Clopidogrel In Human Plasma Research Articles

Development and Validation of a Bioanalytical Method for the Quantification of Clopidogrel in Human Plasma by LC-MS/MS using the Accuracy Profiles

A basic bio-analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of clopidogrel in human plasma. Clopidogrel-d3 was used as an internal standard (IS). Protein precipitation with acetonitrile for extraction of clopidogrel and it IS was used. Chromatographic separation of clopidogrel and IS was performed on a Synergi reverse phase C18 column (150 mm × 2 mm, 4 μm) using an isocratic mode. The mobile phase contained a mixture of 0.1% formic acid and acetonitrile (25 : 75) applying a flow rate of 0.4 mL/min. Ionization was performed by an electrospray ionization source (ESI), in positive MRM (Multiple Reaction Monitoring) mode. This method was validated by accuracy profiles approach using tolerance intervals limits. The calibration curve ranged from 0.0156 ng/mL to 8 ng/mL. This method can be employed effectively in bioequivalence and/or pharmacokinetics. In this method we have avoided the use of methanol in all steps of bioanalysis of clopidogrel to avert the back-conversion of clopidogrel metabolite.

Simultaneous determination of clopidogrel, 2-oxo-clopidogrel, and the thiol metabolite of clopidogrel in human plasma by LC-MS/MS.

Clopidogrel is a pro-drug which needs two-step metabolism to produce the active thiol metabolite. This study aimed to explore an efficient method to simultaneously determine the plasma clopidogrel, 2-oxo-clopidogrel (2-Oxo-CLP), and the clopidogrel active metabolite (CAM). A high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) was therefore developed. The analytes were extracted from plasma by using methyl tert-butyl ether (MTBE). Chromatographic separation was performed on a C18 column under an isocratic elution, accompanied with acetonitrile and deionized water containing 0.1% formic acid. After optimizing the condition of LC-MS/MS, a stable linearity was observed in the standard curves over the concentration ranges of 0.05 to 50.0 ng/mL for clopidogrel, 0.5 to 50.0 ng/mL for 2-Oxo-CLP, and 0.5 to 100 ng/mL for clopidogrel active metabolite derivative (CAMD). The retention time was 4.78 minutes, 3.79 minutes, 3.59 minutes, and 4.82 minutes for clopidogrel, 2-Oxo-CLP, CAMD, and internal standard, respectively. Both the relative standard deviation and the relative error were within the requirement of operating criteria. No significant degradation of clopidogrel, 2-Oxo-CLP, and CAMD occurred under different storage conditions. This method was successfully validated in 3 patients with coronary artery disease. The results showed that the current LC-MS/MS method was efficient for simultaneously detecting clopidogrel, 2-Oxo-CLP, and CAM with fine linearity, accuracy, precision, and stability.

Development and validation of a sensitive and rapid UHPLC–MS/MS method for the simultaneous quantification of the common active and inactive metabolites of vicagrel and clopidogrel in human plasma

Vicagrel, an analog of clopidogrel, is a novel thienopyridine P2Y12 antagonist in clinical development in China for the treatment of acute coronary syndromes. Vicagrel and clopidogrel are prodrugs requiring metabolic activation to produce a pharmacologically active thiol metabolite (H4) in vivo. The formation of H4 from vicagrel in humans is promising more efficient and consistent than that from clopidogrel. After oral dosing of vicagrel or clopidogrel to humans, the active thiol metabolite H4 and two inactive metabolites closely related to H4 formation (the thiol metabolite H3 and the S-methylated metabolite SM3) were observed in plasma; SM3 was the most abundant drug-related component of vicagrel in circulation. In this study, a sensitive, rapid, and reliable UHPLC–MS/MS method was developed for the simultaneous determination of derivatized H3 (MP-H3), derivatized H4 (MP-H4), and SM3 in human plasma to investigate the pharmacokinetics of vicagrel in healthy subjects compared with clopidogrel. After extracted from 50μL of plasma by simple protein precipitation, the analytes and stable isotope-labeled internal standards were separated on an Acquity UPLC BEH C18 column (100mm×2.1mm, 1.7μm). The mobile phase was acetonitrile–water–formic acid (45:55:0.0275, v/v/v) delivered at a flow rate of 0.45mL/min under isocratic elution. The total chromatographic run time was 6min. MP-H3, MP-H4, and SM3 were separated from their corresponding isomers under the chromatographic conditions. Mass spectrometric detection was conducted in multiple reaction monitoring mode on an AB Sciex Qtrap 5500 System using a positive electrospray ionization interface. The calibration curves were linear in the following ranges: 0.100–200ng/mL for MP-H3 and MP-H4 and 1.00–1000ng/mL for SM3. The intra- and inter-day accuracy and precision for each analyte at all concentrations were within acceptable limits (±15%). The validated method was successfully applied to compare the pharmacokinetics of vicagrel and clopidogrel after a single oral administration to healthy subjects in the first-in-human study of vicagrel.

ANALYTICAL VALIDATION OF CLOPIDOGREL IN HUMAN PLASMA THROUGH ULTRAHIGHPERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Objective: This study developed a sensitive, selective, and valid method for analyzing clopidogrel in human plasma using liquid chromatographytandemmass spectrometry (LC-MS/MS).Methods: The chromatography separation was performed on the waters ACQUITY UPLC Class BEH C18 1.7 μm (2.1 × 100 mm) column, consisting of0.1% formic acid in water and 0.1% formic acid in acetonitrile (30-70) as the mobile phase, and an isocratic elution with a flow rate of 0.2 mL/minutes.The mass detection was performed using a Waters Xevo TQD equipped with positive electrospray ionization in the multiple reaction monitoringmodes. The clopidogrel was detected at m/z 322.086>212.097 and irbesartan as an internal standard at m/z 429.233>207.131. The sample wasprepared with protein precipitation method using acetonitrile, vortex mixed for 10 minutes, and centrifuged at 13,000 rpm for 20 minutes.Results: This method showed a linear result at the concentration range of 0.2-10 ng/ml with r≥0.9997.Conclusions: The developed method provides sensitivity, linearity, precision, and accuracy and is suitable for analysis of clopidogrel in human plasmausing LC-MS/MS.

Development and validation of an HPLC–MS/MS method to determine clopidogrel in human plasma

A quantitative method for clopidogrel using online-SPE tandem LC–MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d4) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75mg single dose clopidogrel tablets in 48 healthy male subjects.

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry.

The interest in therapeutic drug monitoring has increased over the last few years. Inter- and intra-patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield(TM) C18 column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)-acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease.

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS/MS

A simple, robust, and rapid LC-MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide-bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel-d4 as an internal standard (IS). The mixtures were separated on a C18 RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI-MS. The detector was operated in selected reaction-monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05-20 and 0.5-200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra- and interday, was less than 8.26% with an accuracy of 87.6-106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.

A validated HPLC–MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: Application to a clinical pharmacokinetic study

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study.

Determination of Clopidogrel in Human Plasma by a Validated LC-MS/MS Method

Clopidogrel (CPD) is prodrug converted endogenously to an unstable active metabolite and an inactive carboxylic acid metabolite. Therefore, CPD is the required analyte in bioequivalence and bioavailability studies. Development and validation of an LC-MS/MS determination of CPD was performed with the aim to be applied for pharmacokinetic analysis. CPD and ticlopidine (TPD, internal standard) were extracted from 200 µL of human plasma with ethylacetate. Chromatographic …

DEVELOPMENT AND VALIDATION OF HPLC-UV METHOD FOR THE DETERMINATION OF CLOPIDOGREL IN PHARMACEUTICAL DOSAGE FORM AND HUMAN PLASMA

A simple, rapid, and sensitive high performance liquid chromatographic method with UV detection has been developed for the quantification of clopidogrel in human plasma and pharmaceutical dosage form using diclofenac sodium as an internal standard and validated according to standard guidelines. The separation of clopidogrel and diclofenac sodium was achieved using Hypersil BDS RP18 (250 × 4.6 mm, 5 µm) at a column oven temperature of 35°C. The methanol and phosphoric acid 25 mM (80:20, v/v) (pH 3.0), used as a mobile phase in the isocratic mode was pumped at a flow rate of 1.2 mL min−1 and eluents were monitored at a wavelength of 220 nm. The retention times for diclofenac sodium and clopidogrel were 4.15 and 4.62 min, respectively. Extraction of drug from plasma was carried out with diethyl ether. The limit of detection (LOD) was 2.0 ng mL−1, while limit of quantification (LOQ) was 5.0 ng mL−1. The calibration curves were linear (r2 = 0.999) over the concentration range of 0.005 to 5.0 µg mL−1 for both standard solutions and spiked plasma. The intra-day and inter-days precision (RSDs) was less than 4.08 and 6.38%, respectively, and the accuracy was more than 99%. The method could be applied for the pharmacokinetic study of clopidogrel in healthy human volunteers as well as to pharmaceutical dosage forms.

Regulatory recommendations for clopidogrel: Focus on validation of a reliable bioanalytical method and its application to a bioequivalence study

A rapid, simple, and sensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the determination of clopidogrel in human plasma was developed and validated using clopidogrel-d4 as the internal standard (IS). The analyte and the IS were extracted from 500 µl aliquots of human plasma via solid phase extraction. The precursor to product ion transitions monitored for clopidogrel and IS were m/z 322.2 → 212.0 and 326.2 → 216.0, respectively. The method was fully validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, dilution integrity, and stability. The Linearity range was 20.4–10,772.6 pg/mL with mean correlation coefficient (r) ≥ 0.9977. The back conversion was also evaluated during method validation as per EMA recommendation. Results proved that clopidogrel was accurately and reliably estimated by the method without any evidence of back conversion of clopidogrel acid. The method was successfully applied to a bioequivalence study of 75 mg clopidogrel bis...

Development and validation of an HPLC–MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion

Quantitative methods using LC–MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm × 2.1 mm, 5 μm), Ascentis Express C8 (10 cm × 2.1 mm, 2.7 μm) and Ascentis Express RP Amide (10 cm × 2.1 mm, 2.7 μm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.

Sensitive Quantification of Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by LC with UV Detection

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.

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.

The validation of a bioanalytical method for the determination of clopidogrel in human plasma

A fast, sensitive and specific LC–MS/MS bioanalytical method for the determination of unchanged clopidogrel in human plasma has been developed and validated over the range of 10–12,000 pg mL −1 ( r 2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 μL of the sample extract was injected onto the LC–MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 °C) by gradient elution at a flow rate of 0.9 mL min −1 over a 3 min run time. Retention times of 1.61 and 1.59 min were observed for clopidogrel and 2H 3-clopidogrel (I.S.), respectively. Detection was achieved using a Sciex API 4000, triple quadrupole mass spectrometer, in positive TurboIonspray™ (electrospray) ionisation mode. Ion transitions were monitored using MRM (multiple reaction monitoring) for clopidogrel ( m/ z 322–212) and for 2H 3-clopidogrel ( m/ z 327–217). This validated method was used to support a pharmacokinetic study in healthy volunteers.

Quantification of clopidogrel in human plasma by sensitive liquid chromatography/tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry method was developed and validated for the assay of clopidogrel in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 322/212 for clopidogrel and m/z 264/154 for the internal standard. The assay exhibited a linear dynamic range of 5-6000 pg/mL for clopidogrel in human plasma. The lower limit of quantification was 5 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.

Determination of the carboxylic acid metabolite of clopidogrel in human plasma by liquid chromatography–electrospray ionization mass spectrometry

A rapid and specific liquid chromatographic–mass spectrometric method has been developed and validated for the determination of the carboxylic acid metabolite of clopidogrel in human plasma. Sulphafurazole was used as internal standard. The samples were subjected to a solid phase extraction procedure using Hypercarb cartridges. The chromatographic separation was performed on a reversed phase porous graphitized carbon column using a mobile phase consisting of 70% methanol in water containing 0.1% (v/v) trifluoroacetic acid, pumped at a flow rate of 0.25mlmin−1. The analytes were detected after positive electrospray ionization using the selected ion monitoring mode of the species at m/z 308 for the carboxylic acid metabolite of clopidogrel, m/z 322 for clopidogrel and m/z 268 for sulphafurazole. Calibration graphs were linear (r>0.9994, n=6), in the range 100–1000ngml−1 for the carboxylic acid metabolite of clopidogrel. The intra- and inter-day R.S.D. values were <3.1%, while the relative error Er was less than −9.6% (n=6). The limits of detection (3.3σ) and quantitation (10σ) for the carboxylic acid metabolite of clopidogrel were found to be 28 and 93ngml−1, respectively. The efficiency of the solid phase extraction procedure for the carboxylic acid metabolite of clopidogrel averaged 74.6%.

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-( S)-α-( o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C 18 cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and α-bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue ( III) of II, was used as internal standard. The 1/ X 2; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.