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)--(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 C₁₈ cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and -bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X²; 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.     

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2–100 μM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 μM for clopidogrel and 1.1 and 3.2 μM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD < 7%), intermediate precision (RSD < 7%) and accuracy (85–96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K_m of 145 ± 30 μM and V_max of 0.4, 1.5 and 3.4 μM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Sensitive method for the determination of chloroquine and its metabolite desethyl-chloroquine in human plasma and urine by high-performance liquid chromatography

A method has been developed for the rapid quantitative analysis of chloroquine and its metabolite desethyl-chloroquine in plasma, blood and urine using high-performance liquid chromatography. An ethylene dichloride extract of the alkalinized biological samples was extracted with dilute acid and chromatographed on a reversed-phase column. Phosphate buffer in acetonitrile was used as the mobile phase with perchlorate as the counter-ion. Ultraviolet absorption at 254 or 340 nm or fluorescence detection was used. The fluorescence spectra and the fluorescence quantum yield of the substances were determined.Chloroquine and desethyl-chloroquine concentrations in the range of 10 nmol/l (UV-detection) and of 0.5 nmol/l (fluorescence detection) could be accurately measured with a relative standard deviation of 12%. The method should be adequate for therapeutic and pharmacokinetic studies.     

Capillary gas chromatographic determination of 1,4-butanediol and gamma-hydroxybutyrate in human plasma and urine

This article describes two methods for the determination of 1,4-butanediol and gamma-hydroxybutyrate in human plasma and urine using capillary gas chromatography. For 1,4-butanediol, plasma or urine samples (500 microl) were extracted by protein precipitation whereas for gamma-hydroxybutyrate, plasma or urine samples (500 microl) were extracted and derivatised with BF3-butanol. The compounds were separated on a Supelcowax-10 column and detection was achieved using a flame ionization detector. The methods are linear over the specific ranges investigated, accurate (with a percentage of the nominal concentration <109.8%) and showed intra-day and inter-day precision within the ranges of 5.0-12.0 and 7.0-10.1%, respectively. No interferences were observed in plasma and urine from hospitalized patients.     

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.     

Estimation of carboxylic acid metabolite of clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic study

A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm x 4.6 mm i.d., 5 microm analytical column maintained at 30 degrees C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0-32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at -70 degrees C for 48 days and at ambient temperature for 24h. In the auto-sampler maintained at 15 degrees C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.     

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) (r2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 microL of the sample extract was injected onto the LC-MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 degrees 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 2H3-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 2H3-clopidogrel (m/z 327-217). This validated method was used to support a pharmacokinetic study in healthy volunteers.     

Simple reversed-phase ion-pair liquid chromatography assay for the simultaneous determination of mycophenolic acid and its glucuronide metabolite in human plasma and urine

A simple and reproducible reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method using isocratic elution with UV absorbance detection is presented for the simultaneous quantitation of mycophenolic acid (MPA) and MPA-glucuronide (MPAG) in human plasma and urine. The sample preparation procedures involved simple protein precipitation for plasma and 10-fold dilution for urine. Each analytical run was completed within 15min, with MPAG and MPA being eluted at 3.8 and 11.4min, respectively. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

High-performance liquid chromatographic determination of iothalamic acid in human plasma and urine

A simple, rapid and sensitive method for the determination of iothalamic acid (IA) in both plasma and urine is reported. After extraction with ethyl acetate, IA was determined by strong anion-exchange high-performance liquid chromatography with ultraviolet detection at 254 nm. The lower limit of detection was 0.5 μg/ml. The average recovery was 73 and 57% from plasma and urine, respectively. Linearity was found over the investigated concentration range (up to 500 μg/ml for plasma and up to 10.0 mg/ml for urine). The reproducibility of the technique was good (coefficient of variation less than 6%) as was the precision and accuracy (coefficient of variation less than 2.5%). No interference from endogenous substances or any of the common drugs tested was found.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Liquid chromatography-mass spectrometry method for determination of ramipril and its active metabolite ramiprilat in human plasma

A fast and robust liquid chromatography-mass spectrometry (LC-MS-MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5-250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Liquid chromatographic method for the determination of plasma itraconazole and its hydroxy metabolite in pharmacokinetic/bioavailability studies

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of itraconazole and its active metabolite, hydroxyitraconazole, in human plasma. Prior to analysis, both compounds together with the internal standard were extracted from alkalinized plasma samples using a 3:2 (v/v) mixture of 2,2,4-trimethylpentane and dichloromethane. The mobile phase comprised 0.02 M potassium dihydrogen phosphate-acetonitrile (1:1, v/v) adjusted to pH 3.0. Analysis was run at flow-rate of 0.9 ml/min with excitation and emission wavelengths set at 260 and 365 nm, respectively. Itraconazole was found to adsorb on glass or plastic tubes, but could be circumvented by prior treating the tubes using 10% dichlorodimethylsilane in toluene. Moreover, rinsing the injector port with acetonitrile helped to overcome any carry-over effect. This problem was not encountered with hydroxyitraconazole. The method was sensitive with limit of quantification of 3 ng/ml for itraconazole and 6 ng/ml for hydroxyitraconazole. The calibration curve was linear over a concentration range of 2.8-720 ng/ml for itraconazole and 5.6-720 ng/ml for the hydroxy metabolite. Mean recovery value of the extraction procedure for both compounds was about 85%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 15%. Hence, the method is suitable for use in pharmacokinetic and bioavailability studies of itraconazole.     

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Simple and fast chromatographic method for the simultaneous determination of vanillylmandelic acid and homovanillic acid in human urine

A high-performance liquid chromatographic method for the determination of vanillylmandelic acid and homovanillic acid is described. The method is fast despite the great polarity differences between the two acids. Moreover the sample pretreatment is quick and it does not need complex or expensive equipment. The only requirement is the disposition of two pumps (or at least two eluent reservoirs) operated alternatively by means of a switching valve placed before the injection device. This makes the method available for most routine laboratories.