the principles of the Declaration of Helsinki. No other prescribed or overthe-counter drugs were not taken from 3 months before the administration of caderofloxacin to the end of the trial. Ten healthy volunteers, including five males and five females, took a single dose of 200 mg caderofloxacin via continuous intravenous infusion. The blood samples were collected before and at 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 36 h post infusion. Blood samples were centrifuged for 5 min at 3000 rpm to obtain plasma, which was kept at 40°C until analysis. The pharmacokinetic parameters were evaluated by DAS software (version 2.1.1, developed by Ruiyuan Sun and Qingshan Zheng). The plasma drug concentration-time profile curve was constructed using Microsoft 2007.
Results and discussion Method development Both positive and negative modes with ESI source were investigated and the signal intensity of caderofloxacin obtained in positive mode was much higher than that of negative mode. In the precursor ion full-scan spectra, the most abundant ions were m/z 412.3 and m/z 393.2 for caderofloxacin and the IS, respectively. The product ion scan spectra showed high-abundance fragment ions at m/z 297.1 and m/z 292.2 for caderofloxacin and the IS, respectively. MS/MS parameters were optimized to obtain the highest sensitivity. The optimal parameters were described above. The product-ion spectra of the two compounds are shown in Fig. 2. Under these conditions, the multiple reaction monitoring transitions were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The chromatographic conditions were optimized to obtain good peak shapes and short retentions. Different types of columns, including Zorbax Eclipse XDB C18, Restek Ultra C18 and Xterra MS C18, were tested to get the best peak shapes. Finally a Zorbax Eclipse XDB C18 column provided satisfactory peak shapes and was hence employed. Methanol and acetonitrile were tested as the organic modifiers of the mobile phase. Methanol was finally adopted as it produced symmetrical sharp peak shapes and higher detection responses. Ammonium acetate was used in the mobile phase to improve the peak shapes. The final mobile phase was ammonium acetate (20 mm, pH 3.0)–methanol (45/55, v/v). The fluent
Copyright © 2015 John Wiley & Sons, Ltd.
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Separation was performed with an Agilent 1100 HPLC system (Agilent Technology, Waldbronn, Germany) consisting of binary pumps, an autosampler and a vacuum degasser. The HPLC system was coupled to an API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source (Applied Biosystems, Foster City, CA, USA), under the control of the Analyst software (version 1.4.1). Chromatographic separation was carried out on a Zorbax XDB Eclipse C18 column (150 × 4.6 mm, 5 μm, Agilent). The isocratic mobile phase consisted of ammonium acetate (20 mm, pH 3.0)–methanol (45/55, v/v). The flow rate was 1.0 mL/min. The effluent was split and 30% was introduced into the mass spectrometer. The injection volume was 10.0 μL. Mass spectrometry detection was performed in positive mode. The main MS/MS parameters were: source temperature, 500°C; heated gas, 7500 (set by the Analyst); ionspray voltage, 5000 V; nebulizer gas, 5; curtain gas, 10; collision gas, 6; collision energy, 39 V for caderofloxacin and 35 V for the IS; collision exit potential, 18 V for caderofloxacin and 13 V for the IS. The multiple reaction monitoring transitions were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The dwell time was 200 ms for each compound.
The method was validated according to the Guidance for Industry Bioanalytical Method Validation of the US Food and Drug Administration (2001). The linearity was evaluated by fitting the peak area ratio (drug vs 2 IS) vs each calibrator concentration with weighted 1/x . Sensitivity was determined by six replicates analysis of the LLOQ. Plasma from six different subjects was tested for possible interferences by endogenous peaks. The intra- and inter-run precisions were evaluated by analyzing QC samples at three levels. Recovery is calculated by peak area ratios of extracted samples/nonextracted samples at three concentration levels. Potential matrix effect was evaluated by the coefficient of variation (CV) of IS-normalized matrix factor in individual plasma from six different subjects at medium QC level. Bench-top stability was investigated by placing QC samples at room temperature before being processed. To evaluate the freeze–thaw stability, QC samples were subjected to three freeze–thaw cycles before processing. Processed stability was evaluated by placing extracted samples at room temperature after preparation. Long term stability was evaluated by storing QC samples at 40°C for 30 days.
X. Zhang et al.
Figure 4. Plasma concentration–time profile of a single dose of 200 mg caderofloxacin via continuous intravenous infusion (n = 10).
Method validation Specificity. Plasma from six individual subjects was tested to evaluate the endogenous interference. No interference was observed at the retention time of caderofloxacin or the IS (see miniatures in Fig. 4). Peak area at the retention time of caderofloxacin was less than 20% that of the LLOQ. Figure 2. Product spectra of (A) caderofloxacin and (B) IS.
was split before being introduced into the MS/MS system. Under these conditions, the typical retention times of caderofloxacin and the IS were 2.11 and 2.22 min (see Fig. 3.), respectively. The total run time was 3.5 min.
Linearity and LLOQ. The calibration curve was linear over the concentration range of 50.0 8000 ng/mL for caderofloxacin in human plasma with correlation coefficient r ≥ 0.9900. A typical weighted (1/x2) regression equation for caderofloxacin in human plasma was: y = 0.548x + 0.0488 (r = 0.9996). The calculated concentrations of the calibrators were within the acceptable limits demonstrated in the US Food and Drug Administration guidance. The LLOQ was 50.0 ng/mL. Sensitivity was evaluated with six replicates of LLOQ. The CV was 3.8% and RE was 5.4%. Precision and accuracy. Data of precisions are presented in Table 1. The CV ranged from 2.0 to 9.4% for the intra-run precision. For the inter-run, the data were from 6.6 to 11.5%. The accuracy was expressed by relative error (RE). The intra-run and inter-run accuracy ranged from 4.4 to 10.0% and from 1.2 to 4.0%, respectively. Recovery and matrix effect. The extraction recovery was evaluated with peak area ratios of extracted to unextracted samples at three levels. The extraction recoveries ranged from 98.7% to 101.7%. The extraction recovery was high because the total volumes of the extracted samples were a little smaller than those of
Table 1. Intra-run and inter-run precision and accuracy for the determination of caderofloxacin in human plasma Nominal concentration (ng/mL) Intra-batch (n = 5)
128
Figure 3. Representative chromatograms of a spiked plasma sample and a blank plasma sample (caderofloxacin concentration, 50.0 ng/mL; IS concentration, 8000 ng/mL).
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Inter-batch (n = 3)
Copyright © 2015 John Wiley & Sons, Ltd.
100 500 5000 100 500 5000
Measured RE (%) concentration, mean ± SD (ng/mL) 110 ± 5 547 ± 11 5222 ± 488 104 ± 9 516 ± 34 4937 ± 570
10.0 9.4 4.4 4.0 3.2 1.2
CV (%)
10.0 9.4 4.4 4.0 3.2 1.2
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Determination of caderofloxacin in human plasma with HPLC/MS/MS Table 2. Recovery and matrix effect for analyzing caderofloxacin and the IS Compound
Nominal concentration (ng/mL)
Recovery (%, n = 3)
Drug matrix factor (n = 6)
CV of matrix factor (n = 6, %)
IS normalized matrix effect (ISMF, n = 6)
CV of ISMF (n = 6, %)
100 500 5000
101.7 98.7 100.1
0.993
0.7
0.995
0.8
Caderofloxacin
ISMF, IS normalized matrix effect.
Table 3. Summary of stability for determination of carderofloxacin in human plasma Conditions
Nominal concentration (ng/mL)
Measured concentration (ng/mL)
RE (%)
Precision (CV, %)
100 500 5000 100 500 5000 100 500 5000 100 500 5000
94.6 ± 0.5 545 ± 19 4933 ± 38 114 ± 1 501 ± 15 4803 ± 203 103 ± 10 529 ± 37 5060 ± 360 87.4 ± 4.2 439 ± 13 5170 ± 156
5.4 9.0 1.3 14 0.2 3.9 3.0 5.8 1.2 12.6 12.2 3.4
0.5 3.5 0.8 0.9 3.0 4.2 9.7 7.0 7.1 4.8 3.0 3.0
Freeze–thaw stability (three cycles) Bench-top stability (RT, 7 h) Processed stability (room temperature, 12 h) Long-term stability ( 40°C, 30 days)
unextracted samples during protein precipitation. Matrix effect was investigated at medium QC with plasma from six individual lots. The mean matrix factor was 0.993 for caderofloxcacin and the CV was 0.7%. The IS-normalized matrix factor (ISMF) was 0.995 and the CV was 0.8%. The results indicate that the matrix effect can be neglected (Table 2).
Table 4. Pharmacokinetic parameters of 10 healthy volunteers after a single dose of 200 mg caderofloxacin (n = 10) Parameter Cmax tmax t1/2α t1/2β K21 K10 K12 Vd CL A α B β AUC(0–t) AUC(0–∞)
Unit
Mean
SD
ng/mL h h h 1/h 1/h 1/h L L/h
2090 2.0 0.213 4.674 0.988 0.787 3.671 26.622 16.923 0.752 5.196 5.573 0.150 12648 13254
520 — 0.217 0.470 0.312 0.501 2.767 13.625 4.598 0.163 3.421 1.700 0.016 3501 3812
ng h/mL ng h/mL
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Application The validated HPLC-MS/MS method was applied to a pharmacokinetic study of caderofloxacin after a single dose of continuous intravenous infusion. Fig. 4 shows the plasma concentration–time profile. The pharmacokinetic parameters were calculated with DAS 2.1.1 software. A two-compartment model was used to analyze the drug concentration–time curve data. The main pharmacokinetic parameters are shown in Table 4. The results of Cmax, t1/2α and Vd were consistent with those in the literature (Bai et al., 2009). However, t1/2β, AUC(0–t) and AUC(0–∞) were a little lower.
Conclusions A simple, selective and rapid HPLC-MS/MS method for the quantification of caderofloxacin in human plasma was developed and fully validated. Simple sample preparation procedures and short run time allowed the high throughput of the assay. Owing to the high sensitivity, only an aliquot of 0.1 mL plasma was needed. The method covered a concentration range of 50.0–8000 ng/mL. It was validated according to the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of caderofloxacin in Chinese healthy volunteers.
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Cmax, peak concentration; tmax, peak time; t1/2α, distribution half time; t1/2β, elimination half time; CL, clearance; Vd, apparent volume of distribution; AUC(0–t), area under curve (0 –t); AUC(0–∞), area under curve (0–∞).
Stability. The results of the stability study aere summarized in Table 3. RE ranged from 5.4 to 9.0% after three freeze–thaw cycles. The results demonstrate that caderofloxacin was stable in human plasma after three freeze–thaw cycles. There is no degradation of the drug in plasma after being kept at room temperature for at least 7 h. Samples were stable at room temperature for at least 12 h after being processed and caderofloxacin was stable in human plasma for at least one month at 40°C.
X. Zhang et al.
References Al-Dgither S, Alvi SN and Hammami MM. Development and validation of an HPLC method for the determination of gatifloxacin stability in human plasma. Journal of Pharmaceutical and Biomedical Analysis 2006; 41(1): 251–255. Bai N, Wang R, Fang Y, Lliu H, Yan M, Jin Y and Li Y. Determination of caderofloxacin concentration in healthy human plasma by RP-HPLC. Chinese Journal of Clinical Pharmacology 2009; 25: 334–337. Bian Z, Tian Y, Zhang Z, Xu F, Li J and Cao X. High performance liquid chromatography-electrospray ionization mass spectrometric determination of balofloxacin in human plasma and its pharmacokinetics. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2007; 850(1–2): 68–73. Biedenbach DJ, Sutton LD and Jones RN. Antimicrobial activity of CS-940, a new trifluorinated quinolone. Antimicrobial Agents in Chemotherapy 1995; 10: 2325–2330. Conte JE Jr, Golden JA, McIver M and Zurlinden E. Intrapulmonary pharmacokinetics and pharmacodynamics of high-dose levofloxacin in healthy volunteer subjects. International Journal of Antimicrobial Agents 2006; 28(2): 114–121. Fang PF, Cai HL, Li HD, Zhu RH, Tan QY, Gao W, Xu P, Liu YP, Zhang WY, Chen YC and Zhang F. Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2010; 878(24): 2286–2291. Hooper D and Wolfson J. Fluoroquinolone antimicrobial agents 1991; 324: 384–394. Huang K, Yang J, Zhang J, Ding Y, Chen L, Xu WY, Xu XJ, Duan R and He Q. Determination of sitafloxacin in human plasma by liquid chromatography– tandem mass spectrometry method: application to a pharmacokinetic study. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014; 957: 36–40. Jin HE, Lee KR, Kang IH, Chung SJ and Shim CK. Determination of zabofloxacin in rat plasma by liquid chromatography with mass spectrometry and its application to pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 2011; 54(4): 873–877. Lin SN, Lamm L, Newton TF, Reid MS, Moody DE and Foltz R. A liquid chromatography–electrospray ionization–tandem mass spectrometry method for quantitation of aripiprazole in human plasma. Journal of Analytical Toxicology 2009; 33(5): 237–242. Liu L, Pang X, Zhang D, Xu X, Liu H, Liang Y, Xie L and Liu X. Determination of caderofloxacin lactate in rat plasma by high-performance liquid chromatography–mass spectrometry and its application in rat
pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis 2007; 45: 799–803. Ma RL, Zhou HH, Liu R, Wang EH and Sheng LS. Analysis of impurity in caderofloxacin. Yao Xue Xue Bao 2003; 12: 950–952. Masuda N, Takahashi Y, Otsuki M, Ibuki E, Miyoshi H and Nishino T. In vitro and in vivo antibacterial activities of CS-940, a new 6-fluoro-8difluoromethoxy quinolone. Antimicrobial Agents and Chemotherapy 1996; 5: 1201–1207. Pranger AD, Alffenaar JW, Wessels AM, Greijdanus B and Uges DR. Determination of moxifloxacin in human plasma, plasma ultrafiltrate, and cerebrospinal fluid by a rapid and simple liquid chromatography–tandem mass spectrometry method. Journal of Analytical Toxicology 2010; 34(3): 135–141. Raju B, Ramesh M, Borkar RM, Padiya R, Banerjee SK and Srinivas R. Development and validation of liquid chromatography–mass spectrometric method for simultaneous determination of moxifloxacin and ketorolac in rat plasma: application to pharmacokinetic study. Biomedical Chromatography 2012; 26(11): 1341–1347. Roy B, Choudhury H, Das A, Das AK and Pal TK. Development and validation of a liquid chromatography-tandem mass spectrometry method to determine ulifloxacin, the active metabolite of prulifloxacin in rat and rabbit plasma: application to toxicokinetic study. Biomedical Chromatography 2011; 25(8): 890–901. US Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research: Rockville, MD, 2001. Vishwanathan K, Bartlett MG and Stewart JT. Determination of gatifloxacin in human plasma by liquid chromatography/electrospray tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2001; 15(12): 915–919. Wang Y, Zhang J, Zhang D, Zou Y and Liu G. Pharmacokinetics of multipledose of caderofloxacin hydrochloride and glucose injections in healthy volunteers. Chinese Pharmaceutical Journal 2009; 3: 217–221. Wang Y, Zhang J, Zhang D, Zou Y and Liu G. Pharmacokinetics of caderofloxacin hydrochloride after intravenous infusion in healthy volunteers. Chinese Journal of New Drugs 2011; 23: 2377–2380. Zheng W, Xu X and Wang E. Direct chiral separation of caderofloxacin enantiomers by HPLC using a glycoprotein column. Journal of Analytical Chemistry 2006; 11: 1090–1092. Zoutendam PH, Gavin M, Martin MJ and Dirr MK. Quantitation of PGE9509924, a novel, nonfluorinated quinolone, in rat plasma using liquid chromatography electrospray–tandem mass spectrometry following solid-phase extraction sample clean-up in a 96-well format. Journal of Pharmaceutical and Biomedical Analysis 2003; 33(5): 1073–1080.
130 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 126–130
Revised: 17 May 2015,
Accepted: 26 May 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3526
A validated HPLC-MS/MS method for the quantification of caderofloxacin in human plasma and its application to a clinical pharmacokinetic study Xianhua Zhang, Suodi Zhai, Jingli Duan and Li. Yang* ABSTRACT: A simple, selective and rapid HPLC-MS/MS method was developed and validated for the determination of caderofloxacin in human plasma. Sparfloxacin was used as the internal standard (IS). After precipitation with methanol and dilution with the mobile phase, the samples were injected into the HPLC-MS/MS system. The chromatographic separation was performed on a Zorbax XDB Eclipse C18 column (150 × 4.6 mm, 5 μm) with a mobile phase of ammonium acetate buffer (20 mm, pH 3.0)–methanol, 45:55 (v/v). The MS/MS analysis was done in positive mode. The multiple reaction monitoring transitions monitored were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The calibration curve was linear over the range of 50.0–8000 ng/mL with an aliquot of 100 μL plasma. The precision of the assay was 2.0–9.4 and 6.6–11.5% for the intra- and inter-run variability, respectively. The intra- and inter-run accuracy (relative error) was 4.4–10.0 and 1.2–4.0%. The total run time was 3.5 min. The assay was fully validated in accordance with the US Food and Drug Administration guidance. It was successfully applied to a pharmacokinetic study of caderofloxacin in healthy Chinese volunteers. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Caderofloxacin; HPLC-MS/MS; Pharmacokinetics; Human plasma
Introduction
126
Caderofloxacin, a newly developed active quinolone carboxylic acid (see the chemical structure in Fig. 1A), is a broad-spectrum antibacterial agent. As a potential member of the quinolone class of antimicrobial agents, it demonstrated activity against Gramnegative and Gram-positive organisms (Hooper and Wolfson, 1991; Biedenbach et al., 1995; Masuda et al., 1996). It is effective for the treatment of acute exacerbation of chronic bronchitis caused by Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae or Staphylococcus aureus bacteria. It is also used for the therapy of infectious pneumonia, acute sinusitis, urinary tract infections, glomerulonephritis and rectal infection (Masuda et al., 1996). Many methods, including HPLC/UV, HPLC/FLD and HPLC/MS/ MS have been reported for the determination of quinolones in biofluids (Huang et al., 2014; Raju et al., 2012; Roy et al., 2011; Jin et al., 2011; Fang et al., 2010; Pranger et al., 2010; Lin et al., 2009; Bian et al., 2007; Conte et al., 2006; Al-Dgither et al., 2006; Zoutendam et al., 2003; Vishwanathan et al., 2001; Zheng et al., 2006; Bai et al., 2009; Ma et al., 2003; Liu et al., 2007; Wang et al., 2009, 2011). However, only a few methods published were to determine caderofloxacin (Bai et al., 2009; Liu et al., 2007; Wang et al., 2011). Using an HPLC/UV method to determine caderofloxacin (Bai et al., 2009), the sensitivity was low and the lower limit of quantitation (LLOQ) was 100 ng/mL. Moreover, a large amount of sample volume (0.5 mL) was used to increase sensitivity. An HPLC/MS method was reported to determine caderofloxacin concentration in rat plasma (Liu et al., 2007); laborious liquid liquid extraction procedures were used to obtain
Biomed. Chromatogr. 2016; 30: 126–130
higher sensitivity and ‘cleaner’ extracts. Furthermore, a relatively long run time was used (10 min) to minimize the interferences. Thus far, there has been no literature about the clinical study of caderofloxacin using an HPLC-MS/MS method that meets the high throughput needs of modern drug development and gives rapid feedback of analytical information for pharmacokinetic studies. Here we developed and validated a simple, rapid and specific method for the determination of caderofloxacin in human plasma with an LLOQ of 50.0 ng/mL. Only an aliquot of 100 μL human plasma was used. Simple protein precipitation was used to prepare samples. Sparfloxacin was used as the internal standard (see the chemical structure in Fig. 1B.). The validated method was successfully applied to a clinical pharmacokinetic study of caderofloxacin in healthy volunteers.
Experimental Chemicals and reagents Caderofloxacin (lot number 130456–200502, purity 82%) and sparfloxacin (IS, lot number 130461–200501, purity 99.64%) were purchased from the National Institute for the Control of Biological Products (Beijing, China).
* Correspondence to: L. Yang, Department of Pharmacy, Peking University Third Hospital, Beijing 100191, People’s Republic of China. Email: [email protected] Department of Pharmacy, Peking University Third Hospital, Beijing100191, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of caderofloxacin in human plasma with HPLC/MS/MS Validation procedures
Figure 1. Chemical structures of (A) caderofloxacin and (B) IS.
HPLC-grade methanol was purchased from Fisher Chemical (So. Norwalk, CT, USA) and ammonium acetate was from Dikma Technologies (Lake Forest, CA, USA). Purified water was prepared using a Milli-Q purification system from Millipore Company (Bedford, MA, USA). Blank human plasma was provided by the hospital blood bank.
Preparation of calibration and quality control standards Two separate caderofloxacin stock solutions and the IS stock solution were prepared with methanol. Calibration and quality control (QC) working solutions were prepared by diluting the two caderofloxacin stock solutions with mobile phase. The IS working solution was obtained by diluting the IS stock solution with mobile phase and the final concentration was 8 μg/mL. All the solutions were stored at 4°C. Calibration and QC standards were prepared by adding 5 μL of the working solutions to 95 μL of blank plasma and mixed. There were eight calibrators ranged from 50.0 to 8000 ng/mL. Concentrations of the QC standards were 100, 500 and 5000 ng/mL.
Sample preparation A 10 μL aliquot of the IS working solution and a 400 μL aliquot of methanol was added to 100 μL of the plasma samples and votexed for 30 s. The mixture was centrifuged for 5 min at 10,000 rpm. Then 100 μL of the upper layer solution was transferred to another tube and diluted with 200 μL of the mobile phase. The mixture was transferred to the sample vials for injection.
Chromatographic and MS/MS conditions
Biomed. Chromatogr. 2016; 30: 126–130
Application to pharmacokinetic study The validated method was applied to a clinical pharmacokinetic study of caderofloxacin. The study was approved by the Institutional Review Board of Peking University Third Hospital. All the volunteers signed the informed consents to participate in the study according to the principles of the Declaration of Helsinki. No other prescribed or overthe-counter drugs were not taken from 3 months before the administration of caderofloxacin to the end of the trial. Ten healthy volunteers, including five males and five females, took a single dose of 200 mg caderofloxacin via continuous intravenous infusion. The blood samples were collected before and at 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 36 h post infusion. Blood samples were centrifuged for 5 min at 3000 rpm to obtain plasma, which was kept at 40°C until analysis. The pharmacokinetic parameters were evaluated by DAS software (version 2.1.1, developed by Ruiyuan Sun and Qingshan Zheng). The plasma drug concentration-time profile curve was constructed using Microsoft 2007.
Results and discussion Method development Both positive and negative modes with ESI source were investigated and the signal intensity of caderofloxacin obtained in positive mode was much higher than that of negative mode. In the precursor ion full-scan spectra, the most abundant ions were m/z 412.3 and m/z 393.2 for caderofloxacin and the IS, respectively. The product ion scan spectra showed high-abundance fragment ions at m/z 297.1 and m/z 292.2 for caderofloxacin and the IS, respectively. MS/MS parameters were optimized to obtain the highest sensitivity. The optimal parameters were described above. The product-ion spectra of the two compounds are shown in Fig. 2. Under these conditions, the multiple reaction monitoring transitions were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The chromatographic conditions were optimized to obtain good peak shapes and short retentions. Different types of columns, including Zorbax Eclipse XDB C18, Restek Ultra C18 and Xterra MS C18, were tested to get the best peak shapes. Finally a Zorbax Eclipse XDB C18 column provided satisfactory peak shapes and was hence employed. Methanol and acetonitrile were tested as the organic modifiers of the mobile phase. Methanol was finally adopted as it produced symmetrical sharp peak shapes and higher detection responses. Ammonium acetate was used in the mobile phase to improve the peak shapes. The final mobile phase was ammonium acetate (20 mm, pH 3.0)–methanol (45/55, v/v). The fluent
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Separation was performed with an Agilent 1100 HPLC system (Agilent Technology, Waldbronn, Germany) consisting of binary pumps, an autosampler and a vacuum degasser. The HPLC system was coupled to an API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source (Applied Biosystems, Foster City, CA, USA), under the control of the Analyst software (version 1.4.1). Chromatographic separation was carried out on a Zorbax XDB Eclipse C18 column (150 × 4.6 mm, 5 μm, Agilent). The isocratic mobile phase consisted of ammonium acetate (20 mm, pH 3.0)–methanol (45/55, v/v). The flow rate was 1.0 mL/min. The effluent was split and 30% was introduced into the mass spectrometer. The injection volume was 10.0 μL. Mass spectrometry detection was performed in positive mode. The main MS/MS parameters were: source temperature, 500°C; heated gas, 7500 (set by the Analyst); ionspray voltage, 5000 V; nebulizer gas, 5; curtain gas, 10; collision gas, 6; collision energy, 39 V for caderofloxacin and 35 V for the IS; collision exit potential, 18 V for caderofloxacin and 13 V for the IS. The multiple reaction monitoring transitions were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The dwell time was 200 ms for each compound.
The method was validated according to the Guidance for Industry Bioanalytical Method Validation of the US Food and Drug Administration (2001). The linearity was evaluated by fitting the peak area ratio (drug vs 2 IS) vs each calibrator concentration with weighted 1/x . Sensitivity was determined by six replicates analysis of the LLOQ. Plasma from six different subjects was tested for possible interferences by endogenous peaks. The intra- and inter-run precisions were evaluated by analyzing QC samples at three levels. Recovery is calculated by peak area ratios of extracted samples/nonextracted samples at three concentration levels. Potential matrix effect was evaluated by the coefficient of variation (CV) of IS-normalized matrix factor in individual plasma from six different subjects at medium QC level. Bench-top stability was investigated by placing QC samples at room temperature before being processed. To evaluate the freeze–thaw stability, QC samples were subjected to three freeze–thaw cycles before processing. Processed stability was evaluated by placing extracted samples at room temperature after preparation. Long term stability was evaluated by storing QC samples at 40°C for 30 days.
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Figure 4. Plasma concentration–time profile of a single dose of 200 mg caderofloxacin via continuous intravenous infusion (n = 10).
Method validation Specificity. Plasma from six individual subjects was tested to evaluate the endogenous interference. No interference was observed at the retention time of caderofloxacin or the IS (see miniatures in Fig. 4). Peak area at the retention time of caderofloxacin was less than 20% that of the LLOQ. Figure 2. Product spectra of (A) caderofloxacin and (B) IS.
was split before being introduced into the MS/MS system. Under these conditions, the typical retention times of caderofloxacin and the IS were 2.11 and 2.22 min (see Fig. 3.), respectively. The total run time was 3.5 min.
Linearity and LLOQ. The calibration curve was linear over the concentration range of 50.0 8000 ng/mL for caderofloxacin in human plasma with correlation coefficient r ≥ 0.9900. A typical weighted (1/x2) regression equation for caderofloxacin in human plasma was: y = 0.548x + 0.0488 (r = 0.9996). The calculated concentrations of the calibrators were within the acceptable limits demonstrated in the US Food and Drug Administration guidance. The LLOQ was 50.0 ng/mL. Sensitivity was evaluated with six replicates of LLOQ. The CV was 3.8% and RE was 5.4%. Precision and accuracy. Data of precisions are presented in Table 1. The CV ranged from 2.0 to 9.4% for the intra-run precision. For the inter-run, the data were from 6.6 to 11.5%. The accuracy was expressed by relative error (RE). The intra-run and inter-run accuracy ranged from 4.4 to 10.0% and from 1.2 to 4.0%, respectively. Recovery and matrix effect. The extraction recovery was evaluated with peak area ratios of extracted to unextracted samples at three levels. The extraction recoveries ranged from 98.7% to 101.7%. The extraction recovery was high because the total volumes of the extracted samples were a little smaller than those of
Table 1. Intra-run and inter-run precision and accuracy for the determination of caderofloxacin in human plasma Nominal concentration (ng/mL) Intra-batch (n = 5)
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Figure 3. Representative chromatograms of a spiked plasma sample and a blank plasma sample (caderofloxacin concentration, 50.0 ng/mL; IS concentration, 8000 ng/mL).
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Inter-batch (n = 3)
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100 500 5000 100 500 5000
Measured RE (%) concentration, mean ± SD (ng/mL) 110 ± 5 547 ± 11 5222 ± 488 104 ± 9 516 ± 34 4937 ± 570
10.0 9.4 4.4 4.0 3.2 1.2
CV (%)
10.0 9.4 4.4 4.0 3.2 1.2
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Determination of caderofloxacin in human plasma with HPLC/MS/MS Table 2. Recovery and matrix effect for analyzing caderofloxacin and the IS Compound
Nominal concentration (ng/mL)
Recovery (%, n = 3)
Drug matrix factor (n = 6)
CV of matrix factor (n = 6, %)
IS normalized matrix effect (ISMF, n = 6)
CV of ISMF (n = 6, %)
100 500 5000
101.7 98.7 100.1
0.993
0.7
0.995
0.8
Caderofloxacin
ISMF, IS normalized matrix effect.
Table 3. Summary of stability for determination of carderofloxacin in human plasma Conditions
Nominal concentration (ng/mL)
Measured concentration (ng/mL)
RE (%)
Precision (CV, %)
100 500 5000 100 500 5000 100 500 5000 100 500 5000
94.6 ± 0.5 545 ± 19 4933 ± 38 114 ± 1 501 ± 15 4803 ± 203 103 ± 10 529 ± 37 5060 ± 360 87.4 ± 4.2 439 ± 13 5170 ± 156
5.4 9.0 1.3 14 0.2 3.9 3.0 5.8 1.2 12.6 12.2 3.4
0.5 3.5 0.8 0.9 3.0 4.2 9.7 7.0 7.1 4.8 3.0 3.0
Freeze–thaw stability (three cycles) Bench-top stability (RT, 7 h) Processed stability (room temperature, 12 h) Long-term stability ( 40°C, 30 days)
unextracted samples during protein precipitation. Matrix effect was investigated at medium QC with plasma from six individual lots. The mean matrix factor was 0.993 for caderofloxcacin and the CV was 0.7%. The IS-normalized matrix factor (ISMF) was 0.995 and the CV was 0.8%. The results indicate that the matrix effect can be neglected (Table 2).
Table 4. Pharmacokinetic parameters of 10 healthy volunteers after a single dose of 200 mg caderofloxacin (n = 10) Parameter Cmax tmax t1/2α t1/2β K21 K10 K12 Vd CL A α B β AUC(0–t) AUC(0–∞)
Unit
Mean
SD
ng/mL h h h 1/h 1/h 1/h L L/h
2090 2.0 0.213 4.674 0.988 0.787 3.671 26.622 16.923 0.752 5.196 5.573 0.150 12648 13254
520 — 0.217 0.470 0.312 0.501 2.767 13.625 4.598 0.163 3.421 1.700 0.016 3501 3812
ng h/mL ng h/mL
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Application The validated HPLC-MS/MS method was applied to a pharmacokinetic study of caderofloxacin after a single dose of continuous intravenous infusion. Fig. 4 shows the plasma concentration–time profile. The pharmacokinetic parameters were calculated with DAS 2.1.1 software. A two-compartment model was used to analyze the drug concentration–time curve data. The main pharmacokinetic parameters are shown in Table 4. The results of Cmax, t1/2α and Vd were consistent with those in the literature (Bai et al., 2009). However, t1/2β, AUC(0–t) and AUC(0–∞) were a little lower.
Conclusions A simple, selective and rapid HPLC-MS/MS method for the quantification of caderofloxacin in human plasma was developed and fully validated. Simple sample preparation procedures and short run time allowed the high throughput of the assay. Owing to the high sensitivity, only an aliquot of 0.1 mL plasma was needed. The method covered a concentration range of 50.0–8000 ng/mL. It was validated according to the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of caderofloxacin in Chinese healthy volunteers.
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Cmax, peak concentration; tmax, peak time; t1/2α, distribution half time; t1/2β, elimination half time; CL, clearance; Vd, apparent volume of distribution; AUC(0–t), area under curve (0 –t); AUC(0–∞), area under curve (0–∞).
Stability. The results of the stability study aere summarized in Table 3. RE ranged from 5.4 to 9.0% after three freeze–thaw cycles. The results demonstrate that caderofloxacin was stable in human plasma after three freeze–thaw cycles. There is no degradation of the drug in plasma after being kept at room temperature for at least 7 h. Samples were stable at room temperature for at least 12 h after being processed and caderofloxacin was stable in human plasma for at least one month at 40°C.
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