- Email: [email protected]
MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers
Journal of Chromatography B, 878 (2010) 2072–2076
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a LC–MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers Zhirong Tan, Dongsheng Ouyang, Yao Chen, Gan Zhou, Shan Cao, Yicheng Wang, Xiujuan Peng, Honghao Zhou ∗ Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Xiangya Road 110, Changsha, Hunan 410078, PR China
a r t i c l e
i n f o
Article history: Received 15 April 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: LC–MS/MS Clebopride Pharmacokinetics
a b s t r a c t A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESIMS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid–liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C18 (5 m, 150 mm × 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]+ ions, m/z 373.9 → m/z184.0 for clebopride, m/z 359.9 → m/z71.5 for itopride. The method was validated over the concentration range of 69.530–4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from −8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride. © 2010 Published by Elsevier B.V.
1. Introduction Orthopramides such as metoclopramide and subpirde are used to treat several gastrointestinal and central nervous system disorders. Clebopride is another orthopramide which produces different pharmacological effects involving both central and peripheral mechanisms. Like other orthopramides, clebopride inhibits the effects of dopamine receptor agonists and displaces radiolabelled ligands such as (3 H)-spiperone from their specific binding sites [1]. The peak concentration of clebopride after oral administration of 0.68 mg clebopride tablet was approximately 1000 pg/ml [2]. The determination of clebopride has been performed by acid decomposition [3], thin-layer chromatographic photodensitometric [4,5], radioimmunoassay (RIA) [6], and GC–MS [7,8]. But the low sensitivity (LLOQ higher than 100 pg/ml [3–8]), long analysis time [7,8] or large volumes of plasma sample [7,8] may not meet the requirement of desired sample throughout, speed and sensitivity in pharmacokinetic and clinical studies of clebopride. To our knowledge, the quantification of clebopride in human plasma using LC–MS/MS has not been reported. In the present work, isolation
∗ Corresponding author. Tel.: +86 731 8448 7233; fax: +86 731 8480 5379. E-mail address: [email protected] (H. Zhou). 1570-0232/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.jchromb.2010.06.006
of clebopride was achieved by a single-step liquid–liquid extraction (LLE) with methyl-tertiary-butyl ether (MTBE), and injection onto the chromatography column. Electrospray ionization (ESI) interface was used because it provided more sensitivity and better reproducibility for clebopride. The analytical procedure was fully validated and successfully used to assess the pharmacokinetics of clebopride in Chinese volunteers. 2. Experimental 2.1. Materials and methods 2.1.1. Standards and reagents Clebopride was supplied by the Hunan Yandi Pharmaceutical Company (Zhuzhou, China); the internal standard, itopride was purchased from Sigma Company Inc. (USA). Acetonitrile, methanol and formic acid were HPLC grade and purchased from Merck Company Inc. (Darmstadt, Germany). Ultra pure water (Millipore, Bedford, MA. USA) was used from a Milli-Q system. The structures of clebopride and internal standard are shown in Fig. 1. 2.1.2. Liquid chromatographic and mass spectrometric conditions A Waters 2695 HPLC system (Waters, USA), equipped with a vacuum degasser and an autosampler was used in the study. The mass spectrometric instrument was a QuttroMicro API mass spec-
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
2073
2.1.4. Sample preparation A volume of 180 l of drug-free human plasma was added to a disposable Eppendorf tube, followed by spiking with 20 l of the standard working solution and 50 l of IS working solution, respectively. Then 50 l 28% ammonium hydroxide was added and the mixture was vortexed for 30 s using a vortex mixer (IKA VIBR AX, Germany). Then, a single-step LLE was adopted to extract both the analytes from the human plasma. For this, 0.6 ml of MTBE was added to each tube followed by vortexing for 10 min. The wellvortexed solutions were then centrifuged at 13,000 rpm for 10 min and 0.4 ml of the upper organic layer was transferred to a new Eppendorf tube and evaporated to dryness set at 40 ◦ C waterbath. The residues were then reconstituted in 200 l mobile phase followed by centrifugation at 13,000 rpm for 5 min before analysis. An aliquot of 10 l was injected into the LC–MS/MS system. 2.2. Method validation The method validation assays were carried out according to the currently accepted the FDA guidelines [9].
Fig. 1. Full-scan parent and product ion spectra of [M+H]+ of (A) clebopride and (B) itopride.
trometer from Waters (Waters, USA) and the chromatographic data system was MassLynx version 4.1 (Waters, USA). The analytical column was a CAPCELL MG-III C18 basic, 150 × 2.1, 5 m, and was purchased from Yamamura Kagaku (Kyoto, Japan), an optiguard (C18 , 4 mm × 2 mm) obtained from Phenomenex Technologies (USA) was used as a guard column. The mobile phase consisted of acetonitrile and 0.1% formic acid with 20 mM ammonium format in a ratio of 90:10 (v/v), and the flow rate was 0.3 ml/min. The effluent from the HPLC column was directed into the ESI probe. Mass spectrometer conditions were optimized to obtain maximal sensitivity. Ionization conditions were optimized as follows: cone and desolvention gas at 50 and 450 L h−1 , capillary at 3.5 kV, RF lens at 0 V, skimmer at 3 V, ion source and desolvation gas temperature at 100 ◦ C and 350 ◦ C, ion energy at 1.0 V, lowand high-mass resolution at 11 for quadrupole 1 and 3, dwell time at 0.1 s, and the inter-scan delay at 0.01 s.The collision activation dissociation (CAD) was set at 20 and 27 eV for clebopride and IS, respectively, using argon gas as the collision gas. MRM detection was employed with a dwell time of 200 ms for each transition. The selection of operating protonated ions is shown in Fig. 1. The scan mode was multiple reaction monitoring using the precursor ion at m/z (M+1)+ (m/z: 373.9, 359.9) and after collision dissociation the product ions m/z: 184.0, 71.5 were used for the quantification of clebopride and IS, respectively.
2.1.3. Standard and quality control sample preparation Stock solutions containing 8.91 mg/ml of each reference compound was prepared in methanol and stored at 4 ◦ C until use. Working solutions, ranging of 695.30–44500 pg/ml, were prepared by serial dilution with methanol. A solution containing 12.85 ng/ml IS was prepared in methanol. The samples for standard calibration curves were prepared by spiking the blank plasma (180 l) with 20 l of the appropriate working solutions to yield the following concentrations: 4450.0, 2225.0, 1112.500, 556.25, 278.13, 139.10 and 69.530 pg/ml. Quality control (QC) samples were prepared from blank plasma at concentrations of 139.10, 556.25 and 4450.0 pg/ml.
2.2.1. Assay specificity Each calibration curve consists of seven calibration points covering the range from 69.530 to 4450.0 pg/ml for clebopride in plasma. The peak area ratios for clebopride and internal standard were measured and a standard curve without the zero concentration was constructed. 2.2.2. Linearity and LLOQ Calibration curves of seven concentrations of clebopride from 69.530 to 4450.0 pg/ml were extracted and assayed with weighted (1/x) least squares linear regression. Blank plasma samples were analyzed to confirm the absence of interferences but were not used to construct the calibration function. According to FDA guidance [9], the LLOQ is defined as the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy, which are 20% of RSD and −20 to 20% of RE in this assay, respectively. 2.2.3. Precision and accuracy The precision of the assay was determined from the QC plasma samples by replicate analyses of four concentration levels of clebopride (139.10, 556.25 and 4450.0 pg/ml). Within-batch precision and accuracy were determined by repeated analyses on three consecutive days (n = 5 series per day). The precision was calculated using the relative standard deviation (RSD) with RSD% = (standard deviation of the mean/mean) × 100. Accuracy was calculated as the relative error (RE) with RE% = (measured concentration–nominal concentration)/nominal concentration × 100. The concentration of each sample was determined using the calibration curve prepared and analyzed on the same batch. 2.2.4. Extraction recovery and matrix effect The extraction recovery was determined by dividing the peak areas of clebopride added into blank plasma and extracted using LLE procedure with those obtained from the compound spiked into equivalent volume of post-extraction supernatant. This procedure was repeated for five replicates at three QC concentration levels of 139.10, 556.25 and 4450.0 pg/ml. The matrix effect was measured by comparing the peak response of sample spiked post-extraction (A) with that of pure standard solution containing equivalent amount of the compound (B). The ratio (A/B × 100)% was used to evaluate the matrix effect. The extraction recovery and matrix effect of IS were also evaluated using the same procedure.
2074
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
Table 1 Accuracy and precision for the analysis of clebopride (n = 3 days, five replicates per day). Spiked concentration (ng/ml)
Measured concentration (ng/ml)
Inter-day RSD (%)
Intra-day RSD (%)
Accuracy percent error (%)
139.10 556.25 4450.0
145.23 ± 23.56 569.68 ± 98.69 4365.24 ± 298.68
6.52 3.78 4.55
6.83 4.12 5.35
1.88 −8.16 −5.27
2.2.5. Stability Freeze and thaw stability. QC plasma samples at three concentration levels were storage temperature (−20 ◦ C) for 24 h and thawed unassisted at room temperature. When completely thawed, the samples were refrozen for 24 h under the same conditions. The freeze–thaw cycles were repeated twice, and the samples were analyzed after three freeze (−20 ◦ C)–thaw (room temperature) cycles. Short-term temperature stability. QC plasma samples at three concentration levels were kept at room temperature for a period that exceeded the routine preparation time of the samples (around 6 h). Long-term stability. QC plasma samples at three concentration levels kept at low temperature (−40 ◦ C) were studied for a period of 30 days. Post-preparative stability. The autosample stability was conducted by reanalyzing extracted QC samples kept under autosampler conditions (4 ◦ C) for 12 h. 2.3. Safety considerations The method required no specific safety precautions. Universal precautions for the handling of chemicals and biofluids were applied. 2.4. Application to pharmacokinetic study The method was applied to evaluate the pharmacokinetics of clebopride tablet in 12 healthy volunteers. The studies were approved by the Human Ethics committee of Hunan Xiangya Hospital. Informed consent was obtained from all subjects after explaining the aims and risks of the study. 12 healthy male volunteers received a single dose of clebopride tablet. An indwelling cannula was placed in one arm for blood sampling. Before sampling, about 0.5 ml blood was discarded; then, 5 ml blood samples were collected in heparinized tubes at the following times on the days of the pharmacokinetic measurements: immediately prior to drug administration (0 h), and at 0.25, 0.50, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10, 12 and 24 h after drug administration, and centrifuged at 3000 rpm for 10 min to separate the plasma fractions. The collected plasma samples were stored at −40 ◦ C until analysis. 3. Results and discussion 3.1. Method development 3.1.1. Optimization of the mass spectrometry condition In the present study, to select an appropriate ionization mode in LC–MS analysis, clebopride and itopride were scanned with ESI and APCI positive and negative ion modes using injection standard solutions. In different ionization modes, the base peak intensity of positive ion was higher than those of negative ion, and the efficiency of ionization in ESI was higher than APCI. The molecular ions with an m/z 373.9 [M+H]+ for clebopride were produced. IS molecule was protonated to form molecule ion with m/z 359.9 [M+H]+ . Therefore, these ions were chosen as parent ions for fragmentation in the multiple reaction monitoring (MRM) mode. The daughter spectra of the parent ions revealed that the predominant daughter fragments
were m/z 184.0 for clebopride and m/z 71.5 for IS. The collision energy in the product MS–MS mode was investigated to optimize the sensitivity, and the optimal values were found to be 27 and 20 eV for the product ion of clebopride and IS, respectively. 3.1.2. Optimization of the chromatographic condition The CAPCELL MG-III C18 column was selected for all analysis since it provided symmetrical peak shape and obtained the highest intensity to clebopride and IS. The separation and ionization of clebopride and IS were affected by compose of mobile phase. The mobile phase pH affected not only the retention time, but also the ionization efficiency of clebopride and IS. The acidity of mobile phase benefited to the ionization of clebopride and IS. 3.1.3. Sample preparation The aim for sample preparation method was to remove interferences from the biological sample and it should be also reproducible with a high recovery involving a minimum number of working steps. LLE gave both high recovery and good chromatography. 28% ammonium hydroxide was added to the plasma to minimize the protein binding. LLE was advantageous because this technique not only extracted the analyte and IS with sufficient efficiency and specificity, but also minimized the experimental cost. Ethyl acetate, diethyl ether MTBE, dichloromethane and trichloromethane were all tested as extraction solvent, and MTBE was finally adopted because of its high extraction efficiency. 3.1.4. IS selection An ideal IS should be a structurally similar analog or stable isotope-labelled compound according to the FDA guideline [9]. However, stable isotope-labelled compound is too expensive for most research institutes. Itopride was chosen for quantification as the IS due to its similarity with the analytes in structure, chromatographic behavior, mass spectrographic behavior and stability. 3.2. Method validation 3.2.1. Specificity The specificity of the method was determined by analyzing six different lots of blank control both with and without the IS. The chromatograms of LLE produced clean extracts with no interference from endogenous compounds at the retention time for clebopride and IS. Fig. 2 shows representative chromatograms of blank human plasma, the LLOQ (69.530 pg/ml) of clebopride and IS in plasma and volunteer’s plasma sample. Typical retention time for clebopride and IS were 1.4 and 1.3 min. And the peak shapes were sharp and symmetrical. The total run time was about 3.0 min. 3.2.2. LLOQ and linearity The calibration curves were prepared daily which showed good linearity in the range 69.530–4450.0 pg/ml for clebopride. The typical regression equation was R = 1.0881C + 0.0404, r = 0.9993, where R corresponds to the peak area ratio of clebopride to the IS, and C (pg/ml) refers to the concentration of clebopride added to the plasma. Linear regressions of the peak area ratios of clebopride to internal standard were weighted by 1/x. The LLOQ of the method was 69.530 pg/ml of clebopride in human plasma, the precision (RSD%) was 15.83% (n = 6), and the accuracy (RE %) was −9.68% (n = 6).
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
2075
Fig. 3. The mean plasma concentration–time profiles of clebopride in human after oral administration of 1.36 mg clebopride, (n = 12).
3.2.3. Within- and between-batch precision and accuracy Data for within-batch and between-batch precision and accuracy of the method for determination of clebopride are presented in Table 1. The accuracy deviation values are within 11% of the actual values. The precision determined at each concentration level does not exceed 6% of the relative standard deviation (RSD). The results revealed good precision and accuracy.
3.2.4. Recovery and matrix effect The extract recoveries of clebopride from human plasma were 88.3 ± 3.2%, 85.9 ± 4.1%, and 82.7 ± 2.7% at concentrations of 139.10, 556.25 and 4450.0 pg/ml, respectively. The mean extraction recovery of IS was 83.9 ± 1.5%. Matrix effect is due to co-elution of some components present in biological samples. These components may not give a signal in MRM of target analyte but can certainly decrease or increase the analyte response dramatically to affect the sensitivity, accuracy and precision of the method. Thus, the evaluation of matrix effect from the influence of co-eluting components on analyte ionization is necessary for an HPLC–MS/MS method. All the ratios defined as in Section 2 were between 85% and 115%. No significant matrix effect for clebopride and IS was observed indicating that no co-eluting substance influenced the ionization of the analytes and IS. Fig. 2. MRM chromatograms of (A) blank plasma; (B) blank plasma spiked with 69.530 pg/ml clebopride (LLOQ) and internal standard; (C) 3 h samples after oral administration of 1.36 mg clebopride tablet. The retention times of clebopride and internal standard were 1.4 and 1.3 min.
3.2.5. Stability Table 2 summarizes the freeze and thaw stability, short-term stability, long-term stability and post-preparative stability data of clebopride. All the results showed good stability during these tests and there were no stability related problems during the routine analysis of samples for pharmacokinetic, bioavailability or bioequivalence studies.
Table 2 Data showing the stability of clebopride in human plasma at different QC levels (n = 5). Accuracy (Mean ± SD) 139.10 pg/ml Freeze and thaw stability Short-term stability Long-term stability Post-preparative stability
125.65 131.29 141.26 128.96
± ± ± ±
9.14 8.92 5.84 8.45
139.10, 556.25 and 4450.0 pg/ml are spiking plasma concentrations.
556.25 pg/ml 526.39 531.95 542.98 540.32
± ± ± ±
32.56 25.35 18.25 29.16
4450.0 pg/ml 4289.30 4329.68 4301.52 4401.92
± ± ± ±
244.65 322.14 258.36 251.56
2076
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
3.3. Pharmacokinetic study The present method was successfully applied to the pharmacokinetics studies after an oral administration of 1.36 mg clebopride tablets to 12 healthy Chinese healthy volunteers. Mean concentration–time profiles of clebopride were displayed in Fig. 3. The parameters were calculated by a non-compartmental model. The values of Cmax and Tmax were obtained directly from experiment observations. The mean of Cmax and Tmax were 2072.78 ± 1321.85 pg/ml and 0.94 ± 0.13 h, respectively. Plasma concentration declined with a t1/2 of 5.45 ± 3.90 h. AUC0−24 and AUC0−∞ values obtained were 8041.25 ± 5420.22 pg h/ml and 8044.77 ± 5420.99 pg h/ml respectively. These pharmacokinetic parameters were in accordance with those reported in the literature [2,7], indicating the applicability of this method to the pharmacokinetics study of clebopride. 4. Conclusions This paper described a sensitive, specific, accurate and precise HPLC–ESI-MS/MS method for the determination of clebopride in human plasma. Good linearity was observed in the range from 69.530 to 4450.0 pg/ml and the LLOQ (69.53 pg/ml) makes this method of real practical use for pharmacokinetics, bioavailability and bioequivalence studies.
The method was successfully applied to a pharmacokinetics study of clebopride in Chinese volunteers. It is the first study, as far as we know, of LC–MS/MS method for determination of clebopride concentration in vivo. Acknowledgement This research is supported by Foundation of Hunan Science and Technology Department general project (2009FJ4008). References [1] S.C. Bittencourt, T.C.M. De Lima, G.S. Morato, Gen. Pharmacol. 26 (1995) 1083. [2] H.W. Ryou, J.H. Lee, Y.H. Chi, Y.H. Hahn, H.K. Tan, K.H. Lee, S.L. Kim, S.Y. Jeon, Y.W. Choi, Yakche Hakhoechi 30 (2000) 179. [3] Y. Hayasaka, S. Murata, K. Umemura, Chem. Pharm. Bull. (Tokyo) 29 (1981) 1478. [4] J. Segura, O.M. Bakke, G. Huizing, A.H. Beckett, Drug Metab. Dispos. 8 (1980) 87. [5] G. Huizing, A.H. Beckett, J. Segura, J. Chromatogr. A 172 (1979) 227. [6] M. Yano, K. Nakamichi, T. Yamaki, T. Fukami, K. Ishikawa, I. Matsumoto, Chem. Pharm. Bull. 32 (1984) 1491. [7] P.R. Robinson, M.D. Jones, J. Maddock, L.W. Rees, J. Chromatogr.: Biomed Appl. 564 (1991) 147. [8] P.R. Robinson, M.D. Jones, J. Maddock, J. Chromatogr.: Biomed. Appl. 432 (1988) 153. [9] Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), May 2001.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a LC–MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers Zhirong Tan, Dongsheng Ouyang, Yao Chen, Gan Zhou, Shan Cao, Yicheng Wang, Xiujuan Peng, Honghao Zhou ∗ Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University, Xiangya Road 110, Changsha, Hunan 410078, PR China
a r t i c l e
i n f o
Article history: Received 15 April 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: LC–MS/MS Clebopride Pharmacokinetics
a b s t r a c t A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESIMS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid–liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C18 (5 m, 150 mm × 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]+ ions, m/z 373.9 → m/z184.0 for clebopride, m/z 359.9 → m/z71.5 for itopride. The method was validated over the concentration range of 69.530–4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from −8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride. © 2010 Published by Elsevier B.V.
1. Introduction Orthopramides such as metoclopramide and subpirde are used to treat several gastrointestinal and central nervous system disorders. Clebopride is another orthopramide which produces different pharmacological effects involving both central and peripheral mechanisms. Like other orthopramides, clebopride inhibits the effects of dopamine receptor agonists and displaces radiolabelled ligands such as (3 H)-spiperone from their specific binding sites [1]. The peak concentration of clebopride after oral administration of 0.68 mg clebopride tablet was approximately 1000 pg/ml [2]. The determination of clebopride has been performed by acid decomposition [3], thin-layer chromatographic photodensitometric [4,5], radioimmunoassay (RIA) [6], and GC–MS [7,8]. But the low sensitivity (LLOQ higher than 100 pg/ml [3–8]), long analysis time [7,8] or large volumes of plasma sample [7,8] may not meet the requirement of desired sample throughout, speed and sensitivity in pharmacokinetic and clinical studies of clebopride. To our knowledge, the quantification of clebopride in human plasma using LC–MS/MS has not been reported. In the present work, isolation
∗ Corresponding author. Tel.: +86 731 8448 7233; fax: +86 731 8480 5379. E-mail address: [email protected] (H. Zhou). 1570-0232/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.jchromb.2010.06.006
of clebopride was achieved by a single-step liquid–liquid extraction (LLE) with methyl-tertiary-butyl ether (MTBE), and injection onto the chromatography column. Electrospray ionization (ESI) interface was used because it provided more sensitivity and better reproducibility for clebopride. The analytical procedure was fully validated and successfully used to assess the pharmacokinetics of clebopride in Chinese volunteers. 2. Experimental 2.1. Materials and methods 2.1.1. Standards and reagents Clebopride was supplied by the Hunan Yandi Pharmaceutical Company (Zhuzhou, China); the internal standard, itopride was purchased from Sigma Company Inc. (USA). Acetonitrile, methanol and formic acid were HPLC grade and purchased from Merck Company Inc. (Darmstadt, Germany). Ultra pure water (Millipore, Bedford, MA. USA) was used from a Milli-Q system. The structures of clebopride and internal standard are shown in Fig. 1. 2.1.2. Liquid chromatographic and mass spectrometric conditions A Waters 2695 HPLC system (Waters, USA), equipped with a vacuum degasser and an autosampler was used in the study. The mass spectrometric instrument was a QuttroMicro API mass spec-
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
2073
2.1.4. Sample preparation A volume of 180 l of drug-free human plasma was added to a disposable Eppendorf tube, followed by spiking with 20 l of the standard working solution and 50 l of IS working solution, respectively. Then 50 l 28% ammonium hydroxide was added and the mixture was vortexed for 30 s using a vortex mixer (IKA VIBR AX, Germany). Then, a single-step LLE was adopted to extract both the analytes from the human plasma. For this, 0.6 ml of MTBE was added to each tube followed by vortexing for 10 min. The wellvortexed solutions were then centrifuged at 13,000 rpm for 10 min and 0.4 ml of the upper organic layer was transferred to a new Eppendorf tube and evaporated to dryness set at 40 ◦ C waterbath. The residues were then reconstituted in 200 l mobile phase followed by centrifugation at 13,000 rpm for 5 min before analysis. An aliquot of 10 l was injected into the LC–MS/MS system. 2.2. Method validation The method validation assays were carried out according to the currently accepted the FDA guidelines [9].
Fig. 1. Full-scan parent and product ion spectra of [M+H]+ of (A) clebopride and (B) itopride.
trometer from Waters (Waters, USA) and the chromatographic data system was MassLynx version 4.1 (Waters, USA). The analytical column was a CAPCELL MG-III C18 basic, 150 × 2.1, 5 m, and was purchased from Yamamura Kagaku (Kyoto, Japan), an optiguard (C18 , 4 mm × 2 mm) obtained from Phenomenex Technologies (USA) was used as a guard column. The mobile phase consisted of acetonitrile and 0.1% formic acid with 20 mM ammonium format in a ratio of 90:10 (v/v), and the flow rate was 0.3 ml/min. The effluent from the HPLC column was directed into the ESI probe. Mass spectrometer conditions were optimized to obtain maximal sensitivity. Ionization conditions were optimized as follows: cone and desolvention gas at 50 and 450 L h−1 , capillary at 3.5 kV, RF lens at 0 V, skimmer at 3 V, ion source and desolvation gas temperature at 100 ◦ C and 350 ◦ C, ion energy at 1.0 V, lowand high-mass resolution at 11 for quadrupole 1 and 3, dwell time at 0.1 s, and the inter-scan delay at 0.01 s.The collision activation dissociation (CAD) was set at 20 and 27 eV for clebopride and IS, respectively, using argon gas as the collision gas. MRM detection was employed with a dwell time of 200 ms for each transition. The selection of operating protonated ions is shown in Fig. 1. The scan mode was multiple reaction monitoring using the precursor ion at m/z (M+1)+ (m/z: 373.9, 359.9) and after collision dissociation the product ions m/z: 184.0, 71.5 were used for the quantification of clebopride and IS, respectively.
2.1.3. Standard and quality control sample preparation Stock solutions containing 8.91 mg/ml of each reference compound was prepared in methanol and stored at 4 ◦ C until use. Working solutions, ranging of 695.30–44500 pg/ml, were prepared by serial dilution with methanol. A solution containing 12.85 ng/ml IS was prepared in methanol. The samples for standard calibration curves were prepared by spiking the blank plasma (180 l) with 20 l of the appropriate working solutions to yield the following concentrations: 4450.0, 2225.0, 1112.500, 556.25, 278.13, 139.10 and 69.530 pg/ml. Quality control (QC) samples were prepared from blank plasma at concentrations of 139.10, 556.25 and 4450.0 pg/ml.
2.2.1. Assay specificity Each calibration curve consists of seven calibration points covering the range from 69.530 to 4450.0 pg/ml for clebopride in plasma. The peak area ratios for clebopride and internal standard were measured and a standard curve without the zero concentration was constructed. 2.2.2. Linearity and LLOQ Calibration curves of seven concentrations of clebopride from 69.530 to 4450.0 pg/ml were extracted and assayed with weighted (1/x) least squares linear regression. Blank plasma samples were analyzed to confirm the absence of interferences but were not used to construct the calibration function. According to FDA guidance [9], the LLOQ is defined as the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy, which are 20% of RSD and −20 to 20% of RE in this assay, respectively. 2.2.3. Precision and accuracy The precision of the assay was determined from the QC plasma samples by replicate analyses of four concentration levels of clebopride (139.10, 556.25 and 4450.0 pg/ml). Within-batch precision and accuracy were determined by repeated analyses on three consecutive days (n = 5 series per day). The precision was calculated using the relative standard deviation (RSD) with RSD% = (standard deviation of the mean/mean) × 100. Accuracy was calculated as the relative error (RE) with RE% = (measured concentration–nominal concentration)/nominal concentration × 100. The concentration of each sample was determined using the calibration curve prepared and analyzed on the same batch. 2.2.4. Extraction recovery and matrix effect The extraction recovery was determined by dividing the peak areas of clebopride added into blank plasma and extracted using LLE procedure with those obtained from the compound spiked into equivalent volume of post-extraction supernatant. This procedure was repeated for five replicates at three QC concentration levels of 139.10, 556.25 and 4450.0 pg/ml. The matrix effect was measured by comparing the peak response of sample spiked post-extraction (A) with that of pure standard solution containing equivalent amount of the compound (B). The ratio (A/B × 100)% was used to evaluate the matrix effect. The extraction recovery and matrix effect of IS were also evaluated using the same procedure.
2074
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
Table 1 Accuracy and precision for the analysis of clebopride (n = 3 days, five replicates per day). Spiked concentration (ng/ml)
Measured concentration (ng/ml)
Inter-day RSD (%)
Intra-day RSD (%)
Accuracy percent error (%)
139.10 556.25 4450.0
145.23 ± 23.56 569.68 ± 98.69 4365.24 ± 298.68
6.52 3.78 4.55
6.83 4.12 5.35
1.88 −8.16 −5.27
2.2.5. Stability Freeze and thaw stability. QC plasma samples at three concentration levels were storage temperature (−20 ◦ C) for 24 h and thawed unassisted at room temperature. When completely thawed, the samples were refrozen for 24 h under the same conditions. The freeze–thaw cycles were repeated twice, and the samples were analyzed after three freeze (−20 ◦ C)–thaw (room temperature) cycles. Short-term temperature stability. QC plasma samples at three concentration levels were kept at room temperature for a period that exceeded the routine preparation time of the samples (around 6 h). Long-term stability. QC plasma samples at three concentration levels kept at low temperature (−40 ◦ C) were studied for a period of 30 days. Post-preparative stability. The autosample stability was conducted by reanalyzing extracted QC samples kept under autosampler conditions (4 ◦ C) for 12 h. 2.3. Safety considerations The method required no specific safety precautions. Universal precautions for the handling of chemicals and biofluids were applied. 2.4. Application to pharmacokinetic study The method was applied to evaluate the pharmacokinetics of clebopride tablet in 12 healthy volunteers. The studies were approved by the Human Ethics committee of Hunan Xiangya Hospital. Informed consent was obtained from all subjects after explaining the aims and risks of the study. 12 healthy male volunteers received a single dose of clebopride tablet. An indwelling cannula was placed in one arm for blood sampling. Before sampling, about 0.5 ml blood was discarded; then, 5 ml blood samples were collected in heparinized tubes at the following times on the days of the pharmacokinetic measurements: immediately prior to drug administration (0 h), and at 0.25, 0.50, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10, 12 and 24 h after drug administration, and centrifuged at 3000 rpm for 10 min to separate the plasma fractions. The collected plasma samples were stored at −40 ◦ C until analysis. 3. Results and discussion 3.1. Method development 3.1.1. Optimization of the mass spectrometry condition In the present study, to select an appropriate ionization mode in LC–MS analysis, clebopride and itopride were scanned with ESI and APCI positive and negative ion modes using injection standard solutions. In different ionization modes, the base peak intensity of positive ion was higher than those of negative ion, and the efficiency of ionization in ESI was higher than APCI. The molecular ions with an m/z 373.9 [M+H]+ for clebopride were produced. IS molecule was protonated to form molecule ion with m/z 359.9 [M+H]+ . Therefore, these ions were chosen as parent ions for fragmentation in the multiple reaction monitoring (MRM) mode. The daughter spectra of the parent ions revealed that the predominant daughter fragments
were m/z 184.0 for clebopride and m/z 71.5 for IS. The collision energy in the product MS–MS mode was investigated to optimize the sensitivity, and the optimal values were found to be 27 and 20 eV for the product ion of clebopride and IS, respectively. 3.1.2. Optimization of the chromatographic condition The CAPCELL MG-III C18 column was selected for all analysis since it provided symmetrical peak shape and obtained the highest intensity to clebopride and IS. The separation and ionization of clebopride and IS were affected by compose of mobile phase. The mobile phase pH affected not only the retention time, but also the ionization efficiency of clebopride and IS. The acidity of mobile phase benefited to the ionization of clebopride and IS. 3.1.3. Sample preparation The aim for sample preparation method was to remove interferences from the biological sample and it should be also reproducible with a high recovery involving a minimum number of working steps. LLE gave both high recovery and good chromatography. 28% ammonium hydroxide was added to the plasma to minimize the protein binding. LLE was advantageous because this technique not only extracted the analyte and IS with sufficient efficiency and specificity, but also minimized the experimental cost. Ethyl acetate, diethyl ether MTBE, dichloromethane and trichloromethane were all tested as extraction solvent, and MTBE was finally adopted because of its high extraction efficiency. 3.1.4. IS selection An ideal IS should be a structurally similar analog or stable isotope-labelled compound according to the FDA guideline [9]. However, stable isotope-labelled compound is too expensive for most research institutes. Itopride was chosen for quantification as the IS due to its similarity with the analytes in structure, chromatographic behavior, mass spectrographic behavior and stability. 3.2. Method validation 3.2.1. Specificity The specificity of the method was determined by analyzing six different lots of blank control both with and without the IS. The chromatograms of LLE produced clean extracts with no interference from endogenous compounds at the retention time for clebopride and IS. Fig. 2 shows representative chromatograms of blank human plasma, the LLOQ (69.530 pg/ml) of clebopride and IS in plasma and volunteer’s plasma sample. Typical retention time for clebopride and IS were 1.4 and 1.3 min. And the peak shapes were sharp and symmetrical. The total run time was about 3.0 min. 3.2.2. LLOQ and linearity The calibration curves were prepared daily which showed good linearity in the range 69.530–4450.0 pg/ml for clebopride. The typical regression equation was R = 1.0881C + 0.0404, r = 0.9993, where R corresponds to the peak area ratio of clebopride to the IS, and C (pg/ml) refers to the concentration of clebopride added to the plasma. Linear regressions of the peak area ratios of clebopride to internal standard were weighted by 1/x. The LLOQ of the method was 69.530 pg/ml of clebopride in human plasma, the precision (RSD%) was 15.83% (n = 6), and the accuracy (RE %) was −9.68% (n = 6).
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
2075
Fig. 3. The mean plasma concentration–time profiles of clebopride in human after oral administration of 1.36 mg clebopride, (n = 12).
3.2.3. Within- and between-batch precision and accuracy Data for within-batch and between-batch precision and accuracy of the method for determination of clebopride are presented in Table 1. The accuracy deviation values are within 11% of the actual values. The precision determined at each concentration level does not exceed 6% of the relative standard deviation (RSD). The results revealed good precision and accuracy.
3.2.4. Recovery and matrix effect The extract recoveries of clebopride from human plasma were 88.3 ± 3.2%, 85.9 ± 4.1%, and 82.7 ± 2.7% at concentrations of 139.10, 556.25 and 4450.0 pg/ml, respectively. The mean extraction recovery of IS was 83.9 ± 1.5%. Matrix effect is due to co-elution of some components present in biological samples. These components may not give a signal in MRM of target analyte but can certainly decrease or increase the analyte response dramatically to affect the sensitivity, accuracy and precision of the method. Thus, the evaluation of matrix effect from the influence of co-eluting components on analyte ionization is necessary for an HPLC–MS/MS method. All the ratios defined as in Section 2 were between 85% and 115%. No significant matrix effect for clebopride and IS was observed indicating that no co-eluting substance influenced the ionization of the analytes and IS. Fig. 2. MRM chromatograms of (A) blank plasma; (B) blank plasma spiked with 69.530 pg/ml clebopride (LLOQ) and internal standard; (C) 3 h samples after oral administration of 1.36 mg clebopride tablet. The retention times of clebopride and internal standard were 1.4 and 1.3 min.
3.2.5. Stability Table 2 summarizes the freeze and thaw stability, short-term stability, long-term stability and post-preparative stability data of clebopride. All the results showed good stability during these tests and there were no stability related problems during the routine analysis of samples for pharmacokinetic, bioavailability or bioequivalence studies.
Table 2 Data showing the stability of clebopride in human plasma at different QC levels (n = 5). Accuracy (Mean ± SD) 139.10 pg/ml Freeze and thaw stability Short-term stability Long-term stability Post-preparative stability
125.65 131.29 141.26 128.96
± ± ± ±
9.14 8.92 5.84 8.45
139.10, 556.25 and 4450.0 pg/ml are spiking plasma concentrations.
556.25 pg/ml 526.39 531.95 542.98 540.32
± ± ± ±
32.56 25.35 18.25 29.16
4450.0 pg/ml 4289.30 4329.68 4301.52 4401.92
± ± ± ±
244.65 322.14 258.36 251.56
2076
Z. Tan et al. / J. Chromatogr. B 878 (2010) 2072–2076
3.3. Pharmacokinetic study The present method was successfully applied to the pharmacokinetics studies after an oral administration of 1.36 mg clebopride tablets to 12 healthy Chinese healthy volunteers. Mean concentration–time profiles of clebopride were displayed in Fig. 3. The parameters were calculated by a non-compartmental model. The values of Cmax and Tmax were obtained directly from experiment observations. The mean of Cmax and Tmax were 2072.78 ± 1321.85 pg/ml and 0.94 ± 0.13 h, respectively. Plasma concentration declined with a t1/2 of 5.45 ± 3.90 h. AUC0−24 and AUC0−∞ values obtained were 8041.25 ± 5420.22 pg h/ml and 8044.77 ± 5420.99 pg h/ml respectively. These pharmacokinetic parameters were in accordance with those reported in the literature [2,7], indicating the applicability of this method to the pharmacokinetics study of clebopride. 4. Conclusions This paper described a sensitive, specific, accurate and precise HPLC–ESI-MS/MS method for the determination of clebopride in human plasma. Good linearity was observed in the range from 69.530 to 4450.0 pg/ml and the LLOQ (69.53 pg/ml) makes this method of real practical use for pharmacokinetics, bioavailability and bioequivalence studies.
The method was successfully applied to a pharmacokinetics study of clebopride in Chinese volunteers. It is the first study, as far as we know, of LC–MS/MS method for determination of clebopride concentration in vivo. Acknowledgement This research is supported by Foundation of Hunan Science and Technology Department general project (2009FJ4008). References [1] S.C. Bittencourt, T.C.M. De Lima, G.S. Morato, Gen. Pharmacol. 26 (1995) 1083. [2] H.W. Ryou, J.H. Lee, Y.H. Chi, Y.H. Hahn, H.K. Tan, K.H. Lee, S.L. Kim, S.Y. Jeon, Y.W. Choi, Yakche Hakhoechi 30 (2000) 179. [3] Y. Hayasaka, S. Murata, K. Umemura, Chem. Pharm. Bull. (Tokyo) 29 (1981) 1478. [4] J. Segura, O.M. Bakke, G. Huizing, A.H. Beckett, Drug Metab. Dispos. 8 (1980) 87. [5] G. Huizing, A.H. Beckett, J. Segura, J. Chromatogr. A 172 (1979) 227. [6] M. Yano, K. Nakamichi, T. Yamaki, T. Fukami, K. Ishikawa, I. Matsumoto, Chem. Pharm. Bull. 32 (1984) 1491. [7] P.R. Robinson, M.D. Jones, J. Maddock, L.W. Rees, J. Chromatogr.: Biomed Appl. 564 (1991) 147. [8] P.R. Robinson, M.D. Jones, J. Maddock, J. Chromatogr.: Biomed. Appl. 432 (1988) 153. [9] Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), May 2001.