Quantification of arginine and its metabolites in human erythrocytes using liquid chromatography-tandem mass spectrometry

Erythrocytes may affect several physiological processes because they are scavengers, vehicles, and (as recently highlighted) a producer of nitric oxide (NO). NO bioavailability is linked to arginine, its metabolic products ornithine and citrulline, and methylarginines. Here we describe a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of analytes involved in the Arg/NO metabolic pathway in erythrocytes. Calibration functions were linear, and the interday coefficients of variation were less than 10%. Limit of quantification values make this method suitable for low concentration samples. The method presented here allows easy sample preparation and provides a valuable tool for the evaluation of the Arg/NO metabolic pathway in erythrocytes.     

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo((R)) C(18) column and the mobile phase was composed of acetonitrile:H(2)O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1-->308.3, 359.0-->308.3, 359.0-->111.2 and 347.0-->312.0 for triazolam, alpha-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05ng/mL for triazolam and 0.1ng/mL for alpha-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was -8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

Simultaneous determination of ebastine and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry

We developed a method for determining ebastine, a new generation of antihistamines, and its three metabolites (hydroxyebastine, carebastine and desalkylebastine) in plasma simultaneously using LC/MS/MS. Four compounds and terfenadine, an internal standard, were extracted from plasma using a mixture of diethylether and dichloromethane in the presence of 1 M HCl. After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:5 mM ammonium acetate, 50:50, v/v) and injected onto a reversed-phase C(18) column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 470.7-->167.1, 486.7-->167.1, 500.6-->167.1, 268.4-->167.1 and 472.7-->436.0 for ebastine, hydroxyebastine, carebastine, desalkylebastine and terfenadine, respectively. The coefficient of variation of the assay precision was less than 12.5%, and the accuracy exceeded 88%. The limit of detection was 0.5 ng/ml for desalkylebastine; 0.2 ng/ml for ebastine, hydroxyebastine and carebastine, respectively. This method was used to measure the plasma concentration of ebastine and its three metabolites from healthy subjects after a single 20 mg oral dose of ebastine. This analytic method is a very simple, sensitive, and accurate to determine the pharmacokinetic profiles of ebastine including its metabolites.     

Quantitative determination of trimebutine maleate and its three metabolites in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective HPLC-MS-MS method was developed for the determination of trimebutine maleate (TM) and its major metabolites N-monodemethyltrimebutine (TM-MPB), N-didemethyltrimebutine (APB) and 3,4,5-trimethoxybenzoic acid (TMBA) in human plasma. The analytes were extracted from plasma samples by liquid-liquid extraction and chromatographed on a YMC J'sphere C(18) column. The mobile phase consisted of 2 mM ammonium acetate buffer (pH 6.5)-methanol (20:80, v/v), and at a flow-rate of 0.2 ml/min. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive-negative switching electrospray ionization (ESI). The method was validated over the concentration range of 1-100 ng/ml for trimebutine maleate and APB, 1-500 ng/ml for MPB, and 50-10,000 ng/ml for TMBA. Inter- and intra-day precision (RSD%) for trimebutine maleate and its three metabolites were all within +/-15% and the accuracy was within 85-115%. The limit of quantitation was 1 ng/ml for trimebutine maleate, TM-MPB and APB, and 50 ng/ml for TMBA. The extraction recovery was on average 58.2% for trimebutine maleate, 69.6% for MPB, 51.2% for APB and 62.5% for TMBA. The method was applied to the pharmacokinetic study of trimebutine maleate and its metabolites in healthy Chinese volunteers.     

Lewisite metabolites detection in urine by liquid chromatography-tandem mass spectrometry

A sensitive and simple method for the quantification and for the detection of 2-chlorovinylarsonous (CVAA) and 2-chlorovinylarsonic (CVAOA) acids was developed. CVAA and CVOA are important biological markers in human and rat urine specific to lewisite (chlorovinylarsonous chloride compounds) exposure. The developed assay was based on the use of solid-phase extraction (SPE) followed by liquid-chromatography coupled to electrospray ionization (negative ion-mode) low-energy collision dissociation-tandem mass spectrometry (ESI-CID-MS/MS). The method demonstrated linearity over at least three orders of magnitude and had a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. The relative standard deviations for the quality control samples ranged from 6 to 11%. Application of this procedure was demonstrated in the lewisite animals exposure model. Rats were exposed intravenously by no lethal doses of lewisite and markers levels in urine samples were analyzed for 21 days post-exposure.     

Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry

Gas chromatography mass spectrometry with selected ion monitoring has been used to develop a method for the quantification of ketotifen and its demethylated, 10-hydroxy and 10-hydroxy demethylated metabolites in human plasma. The minimum detectable concentrations for ketotifen and its demethylated metabolites were 50 pg ml-1 and 300 pg ml-1 for the 10-hydroxy metabolite. The methodology has been applied in studies of the kinetics of the drug in man, and plasma levels of the unchanged drugs and its metabolites in free and conjugated form are reported.     

High-throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma using a monolithic column

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50 mm x 4.6 mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid-liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23s. The isotope-labeled D(6)-bupropion and D(6)-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25-200 ng/ml (bupropion and threo-hydrobupropion), and 1.25-1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.     

Determination of cryptotanshinone and its metabolite in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective LC-MS-MS method has been developed and validated for the determination of cryptotanshinone (CTS) and its active metabolite tanshinone II A (TS II A) in rat plasma using fenofibrate (FOFB) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a Waters symmetry ODS column using methanol and water (85:15) as mobile phase delivered at 1.0 mL/min. LC-MS-MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using atmospheric pressure chemical ionization (APCI) and positive multiple reaction monitoring. Ions monitored were m/z 297.0--> 251.0 for CTS, m/z 295.0--> 249.0 for TS II A, and m/z 361.1--> 233.0 for FOFB with argon at a pressure of 0.2 Pa and collision energy of 25 eV for collision-induced dissociation (CID). The assay was linear over the range 0.1-20 ng/mL for CTS and 0.2-15 ng/mL for TS II A. The average recoveries of CTS and TS II A from rat plasma were 93.7 and 94.7%, respectively. The established method has been applied in a pharmacokinetic study of CTS in rats.     

Identification and quantitative determination of benproperine metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry

Two novel metabolites of benproperine (BPP), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3-OH-BPP) and 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (4-OH-BPP), were confirmed by comparison of retention times and mass spectra with those of synthetic standards using liquid chromatography-tandem mass spectrometry. Selective and sensitive procedures were developed for the simultaneous determination of BPP, 3-OH-BPP and 4-OH-BPP in human plasma and urine. The analytes were extracted from plasma sample and enzymatically hydrolyzed urine samples by liquid-liquid extraction, separated through a Diamonsil C(18) column (150 mm x 4.6 mm i.d.) and determined by tandem mass spectrometry with an electrospray ionization interface in selected reaction monitoring mode. Dextromethorphan was used as internal standard. The mobile phase consisted of acetonitrile-water-formic acid (34:66:1, v/v/v), and flow-rate was 0.5 ml min(-1). This method has a lower limit of quantification (LLOQ) of 60, 4.0 and 4.0 nmol l(-1)for BPP, 3-OH-BPP and 4-OH-BPP in plasma, 4.9, 4.7 and 2.4 nmol l(-1) in urine, respectively. The intra- and inter-run precision were measured to be below 9.2%, and the accuracy was within +/-4.3% for the analytes. The method was successfully used to determine BPP, 3-OH-BPP and 4-OH-BPP in plasma and urine for pharmacokinetic investigation. The results indicated residue of 3-OH-BPP in the body at least 192 h after an oral dose of BPP.     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.     

Simultaneous determination of azelnidipine and two metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A quantitative assay method by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the simultaneous determination of azelnidipine and its two metabolites, M-1 (aromatized form) and M-2 (hydroxylated form), in human plasma was developed and validated. Plasma samples, each of 1.0mL, were extracted by a single step liquid-liquid extraction using a mixture of ethyl acetate and hexane (1:1, v/v), and analyzed by the LC/ESI-MS/MS method. Three analytes were separated by isocratic elution on a C(18) column, and ionized using a positive ion electrospray ionization source. The ion transitions were monitored in selected reaction monitoring (SRM) mode. The chromatographic run time was 11min per injection, with retention time of 3.6, 10.2 and 6.8min for azelnidipine, M-1 and M-2, respectively. The calibration curves for azelnidipine, M-1 and M-2 well fitted to equations by a weighted (1/X(2)) quadratic regression over the range of 0.5-40.0ng/mL (r(2)>0.9979). The intra- and inter-assay precisions (coefficient of variation: C.V.), calculated from quality control (QC) samples, were less than 8.7 and 8.4%, 3.8 and 4.7%, and 11.9 and 13.9%, respectively, for azelnidipine, M-1 and M-2. The accuracy was within +/-9% for azelnidipine, within +/-7% for M-1 and within +/-16% for M-2. The overall recoveries for azelnidipine, M-1 and M-2 were 68.8-78.6%, 54.3-62.9% and 80.4-89.7%, respectively. All analytes evaluated demonstrated acceptable short-term, long-term, auto-sampler and stock solution stabilities. Furthermore, the method developed was successfully applied to pharmacokinetic studies on azelnidipine, M-1 and M-2 after an oral dose of 16mg CALBLOCK tablets (2mgx8mg tablets) to healthy volunteers.     

Simultaneous analysis of THC and its metabolites in blood using liquid chromatography-tandem mass spectrometry

Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy- Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Delta(9)-tetrahydrocannabinol (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid-liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using a XBridge C(18) column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the LLE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using 250 microL of blood. The method was linear over the range investigated (0.5-40 microg/L for THC, 1-40 microg/L for 11-OH-THC, and 2-160 microg/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were <12% for THC and 11-OH-THC and <8% for THC-COOH for certified quality control samples. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. No instability was observed after repeated freezing and thawing or in processed samples. The method was subsequently applied to 63 authentic blood samples obtained from toxicology cases. The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well suited for routine analysis.     

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization

Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid-liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C(8) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL(-1) (r(2)>0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL(-1)), 5.2% (300 ng mL(-1)) and 7.0% (1800 ng mL(-1)). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.     

Determination of dioscin in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm x 4.6 mm i.d.) was packed with Hypersil ODS material (5 microm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.     

Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry

Protodioscin (3-O-[alpha-L-rhamnopyranosyl-(1-->2)-{alpha-L-rhamnopyranosyl-(1-->4)}-beta-D-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-(25 R)-furost-5-ene-3 beta,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20-125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.