330 Original Article
Rapid and Sensitive UPLC-MS/MS Method for the Determination of Domperidone in Human Plasma and its Application to Pharmacokinetic Study
Affiliations
Key words
▶ domperidone ● ▶ UPLC-MS/MS ● ▶ plasma ● ▶ pharmacokinetic study ●
X.-j Qiu1, S.-l. Zheng2, Y.-f. Wang1, R. Wang1, L. Ye3 1
Department of Pharmacology, Medical College of Henan University of Science and Technology, Luoyang, China The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 3 The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 2
Abstract
▼
In this study, a simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method is described for determination of domperidone in human plasma samples using oxcarbazepine as the internal standard (IS). Sample preparation was accomplished through protein precipitation with methanol, and chromatographic separation was performed on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with gradient profile at a flow of 0.45 mL/min. Mass spectrometric
Introduction
▼
received 23.12.2013 accepted 27.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368702 Published online: February 18, 2014 Drug Res 2014; 64: 330–334 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence L. Ye The First Affiliated Hospital of Wenzhou Medical University Fuxue Lane 2 Lucheng District 325000 Wenzhou China Tel.: + 86/577/55976 468 Fax: + 86/577/55976 469
[email protected] Domperidone is a selective peripheral dopamine D2 receptor antagonist which does not penetrate fully into the central nervous system, therefore it is apparently devoid of central sedative and extrapyramidal effects. The drug is principally widely used in many countries for the treatment of motility disorders as well as for its antiemetic properties [1]. Domperidone was for a long time a second choice to cisapride, but the removal of the latter from the market has increased its clinical use. Although domperidone is absorbed rapidly after oral administration, the absolute bioavailability is very low (12.7–17.6 %) owing to its first-pass elimination [2], which may result in very low plasma concentration and substantial inter-subject pharmacokinetics variability. Therefore, it is essential to develop a highly sensitive and fast analytical method to determine domperidone in human plasma, which may also benefit the pharmacokinetic and clinical studies of domperidone. There are some methods that have been published for the determination of domperidone in biological matrices [3, 4]. But, those methods were time-consuming and usually had low sensitivity in several studies. LC-MS provided another
Qiu X-J et al. Determination of Domperidone … Drug Res 2014; 64: 330–334
analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transition of m/z 426.3→175.2 was used to quantify for domperidone. The linearity of this method was found to be within the concentration range of 0.25–100.0 ng/mL for domperidone in human plasma. 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 domperidone in healthy Chinese volunteers after oral administration.
possibility to determine domperidone in various matrices [5, 6]. However, the single ion monitoring mode had low selectivity in the assay. In addition, the low sensitivity and long analysis time may not meet the requirement for sensitivity and high throughput in biosample analysis. Some assays based on LC-MS/MS were also described for determining domperidone in human plasma [7–9]. However, these methods suffer from many negative factors, such as complex sample preparation or obvious matrix effect. Thus, these factors may limit the application of these methods to high-throughput analysis in biological samples. Moreover, an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to determine domperidone with protein precipitation as the method of sample preparation, had a long analysis time (4.0 min per sample) [10]. Thus, the aim of this study was to develop a simple, rapid and sensitive UPLC-MS/MS method for determining domperidone in human plasma. The sample preparation of this method was simple one-step precipitation of plasma protein by methanol, which was time- and effort-saving. The analysis time and the sensitivity could also meet the requirements of high-throughput bioanalysis. Once developed and validated, this method was
Downloaded by: University of Liverpool. Copyrighted material.
Authors
Original Article 331
successfully applied to a pharmacokinetic study in healthy Chinese volunteers after oral administration of a 20 mg domperidone orally disintegrating tablet.
Materials and Methods
▼
Chemicals and reagents Reference standard of domperidone (purity > 98.0 %) and oxcarbazepine (purity > 98.0 %) were both purchased from Sigma (St. Louis, MO, USA). LC-grade acetonitrile and methanol was from Amethyst Chemicals. Formic acid was an analytical grade and purchased from the Beijing Chemical Reagents Company (Beijing, China). Blank human plasma was obtained from The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Ultra-pure water (resistance > 18 mW) was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA).
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 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, USA). The mobile phase consisted of acetonitrile (A) and water containing 1 % formic acid (B). A gradient elution was used. The initial mobile phase composition of 25 % A and 75 % B was maintained for 0.3 min. Between 0.3 and 0.5 min, the percentage of A was changed to 80 % and maintained for 0.2 min. Between 0.7 and 1.0 min, the percentage of A was then return to 25 %. A subsequent re-equilibration time (0.5 min) should be performed before next injection. The flow rate was 0.45 mL/min. The overall run time was 1.5 min. An AB Sciex QTRAP 5500 triple quadruple 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 mass analyzers. The dwell time was set to 200 ms for each MRM transition. The MRM transitions were m/z 426.3→175.2 and m/z 253.4→208.1 ▶ Fig. 1 showed the prodfor domperidone and IS, respectively. ● uct ion mass spectra of the analytes. After optimization, the source parameters were set as follows: curtain gas, 30 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).
Preparation of standard and Quality Control (QC) samples The stock solution of domperidone that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10.00 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The working solutions solutions for calibration and quality controls were made from the stock solution by diluting with methanol. Calibration curve standards were prepared by spiking blank human plasma with appropriate amounts of the working solutions at final drug concentrations of 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 and 100.0 ng/mL for domperidone. The preparation of QC samples were the same, with the 3 levels of plasma concentrations (0.5, 20.0 and 80.0 ng/mL). IS stock solution was made at an initial concentration of 1.00 mg/ mL. The IS working solution (100.0 ng/mL) was made from the
Fig. 1 Full-scan product ion spectra of domperidone a and oxcarbazepine (IS, b).
stock solution using methanol for dilution. All of the solutions were stored in a refrigerator at 4 °C.
Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 μL of the IS working solution (100.0 ng/mL in methanol) was added to 100 μL of collected plasma sample. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 13 000 rmp for 10 min. The supernatant (10 μL) was injected into the UPLC-MS/MS system for analysis.
Method validation Specificity was determined by analysis of blank human plasma samples from 6 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. Plasma samples were quantified using the calibration curve. Calibration curves were constructed validated by analyzing spiked calibration samples on 3 days in a row. Peak area ratio of domperidone to IS was plotted against analyte concentrations, and standard curves were fitted by weighted (1/χ2) least squares linear regression in the concentration of 0.25–100.0 ng/mL for domperidone. A correlation of more than 0.99 was desirable for all the calibration curves. 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. To determine the matrix effect, 6 different blank plasma samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at 3 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 (100.0 ng/mL) in the same manner. The extraction recoveries of domperidone at 3 QC levels (n = 6) were determined by comparing peak area of the analytes in sam-
Qiu X-J et al. Determination of Domperidone … Drug Res 2014; 64: 330–334
Downloaded by: University of Liverpool. Copyrighted material.
Instrumentation and conditions
332 Original Article
Pharmacokinetic study The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University prior to the study. 20 volunteers 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 pharmacokinetics study of domperidone. 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 20 mg domperidone orally disintegrating tablet with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 8, 12, 24 and 36 h after oral administration.
Blood samples were centrifuged at 4 000 × g for 10 min and the plasma was separated and kept frozen at − 20 °C until analysis.
Data analysis Plasma concentration vs. time profiles were analyzed using DAS software (Version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results and Discussion
▼
Method development 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 positive mode than in negative mode. ESI positive 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. A simple one step protein precipitation procedure was chosen to shorten the sample preparation time. The optimization of the procedure was obtained after testing several precipitating solvents and different solvent compositions. The results with the best recoveries and lowest matrix effects for all analytes were achieved with the solvent of methanol. High speed centrifugation at 13 000 rpm helped to remove tiny particles and increase the service life of the column. Chromatographic conditions were optimized to obtain high sensitivity, symmetrical peak shape and suitable retention time. The mobile phase systems of acetonitrile-1 % formic acid in water and methanol-1 % formic acid in water in various proporFig. 2 Representative UPLC-MS/MS chromatograms for domperidone and oxcarbazepine (IS) in human plasma samples: a blank plasma sample; b blank plasma sample spiked with domperidone and IS; c human plasma sample 3.0 h after oral administration of single dosage 20 mg domperidone.
Qiu X-J et al. Determination of Domperidone … Drug Res 2014; 64: 330–334
Downloaded by: University of Liverpool. Copyrighted material.
ples that were spiked with the analytes prior to extraction with those of samples to which the corresponding solution was added after extraction. The extraction recovery of the IS at the working concentration (100.0 ng/mL) was determined in a similar way as a reference. The intra-day precision and accuracy of domperidone were evaluated by analyzing QC samples (0.5, 20.0 and 80.0 ng/mL) with 6 replicates for each concentration. The inter-day precision and accuracy were evaluated by analyzing QC samples with 6 replicates for each concentration over 6 days. The precision was expressed by relative standard deviation (RSD) and the accuracy by relative error (RE). The stabilities of domperidone in human plasma were tested by analyzing 5 replicates of plasma samples at 3 concentration levels (0.5, 20.0 and 80.0 ng/mL) in different conditions. The shortterm 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 evaluated after 3 complete freezethaw 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.
Original Article 333
Specificity UPLC chromatograms of human plasma showed that the retention times for domperidone and IS were approximately 0.93 and ▶ Fig. 2 shows the typical chromatograms 0.52 min, respectively. ● of a blank plasma sample, a blank plasma sample spiked with domperidone and IS, and a plasma sample from a healthy volunteer after an oral administration. No endogenous interferences were observed in the blank plasma samples for the analytes.
Linearity of calibration curve and sensitivity The linear regressions of the peak area ratios vs. concentrations were fitted over the concentration range of 0.25–100.0 ng/mL for domperidone in human plasma. The typical equations of the calibration curve was as follows: y = 2.169x + 0.0119, r = 0.9994, where y represents the ratio of peak area to that of IS, and x represents the plasma concentration. The LLOQ was estimated in the process of calibration curve construction and defined as the concentration giving a signal-noise ratio of 10, was 0.25 ng/mL for domperidone.
Matrix effect and recovery To avoid interference from exogenous compounds co-eluted with the target compound, MS/MS detection, offering unique selectivity against matrix background and requires very limited sample preparation was performed. Ionization of analytes was carried out using the ESI technique with positive polarity and MRM mode. In order to further reduce the matrix effect, we took only 100 μL of the plasma sample was precipitated with acetonitrile, injecting 10 μL. The matrix effect for domperidone at concentrations of 0.5, 20.0 and 80.0 ng/mL and IS (100.0 ng/mL) ▶ Table 1. As a result, matrix effect from plasma were showed in ● was negligible in this method. The recovery was calculated by comparing the mean peak areas of analytes spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100 %. ▶ Table 1. The recovery in plasma ranged Results are shown in ● from 73.6 to 85.4 % for domperidone. The recovery of IS (100.0 ng/ mL) in plasma was 86.9 %.
Table 1 Matrix effect and recovery of domperidone and internal standards from human plasma (n = 6). Analytes
Concentration added (ng/mL) 0.5 20.0 80.0 100.0
Domperidone IS
Matrix effect ( %)
Recovery ( %)
Mean ± SD
RSD ( %) Mean ± SD RSD (%)
97.3 ± 5.1 97.7 ± 2.1 99.2 ± 6.3 96.9 ± 5.5
5.2 2.1 6.4 5.6
73.6 ± 3.7 85.4 ± 4.8 81.3 ± 4.3 86.9 ± 3.5
5.1 5.6 5.2 4.1
Table 2 Precision and accuracy for domperidone of QC samples in human plasma (n = 6). Com-
Concentration
pound
(ng/mL)
Domperidone
RSD %
RE %
intra-day inter-day intra-day
0.5 20.0 80.0
7.8 2.0 3.6
3.8 8.5 4.3
− 9.6 5.7 2.4
inter-day 8.1 − 7.6 4.0
Table 3 Stability of domperidone under various storage conditions (n = 5). Storage
Concentration
Concentration
RSD
conditions
added (ng/mL)
measured (ng/mL)
%
Room temperature/2 h 4 °C/12 h − 20 °C/ 3 freeze-thaw cycles − 20 °C/ 30 days
0.5 20.0 80.0 0.5 20.0 80.0 0.5 20.0 80.0 0.5 20.0 80.0
0.48 ± 0.02 23.98 ± 2.16 85.43 ± 6.65 0.49 ± 0.02 22.22 ± 1.82 89.45 ± 4.97 0.52 ± 0.04 18.53 ± 1.71 78.33 ± 3.67 0.49 ± 0.03 21.56 ± 1.01 77.56 ± 4.75
4.4 9.0 7.8 4.9 8.2 5.6 7.2 9.2 4.7 5.6 4.7 6.1
RE % − 4.6 − 6.5 5.4 − 3.7 8.4 9.4 2.6 − 2.3 − 6.1 − 3.7 5.5 − 2.5
Precision and accuracy The precision of the method was evaluated by calculating RSD for QCs at 3 concentration levels (0.5, 20.0 and 40.0 ng/mL) over 3 validation days. The intra-day precision for domperidone was 7.8 % or less, and the inter-day precision was 8.5 % or less at each QC level. The accuracy of the method ranged from − 9.6 to 8.1 % for domperidone at 3 QC levels. The above results demonstrated that the values were within the acceptable range and the method ▶ Table 2). was accurate and precise (●
Fig. 3 Mean plasma concentration of domperidone after oral administration of single dose of 20 mg of domperidone to 20 healthy human subjects under fasting condition.
Stability All the stability studies of domperidone in plasma were conducted at 3 concentration levels (0.5, 20.0 and 40.0 ng/mL) with 5 determinations for each under different storage conditions. The RSD of the mean test responses was within 10 % in all stabil▶ Table 3). ity tests of domperidone in plasma (●
No effect on the quantitation was observed for plasma samples kept at room temperature for 2 h and at 4 °C for 12 h in an autosampler. There was also no significant degradation when samples of domperidone in plasma were taken through 3 freeze ( − 20 °C)-thaw (25 °C) cycles. And it was also stable at − 20 °C for 30 days. Qiu X-J et al. Determination of Domperidone … Drug Res 2014; 64: 330–334
Downloaded by: University of Liverpool. Copyrighted material.
tions were tested. Acetonitrile-1 % formic acid in water was adopted because it had a much higher response. In the following experiment, the proportion of acetonitrile in the mobile phase was considered. Finally, a gradient elute was chosen owing to the high response of domperidone and suitable retention time it provided.
334 Original Article
This validated UPLC-MS/MS method was successfully applied to a pharmacokinetic study of domperidone in 20 Chinese healthy male volunteers after administration of a 20 mg orally disintegrating tablet. The profile of the mean plasma concentrationtime curve of domperidone in single dose study is shown ▶ Fig. 3. in ● After administration of a single dose of 20 mg domperidone orally disintegrating tablet, the Cmax and Tmax were 33.72 ± 10.78 ng/mL and 0.63 ± 0.13 h, respectively. Plasma concentration declined with a t1/2 of 11.46 ± 2.97 h. The AUC0–t and AUC0→∞ values obtained were 186.80 ± 58.55 and 215.40 ± 56.48 ng/mL · h, respectively. These parameters were in accordance with those reported in the literature [11].
Conclusions
▼
A rapid, sensitive and selective UPLC-MS/MS method for the determination of domperidone in human plasma was developed and validated. Compared with the analytical methods reported in the literatures, the method offered superior sample preparation with a simple one-step precipitation of plasma protein by methanol 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 domperidone orally disintegrating tablet in healthy volunteers.
Declaration of Interest
▼
The authors report no conflicts of interest.
Qiu X-J et al. Determination of Domperidone … Drug Res 2014; 64: 330–334
References 1 Brogden RN, Carmine AA, Heel RC et al. Domperidone. A review of its pharmacological activity, pharmacokinetics and therapeutic efficacy in the symptomatic treatment of chronic dyspepsia and as an antiemetic. Drugs 1982; 24: 360–400 2 Heykants J, Hendriks R, Meuldermans W et al. On the pharmacokinetics of domperidone in animals and man. IV. The pharmacokinetics of intravenous domperidone and its bioavailability in man following intramuscular, oral and rectal administration. Eur J Drug Metab Pharmacokinet 1981; 6: 61–70 3 Michaud V, Simard C, Turgeon J. An improved HPLC assay with fluorescence detection for the determination of domperidone and three major metabolites for application to in vitro drug metabolism studies. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 852: 611–616 4 Dailly E, Drouineau MH, Gournay V et al. Population pharmacokinetics of domperidone in preterm neonates. Eur J Clin Pharmacol 2008; 64: 1197–1200 5 Li Z, Yao J, Zhang Z et al. Simultaneous determination of omeprazole and domperidone in dog plasma by LC-MS method. J Chromatogr Sci 2009; 47: 881–884 6 Wu MS, Gao L, Cai XH et al. Determination of domperidone in human plasma by LC-MS and its pharmacokinetics in healthy Chinese volunteers. Acta Pharmacol Sin 2002; 23: 285–288 7 Zhang D, Chen K, Teng Y et al. Determination of Domperidone in Human Plasma using Liquid Chromatography Coupled to Tandem Mass Spectrometry and its Pharmacokinetic Study. Arzneimittelforschung-Drug Research 2012; 62: 128–133 8 Bose A, Bhaumik U, Ghosh A et al. LC-MS Simultaneous Determination of Itopride Hydrochloride and Domperidone in Human Plasma. Chromatographia 2009; 69: 1233–1241 9 Wu Y, Chu Y, Zhang YH et al. Liquid chromatography-electrospray quadrupole, linear ion trap mass spectrometric method for quantitation of domperidone in Chinese healthy volunteers. Chemical Research in Chinese Universities 2007; 23: 408–411 10 Xu DH, Lou HG, Yuan H et al. Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry. Biomed Chromatogr 2008; 22: 433–440 11 Wang X, Qin F, Jing L et al. Development and validation of UPLC-MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application. Biomed Chromatogr 2013; 27: 371–376
Downloaded by: University of Liverpool. Copyrighted material.
Application of the method