Journal of Chromatography B, 967 (2014) 219–224

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Short Communication

A simple LC–MS/MS method for determination of sitafloxacin in human urine Yuanyuan Wang a , Yang Liu b , Hongwen Zhang a,∗∗ , Yongqing Wang a , Yun Liu a , Libin Wang a , Ning Ou a,∗ a b

Department of Pharmacy, The First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, PR China School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, PR China

a r t i c l e

i n f o

Article history: Received 12 February 2014 Accepted 11 July 2014 Available online 1 August 2014 Keywords: Sitafloxacin LC–MS/MS Urine Urine recovery study

a b s t r a c t Sitafloxacin is a new fluoroquinolone antimicrobial agent with high activity. In this article, we reported a simple, rapid and specific LC–MS/MS method for accurate determination of sitafloxacin concentrations in human urine from healthy volunteers in detail. A two-step dilution method for the analysis of sitafloxacin in human urine using LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines and Chinese State Food and Drug Administration (CFDA) guidelines for the validation of bioanalytical methods. The method uses 50 ␮L of urine and covers a working range from 0.025 to 20 ␮g/mL with a LLOQ of 0.025 ␮g/mL. This new LC–MS/MS assay is sensitive and specific. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Sitafloxacin,7-[(7S)-7-amino-5-azaspiro[2,4]heptan-5-yl]-8chloro-6-fluoro-1-[(1R, 2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro4-oxo-3-quinolinecarboxylic acid sesquihydrate (DU-6859a) is a new fluoroquinolone antimicrobial agent with high activity against Gram-negative and Gram-positive bacteria, and against anaerobic organisms [1–3]. Its chemical structure is shown in Fig. 1. Early clinical studies have been conducted to reveal the pharmacokinetics of sitafloxacin after oral administration [1,2]. These studies showed that sitafloxacin was rapidly and extensively absorbed and that elimination of the drug was largely by renal excretion. Results of sitafloxacin clinical studies [1] in healthy volunteers showed that after 25- to 200-mg sitafloxacin orally administration, Cmax of sitafloxacin in serum ranged from 0.29 to 1.86 ␮g/ml with a tmax of 1.0–1.3 h, and the t1/2 ranged from 4.4 to 5.0 h, the apparent volume of distribution clearly exceeded 1 L/kg, the Cltot ranged from 284 to 328 mL/min. Within 48 h, the cumulative urinary recovery [1,2] of unchanged drug account for approximately 61–74% of the orally administered dose. Nakashima et al. [1] and O’Grady et al. [2] described a method for the quantification of sitafloxacin in human serum and urine using SPE for sample preparation and HPLC with

post-column photolysis and fluorescence detection, which needed considerable time and work. In a sitafloxacin pharmacokinetic study [3] in different animals, sitafloxacin in serum and urine samples were determined by means of radioactivity following the administration of 14 C-labeled sitafloxacin. However, 14 C-labeled sitafloxacin was not considered in this pharmacokinetic study in healthy volunteers. Newly several studies [4–6] on the pharmacokinetics or PK-PD in patients only mentioned the determination of serum concentration of sitafloxacin using LC–MS/MS, and no details about sample preparation were introduced. Another paper [7] introduced a entirely validated LC–MS/MS method to determine sitafloxacin in human plasma using liquid–liquid extraction in sample preparation, which accounting for longer time and more work in sample preparation. So, there were few papers on the determination of sitafloxacin in human urine. The objective of the present study was to develop a simple, selective and sensitive LC–MS/MS method, which only employing two-step dilution for the sample preparation, to determine sitafloxacin in human urine. The assay was validated and applied to a clinical urine recovery study, in which ten healthy Chinese volunteers were orally administered a single 50 mg sitafloxacin. 2. Materials and methods

∗ Corresponding author. Tel.: +86 25 68217377. ∗∗ Corresponding author. Tel.: +86 25 68216976. E-mail addresses: [email protected] (H. Zhang), ningou [email protected] (N. Ou). http://dx.doi.org/10.1016/j.jchromb.2014.07.015 1570-0232/© 2014 Elsevier B.V. All rights reserved.

2.1. Chemicals and reagents Sitafloxacin (purity 99.68%), sitafloxacin tablet (50 mg) and moxifloxacin hydrochloride (as internal standard, IS, purity 100.1%,

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Y. Wang et al. / J. Chromatogr. B 967 (2014) 219–224

O O F

F

CO2H

3/2H2O

N

N Cl

COOH

N

N HN

.HCl

OCH3

F

H2N

a

b

Fig. 1. Chemical structures of sitafloxacin (a) and moxifloxacin hydrochloride (b).

chemical structure is shown in Fig. 1) were supplied by Nanjing Yoko Biomedical R&D Ltd. China, and HPLC-grade methanol was purchased from Merck (Merck & Co. Inc., Germany). Formic acid (analytical grade) was purchased from Lingfeng Chemical Reagents Limited Company (Shanghai, China). Water was obtained from a Milli-Q water purification system (Millipore Corp., USA). 2.2. Instrumentation and conditions The LC–MS/MS equipment consisted of two LC-20AD pumps, a SIL-20ACHT autosampler, a CTO-20AC column oven (SHIMADZU Corporation, Japan) and a Qtrap® 5500 mass spectrometer (AB Sciex, USA), equipped with an ESI ion source. Analyst software (Version 1.6.1) and MultiQuant software (Version 1.6.1) were used for data acquisition and analysis, respectively. LC separation was performed on a Agilent Proshell 120 SB-C18 column (50 mm × 2.1 mm, 2.7 ␮m) using isocratic elution with a mobile phase of methanol–0.1% formic acid (38/62, v/v) at a flow rate of 0.3 mL/min. The column temperature was maintained at 40 ◦ C. The autosampler temperature was 10 ◦ C. The mass spectrometer was operated in the positive ionization mode. The injection volume was 5 ␮L. The ionspray voltage was set to 5500 V and the ion source temperature was set at 600 ◦ C. The curtain gas and collision gas were 35.0 psi and set at medium level, respectively. The ion source gas 1 and ion source gas 2 were all set at 50 psi. The declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) for sitafloxacin were 140, 10, 27 and 30 V, respectively. For moxifloxacin (IS), the declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) were 130, 12, 28 and 26 V, respectively. Detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 410.1 precursor ion to the m/z 392.1 product ion for sitafloxacin. Moxifloxacin was monitored with m/z 402.2 precursor ion to the m/z 384.2 product ion. 2.3. Preparation of calibration standards and quality control samples Primary stock solutions of sitafloxacin and IS were prepared by dissolving sitafloxacin and IS reference standards in methanol at a concentration of 1 mg/mL after correction for impurity, water of crystallization and hydrochloric acid. The primary stock solution of sitafloxacin was successively diluted with methanol to prepare working solutions to prepare calibration standards for sitafloxacin. Another primary stock solution (1 mg/mL) of sitafloxacin was made in methanol for preparation of quality control (QC) samples. The stock solutions of sitafloxacin and IS were stored at −20 ◦ C, which were found to be stable for 40 days. Working solutions of sitafloxacin to prepare calibration curve were prepared by serial

dilutions with methanol, at final concentrations of 0.25, 1, 3, 10, 25, 50, 100 and 200 ␮g/mL from the primary stock solution. QC working solutions of sitafloxacin were diluted with methanol, at final concentrations of 0.5, 1, 16 ␮g/mL. IS working solution was at final concentration of 2 ␮g/mL by dilutions with methanol. Calibration standards and QC samples in urine were prepared by diluting corresponding working solutions with fresh drug-free human urine (no any stabilizers were added), respectively. The final concentrations of calibration standards were 0.025, 0.1, 0.3, 1, 2.5, 5, 10, 20 ␮g/mL. The final concentrations of QC samples were 0.05, 1 and 16 ␮g/mL. Urine samples were stored at −70 ◦ C. 2.4. Sample preparation Urine samples were prepared by two-step of dilution method. Aliquots (50 ␮L) of human urine were pipetted into 1.5 mL plastic centrifuge tubes with the addition of 20 ␮L of internal standard (2 ␮g/mL) and 350 ␮L of 0.1% formic acid in methanol, The samples were vortex mixed for 2 min and centrifuged at 13,800 × g for 10 min at 4 ◦ C. 25 ␮L aliquots of the supernatant were added with 975 ␮L of 0.1% formic acid, then, were finally mixed for 2 min. Sample preparation was done under light-free condition. 2.5. Method validation The method was validated for selectivity, sensitivity, linearity, recovery, matrix effects, precision, accuracy, stability and dilution integrity according to the US Food and Drug Administration (FDA) [8] and Chinese State Food and Drug Administration (CFDA) guidelines [9] for the validation of bioanalytical methods. 2.5.1. Specificity The specificity of this method was evaluated by comparison of LC–MS/MS chromatograms of sitafloxacin at the LLOQ to those of six individual human blank urine samples. 2.5.2. Linearity Calibration standards in human urine were prepared and analyzed in three independent runs. The following assay procedures were the same as those described above. In each run, a blank urine sample was analyzed to confirm the absence of interference, but not used to construct the calibration function. The calibration curves were constructed by weighted (1/x2 ) least-square linear regression analysis of the peak area ratio of analyte to its internal standard against nominal analyte concentration. The LLOQ is defined as the lowest concentration on the calibration curve at which precision (relative standard deviation, RSD%) was within 20% and accuracy (relative deviation, RE%) was within ±20%, and it was established using five samples independent of standards. The deviations of back-calculated concentrations of calibration standards, except for

Y. Wang et al. / J. Chromatogr. B 967 (2014) 219–224

LLOQ, from their nominal values should be within ±15% for calibration levels. 2.5.3. Extraction recovery The extraction recovery of sitafloxacin was calculated at three levels (0.05, 1 and 16 ␮g/mL) by comparing two groups of control samples: (A) drug spiked to urine and prepared normally (pre-extraction); (B) drug spiked after extraction of blank urine (post-extraction). The ratio (A/B × 100) is defined as the extraction recovery. The reproducibility of the extraction procedure was determined as RSD%.

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any acute or chronic disease, or any allergy to any drugs; no using any kind of drugs within two weeks before the trial. All subjects provided written informed consent prior to participation. Ten healthy Chinese volunteers were orally administrated a single dose 50 mg sitafloxacin tablet with 200 mL of water after fasting for 12 h. Urine samples were collected prior to and in the following intervals: 0–3, 3–6, 6–12, 12–24, 24–36, and 36–48 h after dosing. Sample aliquots were stored frozen at −70 ◦ C until analysis. 3. Results and discussion 3.1. LC–MS/MS optimization

2.5.4. Matrix effect The matrix effect (ME) on the ionization of the analyte was evaluated by comparing the peak areas of post-extraction blank urine samples from six different subjects spiked with analyte with that of the standards in mobile phase at equivalent concentrations. The ME of the method was evaluated at all QC levels (0.05, 1 and 16 ␮g/mL). The inter-subject variability (RSD%) of matrix effects at every concentration level was assessed. 2.5.5. Precision and accuracy Precision and accuracy were determined by measuring the concentrations of analyte in urine in five replicates of QC samples at three different concentrations for three separate batches. Assay precision was calculated using the relative standard deviation (RSD%). Accuracy is defined as the relative deviation in the calculated value (E) of a standard from that of its true value (T), expressed as a percentage (RE%). It was calculated by using the formula RE% = (E − T)/T × 100. 2.5.6. Stability The short-term stability of sitafloxacin was assessed by determining QC samples kept at room temperature for 2 h, which exceeded the routine preparation time of samples. The long-term stability was evaluated by determining QC samples kept at low temperature (−70 ◦ C) for 29 days. The processed samples stability was measured by determining QC samples kept under autosampler condition (10 ◦ C) for 24 h. The freeze–thaw stability was tested by analyzing QC samples undergoing three freeze (−70 ◦ C) and thaw (room temperature) cycles on consecutive days. 2.5.7. Dilution integrity Dilution integrity was investigated to ensure that samples could be diluted with blank matrix without affecting the final concentration. Dilution integrity experiment was performed for study sample concentrations crossing the ULOQ (the highest standard of the calibration curve). Sitafloxacin spiked human urine samples prepared at five-times above the ULOQ concentration. Urine samples at the concentration of 100 ␮g/mL were diluted tenfold with human blank urine to obtain the final test concentrations of 10 ␮g/mL (n = 5), then analyzed by LC–MS/MS. The back-calculated concentration should have precision of