Determination of ambroxol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI)

A rapid, sensitive and specific method to determination of ambroxol in human plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/ESI) was described. Ambroxol and the internal standard (I.S.), fentanyl, were extracted from plasma by N-hexane-diethyl ether (1:1, v/v) after alkalinized with ammonia water. A centrifuged upper layer was then evaporated and reconstituted with 100 microl mobile phase. Chromatographic separation was performed on a BDS HYPERSIL C18 column (250 mmx4.6 mm, 5.0 microm, Thermo electron corporation, USA) with the mobile phase consisting of 30 mM ammonium acetate (0.4% formic acid)-acetonitrile (64:36, v/v) at a flow-rate of 1.2 mL min(-1). The total run time was 5.8 min for each sample. Detection and quantitation was performed by the mass spectrometer using selected ion monitoring at m/z 261.9, 263.8 and 265.9 for ambroxol and m/z 337.3 for fentanyl. The calibration curve was linear within the concentration range of 1.0-100.0 ng mL(-1) (r=0.9996). The limit of quantification was 1.0 ng mL(-1). The extraction recovery was above 83.3%. The methodology recovery was higher than 93.8%. The intra- and inter-day precisions were less than 6.0%. The method is accurate, sensitive and simple for the study of the pharmacokinetics and metabolism of ambroxol.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of balofloxacin in human plasma and its pharmacokinetics

A selective and sensitive high performance liquid chromatography-electrospray ionisation-mass spectrometry method has been developed for the determination of balofloxacin (BLFX) in human plasma. The sample preparation was a liquid-liquid extraction, and chromatographic separation was achieved with an Agilent ZORBAX 300SB C18 2.1 mm x 150 mm column using a mobile phase comprised of methanol-water (10 mM CH(3)COONH(4), pH 3.0)=40:60 (v/v). Standard curves were linear (r=0.9992) over the concentration range of 0.03-3 microg/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation (R.S.D.). The limit of detection (LOD) was 0.02 microg/ml. The validated HPLC-electrospray ionization (ESI)-MS method has been used successfully to study balofloxacin pharmacokinetics in healthy volunteers.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of tolterodine tartrate in human plasma

A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the determination of tolterodine tartrate in human plasma. With oxybutynin as internal standard, tolterodine tartrate was extracted from plasma with n-hexane: isopropanol (95:5, v/v). The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile-water (10 mM CH3COONH4, pH 3.0)=50:50 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu Shim-pack VP-ODS C18 column (150 mmx2.0 mm I.D.) by means of selected-ion monitoring (SIM) mode mass spectrometry. Standard curves were linear (r=0.9993) over the concentration range of 0.1-30.0 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection (LOD) was 0.05 ng/ml. The validated LC-ESI-MS method has been used successfully to study tolterodine tartrate pharmacokinetic, bioavailability and bioequivalence in 20 healthy male volunteers.     

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma

A novel, selective and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC BEH C(18) column (100 mm x 2.1mm, 1.7 microm) with mobile phase consisting of acetonitrile-water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r(2)> or =0.99) over the concentration range of 1.04-1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within +/-0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.     

Determination of alachlor and its metabolites in rat plasma and urine by liquid chromatography-electrospray ionization mass spectrometry

A method based on liquid chromatography (LC) in combination with mass spectrometry (MS) for the analysis of alachlor (ALA) and its metabolites, 2-chloro-N-[2,6-diethylphenyl]acetamide (CDEPA) and 2,6-diethylaniline (DEA), in rat plasma and urine has been developed. 13C-labeled ALA was used as the internal standard for quantitation. The analyte in plasma or urine was isolated using a Waters Oasis HLB extraction plate. The mass spectrometer was operated in the ESI MS-SIM mode with a programming procedure. The retention times for ALA, CDEPA and DEA were 1.84, 3.11 and 4.12 min, respectively. The limits of quantification (LOQ) for ALA, CDEPA and DEA were 2.3, 0.8 and 0.8 ng per injection, respectively. The linear fit of analyte to mass response had an R2 of 0.99. Reproducibility of the sample handling and LC-MS analysis had a RSD of < or = 10%. The average recoveries for these analytes in rat plasma were better than 90%. Similar results were obtained with rat urine.     

Quantitative determination of azithromycin in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of azithromycin in human plasma and its application in a pharmacokinetic study. With roxithromycin as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The analysis was carried out on an ACQUITY UPLC BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with gradient elution at flow rate of 0.35 mL/min. The mobile phase was 50 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 1-1000 ng/mL, with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was -1.3% to 5.7% at all QC levels. The method was applicable to clinical pharmacokinetic study of azithromycin in healthy volunteers following oral administration.     

Sensitive determination of vinorelbine and its metabolites in human serum using liquid chromatography-electrospray mass spectrometry

A liquid chromatography-electrospray mass spectrometry method was developed for the quantitation of vinorelbine (VNB) and two metabolites, vinorelbine N-oxide (VNO) and deacetyl vinorelbine (DAV) in human serum. The limits of quantitation (LOQ) reached 0.5 ng/ml for both VNB and VNO and 1 ng/ml for DAV. The method was proved linear in the range of LOQs up to 1000 ng/ml, and extraction recovery was 80% on average for the three compounds. It was applied to the pharmacokinetic monitoring of vinorelbine and, for the first time, to the detection of VNO in the serum of patients suffering from non-small-cell lung cancer.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid-liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQDUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0-500.0 ng/ml with a coefficient of determination (r2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.     

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.     

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.     

A novel analysis method for diterpenoids in rat plasma by liquid chromatography-electrospray ionization mass spectrometry

A sensitive, specific, and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of lasiodonin, oridonin, ponicidin, and rabdoternin A in rat plasma using sulfamethoxazole as an internal standard (IS). The plasma samples were pretreated and extracted by liquid-liquid extraction. Chromatographic separation was performed on a C₁₈ column with linear gradient elution using water and methanol, both of which were acidified with 0.1% aqueous formic acid, at a flow rate of 0.8 ml/min. Detection was accomplished by scanning with multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion pairs (m/z) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for all analytes in the rat plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon rubescens extract to rats.     

Determination of green tea catechins in human plasma using liquid chromatography-electrospray ionization mass spectrometry

A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC-ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1M aqueous acetic acid and 0.1M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z=289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2-19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 degrees C within 24h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.     

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.     

Liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of nine chlorogenic acids (CGAs), three isomers each of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs) and dicaffeoylquinic acids (dCQAs), and their two metabolites, caffeic acid (CA) and ferulic acid (FA), in human plasma. In simultaneous multiple reaction monitoring (MRM) measurements using ESI-MS/MS with a negative ion mode, a deprotonated molecular ion derived from each of the 11 molecules was used as a precursor ion while three diagnostic product ions characteristic for each were selected for the qualitative analysis. To obtain maximal intensities for all diagnostic product ions, the collision energy was optimized for each one. LC separation was achieved under conditions of a reversed-phase Inertsil ODS-2 column combined with a gradient elution system using 50mM acetic acid with 3% acetonitrile aqueous solution and 50 mM acetic acid with 100% acetonitrile. In the quantitative analysis, one of the three diagnostic product ions for each of the 11 molecules was selected. Application of simultaneous LC-ESI-MS/MS MRM measurements to analyze the 11 standards spiked into blank human plasma indicated that all diagnostic product ions were detected without any interference, and that the sensitivity, linearity and recovery of this method were acceptable. When using this method to analyze those 11 molecules in the plasma after oral ingestion of 250 ml of a drink containing a green coffee bean extract (300 mg CGAs), all 11 molecules were identified and CQAs, FQAs and FA were quantified. CQAs, FQAs and dCQAs in human plasma were detected for the first time. This method should be useful to understand the biological and pharmacological effects of CGAs, such as improvement of human hypertension.     

Simple liquid chromatography-electrospray ionization mass spectrometry method for the routine determination of salmon calcitonin in serum

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the quantification of salmon calcitonin (sCT) in serum. Serum samples from rats and dogs were deproteinized and freeze-dried. The residue was then reconstituted with 57% acetonitrile in water containing 0.1% trifluoroacetic acid and 0.005% benzalkonium chloride. A 20-microl aliquot of the reconstituted solution was injected onto a polymer based RP-C18 column, The outlet was connected to an ion-trap mass spectrometer equipped with an ESI source, and spectra were recorded in a positive-ion, selected-ion monitoring mode. The limit of quantification of the method was 10 ng/ml. Biexponential curves were observed for the temporal serum concentration of sCT following intravenous administration of sCT to rats (100 microg/kg) and dogs (250 microg/kg), resulting in reasonable pharmacokinetic parameters. The present method appears applicable to routine analysis of serum sCT in pharmacokinetic studies with good selectivity, accuracy and precision.     

Determination of nitrofuran metabolites in milk by liquid chromatography-electrospray ionization tandem mass spectrometry

An LC-ESI-MS-MS method for the analysis of metabolites of four nitrofurans (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) in raw milk has been developed. The samples were achieved by hydrolysis of the protein-bound drug metabolites, derivatization with 2-nitrobenzaldehyd (2-NBA) and clean-up extraction liquid-liquid with ethyl acetate. LC separation was achieved by using a Phenomenex Luna C-18 column. The mass spectrometer operated in multiple reaction monitoring mode (MRM) with positive electro-spray interface (ESI). The method validation was done according to the criteria laid down in Commission Decision No. 2002/657 EC. The validation includes the determination of linearity, repeatability, within-laboratory reproducibility, accuracy, decision limit (CCalpha) and detection capability (CCbeta). The calibration curves were linear, with typical (R(2)) values higher than 0.991. The coefficient of variation (CV, %) was lower than 9.3% and the accuracy (RE, %) ranged from -9.0% to 7.0%. CV within-laboratory reproducibility was lower than 13%. The limits of decision (CCalpha) and detection capability (CCbeta) were 0.12-0.29 microg/kg and 0.15-0.37 microg/kg, thus below the minimum required performance limit (MRPL) set at 1 microg/kg by the UE. This validated method was successfully applied for the determination of nitrofuran metabolites in a large number of milk samples.     

Quantitation of paclitaxel and its two major metabolites using a liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid-liquid extraction and analyzed on a C(18) column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5-500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.