Journal of Chromatography B, 967 (2014) 235–239
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
UPLC–MS/MS determination of thiamphenicol in human plasma and its application to a pharmacokinetic study Zhe Wang a , Hui Yang b , Wei Sun a , Cheng-ke Huang a , Xiao Cui a , Xiang-jun Qiu b , Qing-quan Lian c,d , Zeng-shou Wang a,∗ a
Department of Pharmacy, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China Medical College of Henan University of Science and Technology, Luoyang 471003, China c Zhejiang Province Key Laboratory of Anesthesiology, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China d Department of Anesthesiology, Critical Care and Pain Medicine, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China b
a r t i c l e
i n f o
Article history: Received 16 February 2014 Received in revised form 10 July 2014 Accepted 15 July 2014 Available online 4 August 2014 Keywords: Thiamphenicol UPLC–MS/MS Human plasma Pharmacokinetics
a b s t r a c t A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine thiamphenicol (TAP) in human plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to precipitation of plasma protein, and to a 0.1 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 354.3 → 185.1 for TAP and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 10–8000 ng/mL with a lower limit of quantification of 10 ng/mL. Only 1.5 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of TAP in healthy Chinese volunteers after oral administration. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Thiamphenicol (d(+)-threo-2-di-chloroacetamido-1-(4methylsulphonylphenyl) propane-1,3-diol, TAP, Fig. 1), an analogue of chloramphenicol, is developed by replacing the aromatic nitro group with a methylsulphonyl group [1]. Its broad antibiotic spectrum includes both Gram-positive and Gram-negative pathogens, especially effective against anaerobic organisms [2]. At a sub-cellular level, TAP binds to the 50S subunits of the 70S ribosomes to block peptidyl transferase, hence inhibiting the extension of peptide chain and synthesis of bacterial protein. However, the administration of TAP to rats, in relatively high doses shows haematological toxicity [3,4]. Nevertheless, due to its low price and steady antibiosis effectiveness, the use of TAP in human, livestock and aquaculture still exists. So, it is very
∗ Corresponding author. Tel.: +86 577 88002658. E-mail address: [email protected] (Z.-s. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.07.033 1570-0232/© 2014 Elsevier B.V. All rights reserved.
important to develop a sensitive, rapid and simple method for the determination of TAP in human plasma. Up to now, the main approaches for the detection of TAP in honey, meat, seafood, milk, egg, plasma and urine include immunoassay [5], HPLC [6–9], LC–MS/MS [10–13] and GC [14–16]. However, these methods have their own disadvantages, such as complex analyzing processes, long time requirement for preparation of samples, and long chromatographic run times, which are not preferable when dealing with a large number of samples. Moreover, no publication has described the quantitative analysis of TAP in the biological fluid using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Therefore, a novel, simple and rapid method for measurement of TAP concentration in human plasma sample is still required for analyses involving TAP monitoring. UPLC–MS/MS has been evaluated as a faster and more efficient analytical tool compared with current chromatography [17]. In the present study, a UPLC–MS/MS method for the determination of TAP using chlorzoxazone as an internal standard (IS) was developed.
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20, 500, 6400 ng/mL for TAP. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (1 g/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C. 2.4. Sample preparation Fig. 1. The chemical structures of TAP and IS in the present study: (A) TAP; (B) chlorzoxazone (IS).
This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers. 2. Materials and methods 2.1. Chemicals materials TAP and chlorzoxazone (internal standard, IS) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC grade and purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Blank human plasma used in this study was supplied by The Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). 2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid) as follows: 0–0.3 min (18% A), 0.3–1.5 min (18–98% A). The flow rate was 0.40 mL/min. The overall run time was 1.5 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 200 ms for each MRM transition. The MRM transitions were m/z 354.3 → 185.1 and m/z 168.1 → 132.1 for TAP and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 3.5 kV; and temperature, 350 ◦ C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex). 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of TAP that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 10, 20, 50, 100, 400, 1000, 2000, 4000 and 8000 ng/mL for TAP in human plasma. The concentrations of QC samples in plasma were
Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (1 g/mL) and 1 mL ethyl acetate was added to 100 L of collected plasma sample. After vortexing for 2 min, the samples were centrifuged at 13,000 rpm for 5 min, the organic phase was transferred into a clean tube and evaporated under nitrogen stream at 40 ◦ C. The residue was dissolved in 100 L of mobile phase. The supernatant (5 L) was injected into the UPLC–MS/MS system for analysis. 2.5. Method validation Before using this method to determinate TAP in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances [18]. Specificity was determined by analysis of blank human plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of nine concentrations of TAP (10–8000 ng/mL) were separately extracted and assayed on three separate days. The linearity for TAP was investigated by weighted (1/x2 ) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (20, 500, 6400 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy between −15 and 15%. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (100 ng/mL) similarly. To determine the matrix effect, six different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (100 ng/mL) in the same manner. The stabilities in human plasma were tested by analyzing five replicates of plasma samples at three concentration levels (20, 500, 6400 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 ◦ C for 12 h. The freeze–thaw stability was
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Fig. 2. Representative chromatograms of TAP and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with TAP (1) and IS (2); (C) a plasma sample from a person 1 h after an oral administration 500 mg.
evaluated after three complete freeze–thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 30 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15%) and precision (RSD % ≤15%).
0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 15 h after oral administration. Blood samples were centrifuged at 4000 × g for 8 min and the plasma was separated and kept frozen at −20 ◦ C until analysis. 2.7. Data analysis Plasma concentration vs. time profiles were analyzed using DAS (Drug and statistics) software (Version 3.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
2.6. Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 500 mg TAP tablets (Fujian Haiwang Pharmaceutical Co., Ltd.) to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of Tonji Medical College, Huazhong University of Science and Technology prior to the study. Twenty volunteers (aged 21–28 years, weighing 50–70 kg, and height 156–180 cm) were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetic study of TAP. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral dose of 500 mg TAP with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes before and
3. Results and discussion 3.1. Method development and optimization Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological samples in order to remove the possible interfering matrix components and increase selectivity and sensitivity. In this study, liquid–liquid extraction with organic solvent was used for the sample preparation. This simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the analytes. In this work, was added in the process of sample preparation. Internal standard plays an important role in biopharmaceutical analysis, and chlorzoxazone was found to be suitable
Table 1 Precision and accuracy of method for the determination of TAP in human plasma (n = 6). Analytes
Concentration added (ng/mL)
TAP
20 500 6400
Intra-day precision
Inter-day precision
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
21.03 ± 2.11 504.88 ± 15.43 6430.40 ± 68.99
11.18 3.17 1.08
5.17 0.98 0.47
21.20 ± 2.45 509.83 ± 24.75 6106.83 ± 281.21
13.55 4.60 0.80
5.98 −0.29 −1.04
Table 2 Recovery and matrix effect of TAP and internal standards (n = 6). Analytes
Concentration added (ng/mL)
Recovery (%) Mean ± SD
TAP IS
20 500 6400 100
78.74 75.89 79.31 79.91
± ± ± ±
4.76 1.29 0.65 2.10
Matrix effect (%) RSD (%)
Mean ± SD
6.04 1.70 0.65 2.10
97.72 101.39 104.69 103.42
± ± ± ±
6.40 5.07 1.44 2.72
RSD (%) 6.55 5.00 1.37 2.63
RSD (%)
7.86 5.43 4.60 20.06 ± 1.77 522.53 ± 27.81 6439.02 ± 303.48 7.65 1.13 −0.45 12.40 4.12 4.75 21.53 ± 2.42 505.64 ± 19.92 6371.01 ± 300.09 14.46 2.66 2.15 20.28 ± 2.87 508.4 ± 13.22 6425.12 ± 136.16 20 500 6400
1.40 1.68 0.39
Mean ± SD RE (%) RSD (%) Mean ± SD
TAP
The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100. Results are shown in Table 2. The recovery in plasma ranged from 75.89 to 79.31% for TAP. The recovery of IS (100 ng/mL) in plasma was 79.91%. The matrix effect in human plasma were all between 97.72 and 104.69% for TAP at different QC levels (Table 2). The matrix effect for IS (100 ng/mL) was 103.42%. No apparent matrix effect was found
Concentration added (ng/mL)
3.5. Recovery and matrix effect
Analytes
The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations for each biological matrix. The results are summarized in Table 1. The method was reliable and reproducible since RSD % was below 15% and RE % was between −1.04 and 5.98% for all the investigated concentrations of TAP in human plasma.
Table 3 Stability results of TAP in human plasma in different conditions (n = 5).
3.4. Precision and accuracy
4 ◦ C, 12 h
The peak area ratios of TAP/IS versus the nominal concentrations of TAP showed a good linear relationship over the concentration ranges of 10–8000 ng/mL in human plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.073101x ± 0.0146677, where y represents the peak area ratios of TAP to the IS and x represents plasma concentrations of analyte. The LLOQ in human plasma was 10 ng/mL with the RSD and RE of 9.25 and 3.64%, respectively.
Room temperature, 2 h
3.3. Linearity and sensitivity
4.73 −2.81 1.21
UPLC–MS/MS chromatogram of the analytes in human plasma samples were shown in Fig. 2. The retention times of TAP and IS are 0.84 and 1.25 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
9.59 6.61 2.21
Mean ± SD RE (%)
3.2. Specificity
20.95 ± 1.85 485.96 ± 32.61 6477.32 ± 143.76
Three freeze–thaw
RSD (%)
RE (%)
−20 ◦ C, 30 days
RSD (%)
RE (%)
for the present work and finally used as the internal standard for its stability and high recovery. Other chromatographic conditions, especially the composition of mobile phase, were tested to achieve good resolution and symmetric peak shapes of analytes as well as a short run time. It was found that with the acetonitrile and 1% formic acid in water with gradient elution could achieve our purpose and were finally adopted as the mobile phase for chromatographic separation. The retention time was 0.84 min for TAP and 1.25 min for IS. The run time was 1.5 min. The advantage of this method is that a relatively larger number of samples can be analyzed in a short time, thus increasing output [19–21]. Analysis method was set up by optimizing UPLC and MS/MS conditions to obtain the best possible sensitivity. The analytes were analyzed firstly using MS by syringe infusion of individual standard solution and they were all more efficiently ionized in ESI negative mode than in positive mode. ESI negative mode was therefore employed. Parameters such as ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecules of analytes. The collision energy of collision-induced decomposition (CID) was optimized for maximum response of the fragmentation of analytes. MRM was used to monitor precursor ion and production, which could reduce interference and enhance selectivity. The MRM transitions were m/z 354.3 → 185.1 and m/z 168.1 → 132.1 for TAP and IS, respectively
0.32 4.51 0.61
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Mean ± SD
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administration of 500 mg TAP tablets, the main pharmacokinetic parameters of TAP for twenty volunteers were estimated. The mean plasma concentration–time curve of TAP was displayed in Fig. 3, and the main pharmacokinetic parameters of TAP were calculated and are summarized in Table 4. Compared with recently published papers describing the pharmacokinetic profiles of TAP in healthy volunteers, the pharmacokinetic parameters of TAP obtained in the study were generally similar [22]. 4. Conclusions
Fig. 3. Plasma concentration versus time curves of TAP after a single oral administration 500 mg to 20 Chinese healthy male volunteers. Table 4 Pharmacokinetic parameters of TAP after oral administration 500 mg to 20 Chinese healthy male volunteers. Parameters
Mean ± SD
t1/2 (h) Cmax (g/mL) Tmax (h) AUC0→15 (g/mL h) AUC0→∞ (g/mL h)
3.287 5.247 2.075 29.043 30.736
± ± ± ± ±
0.401 1.218 0.545 4.926 5.529
to affect the determination of TAP and IS in plasma. As a result, the matrix effect from plasma was negligible in this method. 3.6. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. Table 3 shows the stability data for TAP in plasma under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 2 h and at 4 ◦ C for 12 h. No significant degradation was observed when samples of TAP were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, TAP in samples were stable at −20 ◦ C for 30 days. 3.7. Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of TAP in human plasma. After an oral
An UPLC–MS/MS method for the determination of TAP in human plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of TAP level in human plasma using an UPLC–MS/MS method. Compared with the analytical methods reported in the literatures, the method offered superior sample preparation with liquid–liquid extraction with organic solvent to precipitation of plasma protein and shorter run time of 1.5 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of TAP tablets in healthy volunteers. References [1] J. Fuglesang, T. Bergan, Antibiot. Chemother. (1971) 31 (1982) 1–21. [2] V. Tullio, A. Cuffini, N. Mandras, J. Roana, G. Banche, D. Ungheri, N. Carlone, Int. J. Antimicrob. Agents 24 (2004) 381–385. [3] J.A. Turton, C.M. Andrews, A.C. Havard, S. Robinson, M. York, T.C. Williams, F.M. Gibson, Food Chem. Toxicol. 40 (2002) 1849–1861. [4] J.P. Kaltwasser, B. Simon, E. Werner, U. Leuschner, M.H. Khan, H.J. Becker, W. Stille, Klin. Wochenschr. 51 (1973) 347–350. [5] T.L. Fodey, S.E. George, I.M. Traynor, P. Delahaut, D.G. Kennedy, C.T. Elliott, S.R. Crooks, J. Immunol. Methods 393 (2013) 30–37. [6] J. Li, H. Chen, H. Chen, Y. Ye, J. Sep. Sci. 35 (2012) 137–144. [7] H. Chen, H. Chen, L. Liao, J. Ying, J. Huang, J. Chromatogr. Sci. 48 (2010) 450–455. [8] H. Chen, H. Chen, J. Ying, J. Huang, L. Liao, Anal. Chim. Acta 632 (2009) 80–85. [9] B. Yang, Y. Lu, J. Zheng, J. Zhu, J. He, D. Zhao, P. Song, X. Chen, Xenobiotica 41 (2011) 226–231. [10] D. Tian, F. Feng, Y. Li, J. Yang, X. Tian, F. Mo, J. Pharm. Biomed. Anal. 48 (2008) 1015–1019. [11] P. Luo, X. Chen, C. Liang, H. Kuang, L. Lu, Z. Jiang, Z. Wang, C. Li, S. Zhang, J. Shen, J. Chromatogr, B. Analyt, Technol. Biomed. Life Sci. 878 (2010) 207–212. [12] S. Zhang, Z. Liu, X. Guo, L. Cheng, Z. Wang, J. Shen, J. Chromatogr, B. Analyt, Technol. Biomed. Life Sci. 875 (2008) 399–404. [13] X. Chen, Z. Yue, C. Ji, S. Liang, Se Pu 23 (2005) 92–95. [14] J. Shen, X. Xia, H. Jiang, C. Li, J. Li, X. Li, S. Ding, J. Chromatogr, B. Analyt, Technol. Biomed. Life Sci. 877 (2009) 1523–1529. [15] P. Li, Y. Qiu, H. Cai, Y. Kong, Y. Tang, D. Wang, M. Xie, Se Pu 24 (2006) 14–18. [16] A.P. Pfenning, J.E. Roybal, H.S. Rupp, S.B. Turnipseed, S.A. Gonzales, J.A. Hurlbut, J. AOAC Int. 83 (2000) 26–30. [17] A. de Villiers, F. Lestremau, R. Szucs, S. Gelebart, F. David, P. Sandra, J. Chromatogr. A 1127 (2006) 60–69. [18] S. Jamalapuram, P.K. Vuppala, A.H. Abdelazeem, C.R. McCurdy, B.A. Avery, Biomed. Chromatogr. 27 (2013) 1034. [19] K.Y. Chou, T.Y. Cheng, C.M. Chen, P.L. Hung, Y.Y. Tang, Y.J. Chung-Wang, Y.C. Shih, J. AOAC Int. 92 (2009) 1225–1232. [20] K. Xie, L. Jia, Y. Yao, D. Xu, S. Chen, X. Xie, Y. Pei, W. Bao, G. Dai, J. Wang, Z. Liu, J. Chromatogr. B 879 (2011) 2351–2354. [21] R.L. Pentecost, A.J. Niehaus, N.A. Werle, J. Lakritz, Res. Vet. Sci. 95 (2013) 594–599. [22] X. Chen, B. Yang, L. Ni, G. Wang, J. Pharm. Biomed. Anal. 41 (2006) 943–949.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
UPLC–MS/MS determination of thiamphenicol in human plasma and its application to a pharmacokinetic study Zhe Wang a , Hui Yang b , Wei Sun a , Cheng-ke Huang a , Xiao Cui a , Xiang-jun Qiu b , Qing-quan Lian c,d , Zeng-shou Wang a,∗ a
Department of Pharmacy, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China Medical College of Henan University of Science and Technology, Luoyang 471003, China c Zhejiang Province Key Laboratory of Anesthesiology, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China d Department of Anesthesiology, Critical Care and Pain Medicine, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China b
a r t i c l e
i n f o
Article history: Received 16 February 2014 Received in revised form 10 July 2014 Accepted 15 July 2014 Available online 4 August 2014 Keywords: Thiamphenicol UPLC–MS/MS Human plasma Pharmacokinetics
a b s t r a c t A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine thiamphenicol (TAP) in human plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to precipitation of plasma protein, and to a 0.1 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 354.3 → 185.1 for TAP and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 10–8000 ng/mL with a lower limit of quantification of 10 ng/mL. Only 1.5 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of TAP in healthy Chinese volunteers after oral administration. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Thiamphenicol (d(+)-threo-2-di-chloroacetamido-1-(4methylsulphonylphenyl) propane-1,3-diol, TAP, Fig. 1), an analogue of chloramphenicol, is developed by replacing the aromatic nitro group with a methylsulphonyl group [1]. Its broad antibiotic spectrum includes both Gram-positive and Gram-negative pathogens, especially effective against anaerobic organisms [2]. At a sub-cellular level, TAP binds to the 50S subunits of the 70S ribosomes to block peptidyl transferase, hence inhibiting the extension of peptide chain and synthesis of bacterial protein. However, the administration of TAP to rats, in relatively high doses shows haematological toxicity [3,4]. Nevertheless, due to its low price and steady antibiosis effectiveness, the use of TAP in human, livestock and aquaculture still exists. So, it is very
∗ Corresponding author. Tel.: +86 577 88002658. E-mail address: [email protected] (Z.-s. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.07.033 1570-0232/© 2014 Elsevier B.V. All rights reserved.
important to develop a sensitive, rapid and simple method for the determination of TAP in human plasma. Up to now, the main approaches for the detection of TAP in honey, meat, seafood, milk, egg, plasma and urine include immunoassay [5], HPLC [6–9], LC–MS/MS [10–13] and GC [14–16]. However, these methods have their own disadvantages, such as complex analyzing processes, long time requirement for preparation of samples, and long chromatographic run times, which are not preferable when dealing with a large number of samples. Moreover, no publication has described the quantitative analysis of TAP in the biological fluid using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Therefore, a novel, simple and rapid method for measurement of TAP concentration in human plasma sample is still required for analyses involving TAP monitoring. UPLC–MS/MS has been evaluated as a faster and more efficient analytical tool compared with current chromatography [17]. In the present study, a UPLC–MS/MS method for the determination of TAP using chlorzoxazone as an internal standard (IS) was developed.
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20, 500, 6400 ng/mL for TAP. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (1 g/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C. 2.4. Sample preparation Fig. 1. The chemical structures of TAP and IS in the present study: (A) TAP; (B) chlorzoxazone (IS).
This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers. 2. Materials and methods 2.1. Chemicals materials TAP and chlorzoxazone (internal standard, IS) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC grade and purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Blank human plasma used in this study was supplied by The Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). 2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid) as follows: 0–0.3 min (18% A), 0.3–1.5 min (18–98% A). The flow rate was 0.40 mL/min. The overall run time was 1.5 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 200 ms for each MRM transition. The MRM transitions were m/z 354.3 → 185.1 and m/z 168.1 → 132.1 for TAP and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 3.5 kV; and temperature, 350 ◦ C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex). 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of TAP that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 10, 20, 50, 100, 400, 1000, 2000, 4000 and 8000 ng/mL for TAP in human plasma. The concentrations of QC samples in plasma were
Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (1 g/mL) and 1 mL ethyl acetate was added to 100 L of collected plasma sample. After vortexing for 2 min, the samples were centrifuged at 13,000 rpm for 5 min, the organic phase was transferred into a clean tube and evaporated under nitrogen stream at 40 ◦ C. The residue was dissolved in 100 L of mobile phase. The supernatant (5 L) was injected into the UPLC–MS/MS system for analysis. 2.5. Method validation Before using this method to determinate TAP in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances [18]. Specificity was determined by analysis of blank human plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of nine concentrations of TAP (10–8000 ng/mL) were separately extracted and assayed on three separate days. The linearity for TAP was investigated by weighted (1/x2 ) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (20, 500, 6400 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy between −15 and 15%. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (100 ng/mL) similarly. To determine the matrix effect, six different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (100 ng/mL) in the same manner. The stabilities in human plasma were tested by analyzing five replicates of plasma samples at three concentration levels (20, 500, 6400 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 ◦ C for 12 h. The freeze–thaw stability was
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Fig. 2. Representative chromatograms of TAP and IS in human plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with TAP (1) and IS (2); (C) a plasma sample from a person 1 h after an oral administration 500 mg.
evaluated after three complete freeze–thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 30 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15%) and precision (RSD % ≤15%).
0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 15 h after oral administration. Blood samples were centrifuged at 4000 × g for 8 min and the plasma was separated and kept frozen at −20 ◦ C until analysis. 2.7. Data analysis Plasma concentration vs. time profiles were analyzed using DAS (Drug and statistics) software (Version 3.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
2.6. Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 500 mg TAP tablets (Fujian Haiwang Pharmaceutical Co., Ltd.) to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of Tonji Medical College, Huazhong University of Science and Technology prior to the study. Twenty volunteers (aged 21–28 years, weighing 50–70 kg, and height 156–180 cm) were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetic study of TAP. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral dose of 500 mg TAP with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes before and
3. Results and discussion 3.1. Method development and optimization Owing to the complex matrices, sample preparation is usually required for the determination of the analytes in biological samples in order to remove the possible interfering matrix components and increase selectivity and sensitivity. In this study, liquid–liquid extraction with organic solvent was used for the sample preparation. This simple procedure produced a clean chromatogram for the blank plasma sample and yielded satisfactory recoveries for the analytes. In this work, was added in the process of sample preparation. Internal standard plays an important role in biopharmaceutical analysis, and chlorzoxazone was found to be suitable
Table 1 Precision and accuracy of method for the determination of TAP in human plasma (n = 6). Analytes
Concentration added (ng/mL)
TAP
20 500 6400
Intra-day precision
Inter-day precision
Mean ± SD
RSD (%)
RE (%)
Mean ± SD
RSD (%)
RE (%)
21.03 ± 2.11 504.88 ± 15.43 6430.40 ± 68.99
11.18 3.17 1.08
5.17 0.98 0.47
21.20 ± 2.45 509.83 ± 24.75 6106.83 ± 281.21
13.55 4.60 0.80
5.98 −0.29 −1.04
Table 2 Recovery and matrix effect of TAP and internal standards (n = 6). Analytes
Concentration added (ng/mL)
Recovery (%) Mean ± SD
TAP IS
20 500 6400 100
78.74 75.89 79.31 79.91
± ± ± ±
4.76 1.29 0.65 2.10
Matrix effect (%) RSD (%)
Mean ± SD
6.04 1.70 0.65 2.10
97.72 101.39 104.69 103.42
± ± ± ±
6.40 5.07 1.44 2.72
RSD (%) 6.55 5.00 1.37 2.63
RSD (%)
7.86 5.43 4.60 20.06 ± 1.77 522.53 ± 27.81 6439.02 ± 303.48 7.65 1.13 −0.45 12.40 4.12 4.75 21.53 ± 2.42 505.64 ± 19.92 6371.01 ± 300.09 14.46 2.66 2.15 20.28 ± 2.87 508.4 ± 13.22 6425.12 ± 136.16 20 500 6400
1.40 1.68 0.39
Mean ± SD RE (%) RSD (%) Mean ± SD
TAP
The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100. Results are shown in Table 2. The recovery in plasma ranged from 75.89 to 79.31% for TAP. The recovery of IS (100 ng/mL) in plasma was 79.91%. The matrix effect in human plasma were all between 97.72 and 104.69% for TAP at different QC levels (Table 2). The matrix effect for IS (100 ng/mL) was 103.42%. No apparent matrix effect was found
Concentration added (ng/mL)
3.5. Recovery and matrix effect
Analytes
The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations for each biological matrix. The results are summarized in Table 1. The method was reliable and reproducible since RSD % was below 15% and RE % was between −1.04 and 5.98% for all the investigated concentrations of TAP in human plasma.
Table 3 Stability results of TAP in human plasma in different conditions (n = 5).
3.4. Precision and accuracy
4 ◦ C, 12 h
The peak area ratios of TAP/IS versus the nominal concentrations of TAP showed a good linear relationship over the concentration ranges of 10–8000 ng/mL in human plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.073101x ± 0.0146677, where y represents the peak area ratios of TAP to the IS and x represents plasma concentrations of analyte. The LLOQ in human plasma was 10 ng/mL with the RSD and RE of 9.25 and 3.64%, respectively.
Room temperature, 2 h
3.3. Linearity and sensitivity
4.73 −2.81 1.21
UPLC–MS/MS chromatogram of the analytes in human plasma samples were shown in Fig. 2. The retention times of TAP and IS are 0.84 and 1.25 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
9.59 6.61 2.21
Mean ± SD RE (%)
3.2. Specificity
20.95 ± 1.85 485.96 ± 32.61 6477.32 ± 143.76
Three freeze–thaw
RSD (%)
RE (%)
−20 ◦ C, 30 days
RSD (%)
RE (%)
for the present work and finally used as the internal standard for its stability and high recovery. Other chromatographic conditions, especially the composition of mobile phase, were tested to achieve good resolution and symmetric peak shapes of analytes as well as a short run time. It was found that with the acetonitrile and 1% formic acid in water with gradient elution could achieve our purpose and were finally adopted as the mobile phase for chromatographic separation. The retention time was 0.84 min for TAP and 1.25 min for IS. The run time was 1.5 min. The advantage of this method is that a relatively larger number of samples can be analyzed in a short time, thus increasing output [19–21]. Analysis method was set up by optimizing UPLC and MS/MS conditions to obtain the best possible sensitivity. The analytes were analyzed firstly using MS by syringe infusion of individual standard solution and they were all more efficiently ionized in ESI negative mode than in positive mode. ESI negative mode was therefore employed. Parameters such as ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecules of analytes. The collision energy of collision-induced decomposition (CID) was optimized for maximum response of the fragmentation of analytes. MRM was used to monitor precursor ion and production, which could reduce interference and enhance selectivity. The MRM transitions were m/z 354.3 → 185.1 and m/z 168.1 → 132.1 for TAP and IS, respectively
0.32 4.51 0.61
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administration of 500 mg TAP tablets, the main pharmacokinetic parameters of TAP for twenty volunteers were estimated. The mean plasma concentration–time curve of TAP was displayed in Fig. 3, and the main pharmacokinetic parameters of TAP were calculated and are summarized in Table 4. Compared with recently published papers describing the pharmacokinetic profiles of TAP in healthy volunteers, the pharmacokinetic parameters of TAP obtained in the study were generally similar [22]. 4. Conclusions
Fig. 3. Plasma concentration versus time curves of TAP after a single oral administration 500 mg to 20 Chinese healthy male volunteers. Table 4 Pharmacokinetic parameters of TAP after oral administration 500 mg to 20 Chinese healthy male volunteers. Parameters
Mean ± SD
t1/2 (h) Cmax (g/mL) Tmax (h) AUC0→15 (g/mL h) AUC0→∞ (g/mL h)
3.287 5.247 2.075 29.043 30.736
± ± ± ± ±
0.401 1.218 0.545 4.926 5.529
to affect the determination of TAP and IS in plasma. As a result, the matrix effect from plasma was negligible in this method. 3.6. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. Table 3 shows the stability data for TAP in plasma under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 2 h and at 4 ◦ C for 12 h. No significant degradation was observed when samples of TAP were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, TAP in samples were stable at −20 ◦ C for 30 days. 3.7. Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of TAP in human plasma. After an oral
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