Sensitive Quantification of Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by LC with UV Detection 2009, 70, 953–956

Mohammad-Reza. Rouini1,2,&, Yalda H. Ardakani1, Alireza Foroumadi2, Hoda Lavasani1, Lida Hakemi1 1

2

Biopharmaceutics and Pharmacokinetics Division, Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14155-6451, Iran; E-Mail: [email protected] Faculty of Pharmacy, Pharmaceutical Sciences Research Centre, Tehran University of Medical Sciences, Tehran 14155-6451, Iran

Received: 3 February 2009 / Revised: 16 May 2009 / Accepted: 10 June 2009 Online publication: 28 July 2009

Abstract A rapid LC method with UV detection was developed for the quantification of carboxylic acid metabolite of clopidogrel in human plasma. Following a simple protein precipitation using a mixture of methanolic solution of ZnSO4, the analyte and commercially available internal standard were separated using a mobile phase of water–acetonitril (85:15, v/v) adjusted to pH 3.5 on a Chromolith C18 column at a flow rate of 2.5 mL min-1 with a total retention time of 4 min. Linearity was verified over the range of 20–3,000 ng mL-1 where the LOQ was 20 ng mL-1. This method was applied in a pharmacokinetic study.

Keywords Column liquid chromatography-UV dection Clopidogrel carboxylic acid metabolite Human plasma

Introduction Clopidogrel, a new therapeutic drug chemically known as methyl (1)-(S)a-(2-chlorophenyl)-6,7dihydrothieno[3, 2-c]pyridin-5(4H)-acetate hydrogen sulfate, is an antiplatelet agent which

Full Short Communication DOI: 10.1365/s10337-009-1245-2 0009-5893/09/09

selectively inhibits the binding of adenosine diphosphate (ADP) to its platelet receptor and blocks the subsequent platelet aggregation [1]. This drug is indicated for the reduction of atherosclerotic events (myocardial infarction, stroke, and vascular death) in patients

with atherosclerosis documented by recent stroke, myocardial infarction, or established peripheral arterial disease [1]. Clopidogrel a prodrug and hepatic biotransformation is essential for its in vivo antiplatelet activity. The active metabolite is formed by the oxidation of clopidogrel to 2-oxo clopidogrel and subsequent hydrolysis [2]. Following an oral administration in human, the plasma levels of clopidogrel are very low due to extensive metabolism and difficult to quantify. The main circulating metabolite (the carboxylic acid derivative) (Fig. 1) is pharmacologically inactive and represents 85% of circulating metabolites in human plasma. Since, neither the parent drug nor the active metabolite is detected in plasma, the measurement of pharmacodynamic effects by estimating platelet aggregation is believed to be a better measure for in vivo estimations [2]. However, the quantification of inactive carboxylic acid metabolite of clopidogrel which is the most abundant species circulating in blood has immerged as an indirect approach for studying the pharmacokinetics of clopidogrel [3].

Chromatographia 2009, 70, September (No. 5/6) Ó 2009 Vieweg+Teubner | GWV Fachverlage GmbH

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H

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selective and sensitive determination of CCA in human plasma.

N

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S Cl OH

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Materials O

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Fig. 1. Chemical structure of (a) Clopidogrel carboxylic acid (CCA), (b) 2,4,6-trihydroxyacetaphenon (IS)

A few analytical methods including gas chromatography–mass spectrometry (GC–MS) [4], liquid chromatography– mass spectrometry (LC–MS) [5, 6] and LC with UV detection [4, 7, 8] have been published for determination of the inactive metabolite of clopidogrel in biological fluids. Although using MS detection even with GC or LC improves the limit of quantification, these equipments are not routinely available in most analytical research labs. Among all reported LC methods with UV detection, complex liquid-liquid extraction methods and/or long analytical run time are required [4, 7, 8]. While LOQ of 100 ng mL-1 [4] and 200 ng mL-1 [8] have been reported in published papers, in human single dose pharmacokinetic studies an LOQ of less than 100 ng mL-1 is required for measuring of the analyte up to three half lives post-dose [7]. Most recently Bahrami et al. [7] reported an LC-UV method for determination of CCA in human serum. Although the analyte is eluted in relatively short time (about 5 min), the method needs a time consuming LLE method and temperature control of 50 °C which reduces the LC column life. Therefore in this study, we developed and validated a new rapid LC-UV method based on one step protein precipitation sample preparation for

954

Clopidogrel carboxylic acid (USP reference standard) was donated by Osvah Pharmaceutical Co. (Tehran, Iran). The internal standard of 2,4,6-trihydroxyacetaphenon (Fig. 1) was from Sigma-Aldrich Chemical Company (Milwaukee, USA). LC grade acetonitrile, methanol, analytical grade phosphoric acid (85%) and zinc sulphate were supplied by Merck (Darmstadt, Germany). Water used in all experiments was of Direct-QE quality (Millipore, France).

Standard Solutions Stock solutions of CCA and IS containing 1 mg mL-1 of compounds were prepared in methanol and stored at 4 °C and the intermediate stock standards were prepared by appropriate dilution in water of the stock solution. Appropriate aliquots of the standard solutions were added into human drug free plasma obtained from healthy volunteers as diluent. Starting from stock solution of clopidogrel carboxylic acid (100 lg mL-1) in water, final concentrations of 20, 50, 75, 100, 150, 250, 500, 1,000, 1,500, 2,000, 2,500, 3,000 ng mL-1 were obtained for the calibration curve. Plasma concentrations of 20, 100, 500, 1,000 ng mL-1 were used as quality control (QC) samples.

Apparatus and Chromatographic Conditions The chromatographic apparatus consisted of a low pressure gradient LC pump (smart line 1000), a UV detector (smart line 2600) operated at a wavelength of 230 nm and an online degasser and manager (smart line 5000, all from Knauer (Berlin, Germany). A Rheodyne

model 7725i injector with a 100 lL loop was used. The data was acquired and processed by means of ChromGate chromatography software (Knauer). Chromatographic separation was achieved by a Chromolith Performance RP-8e 100 mm 9 4.6 mm column (Merck) protected by a Chromolith Guard cartridge RP-18e 5 mm 9 4.6 mm. The mobile phase consisted of a mixture of water–acetonitrile (85:15 v/v) adjusted to pH 3.5 by phosphoric acid and was delivered in isocratic mode at 2.5 mL min-1 flow rate.

Sample Preparation 200 lL plasma sample were transferred into a 1.5 mL Eppendorf polypropylene tube and 10 lL of each IS solution and HCl (0.2 N) were added and mixed for 30 s. Subsequently, 200 lL of zinc sulfate solution in methanol (5 g zinc sulfate was dissolved in 50 mL methanol) were added and the mixture was then shaken vigorously for 5 min. After centrifugation at 12,0009g for 10 min, a 100 lL aliquot of clear supernatant was injected into the LC system.

Accuracy and Precision Accuracy was calculated as deviation of the mean from the nominal concentration, and defined as the percentage difference between the back-calculated concentrations of QCs. The accuracy and precision were determined from between and within day repetition of the low four QC plasma samples.

Lower Limit of Quantification and Recovery Lower limit of quantification (LLOQ) was calculated according to the FDA guidance for bioanalytical method validation [9]. The LLOQ was defined as the lowest concentration showing relative standard deviation of less than 20%.

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The recovery was determined by comparing the AUCs obtained from plasma treated QC spiked samples with the same concentrations of drug in distilled water.

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The present method was applied in a pharmacokinetic study. The protocol was approved by the Ethics Committee of Tehran University of Medical Sciences and written informed consent was obtained from a volunteer who participated in the study. The volunteer was not allowed to take any other medication for 2 weeks before and throughout the study. After an overnight fasting, a 75 mg Plavix tablet (Sanofi Aventis, Australia) was administered and peripheral venous blood samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 24 h in heparinized tubes. After centrifugation, plasma samples were separated and stored at -20 °C until analysis.

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Linearity The linearity of the relationship between peak area ratios and corresponding concentrations (which were needed in this study) were demonstrated by the correlation coefficient obtained for the Full Short Communication

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Chromatograms of plasma samples, depicting the separation and detection of CCA are presented in Fig. 2. Selectivity was indicated by absence of any endogenous as well as clopidogrel and thiolic metabolite interference at retention times of peaks of interest as evaluated by chromatograms of control human plasma and plasma spiked with compounds. These samples were prepared according to the sample preparation procedure. No change in column efficiency and back pressure was observed after several injections of protein precipitated samples. The retention times for CCA and IS were 3.3 and 4.0 min respectively.

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Fig. 2. Chromatograms of (a) a blank human plasma, (b) a blank human plasma spiked with CCA (2,000 ng mL-1) and (c) plasma sample taken from a volunteer 1.5 h after a single oral dose (75 mg) of Clopidogrel

regression line. The calibration curve showed equation of y = 0.0005x - 0.0169 with correlation coefficient (r2) of 0.999.

Lower Limit of Quantification The lower limit of quantification (LLOQ) was calculated as reported 20 ng mL-1.

Recovery, Accuracy and Precision The results from the validation of the method in human plasma are listed in Table 1. The method proved to be accurate and precise. Accuracy values were within acceptable limits for analyte. The values of within-day and between-day

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Table 1. Between- and within-day variability and recovery for determination clopidogrel carboxylic acid (CCA) (n = 5) Conc. (ng mL-1)

Between-day RSD (%)

CCA 20 100 500 1000

Within-day Accuracy (%)

Accuracy (%)

(%)

RSD (%)

Acknowledgments 7.9 11.3 6.3 2.9

97.4 100.3 100.1 100.1

Fig. 3. Concentration–time profile of clopidogrel carboxylic acid (CCA) after administration of 75 mg single oral dose of clopidogrel

precision ranged from 2.16 to 13.34%. Accuracy at QC concentrations was reported 97.4–100.1% for CCA and the absolute recoveries ranged from 72.3 to 98.8%.

Application of the Method The method was applied for determination of CCA in plasma of a volunteer who received a 75 mg Plavix tablet. The concentration-time profile obtained from this volunteer is shown in Fig. 3. This volunteer shows a maximum plasma concentration of 980 ng mL-1 for CCA which appears in 1 h after administration.

Discussion The limitation of flow rate applied to routine LC columns may result in long run time in most reported LC methods. Flow limitation may be resolved by using monolithic LC columns. The Chromolith column, as a monolithic LC column, has a biporous structure which offers a high porosity compared to usual columns (http://www.chromolith.com) which allows high flow rates without loss of performance or limitations due to

956

RSD (%)

Recovery

which could be applied in pharmacokinetic, bioequivalence studies as well as TDM of clopidogrel in patients.

7.6 11.7 7.1 8.6

95.2 100.1 100.1 100.1

92.3 95.7 94.9 98.8

5.5 3.9 1.3 1.4

increased pressure. Monolithic columns, may, therefore provide faster separation than those of conventional LC columns. The described method was established as a rapid analytical tool in a pharmacokinetic study requiring short retention time, high precision, sensitivity and small volumes of plasma for analysis. The parameters of the assay obtained in the course of validation processes presented above in the results section were considered satisfactory for its clinical application. A simple analytical procedure based on one-step protein precipitation with zinc sulphate and a total run time of 4.5 min allowed the possibility of determination of several samples a day. The developed method does not need high column temperature as applied by Bahrami et al. [7], the run time is shorter than previously published LC-UV methods [4, 8] and the sensitivity is suitable for pharmacokinetic studies. The stability of retention times and robustness of the methods allowed injection of more than 600 treated plasma samples without any loss in column efficiency as required in most bioequivalence studies. Mani et al. [10] stated that determining clopidogrel metabolite plasma concentrations could be a useful tool for identifying poor compliance and variable metabolism in clopidogrel-treated patients. Our method with one step sample preparation, short run time, availability of UV detector and very low LOQ allowed the TDM of clopidogrel in treated patients.

This work was fully supported by a grant from the Pharmaceutical Sciences Research Centre, Tehran University of Medical Sciences. The authors wish to thank Osvah Pharmaceutical Co. for their kind donation of CCA.

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Conclusion A sensitive, accurate and precise bioanalytical method for determination of CCA in human plasma was developed Chromatographia 2009, 70, September (No. 5/6)

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