Challenges in Simultaneous Determination of Hydrochlorothiazide and Ramipril in Human Plasma: Application to a Bioequivalence Study.

An isotope dilution selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of hydrochlorothiazide (HCTZ) and ramipril in human plasma through a new concept of periodical polarity switching. Extraction of HCTZ, ramipril and their deuterated analogs as internal standards (ISs) was carried out from 150 µL of human plasma by solid-phase extraction method. Chromatographic separation of analytes was performed on Hypurity C18 (150 mm × 4.6 mm, 5 µ) column under gradient conditions with methanol:0.2% (v/v) formic acid in water as the mobile phase. The method was validated over a concentration range of 0.750-300 ng/mL for HCTZ and 0.125-80.0 ng/mL for ramipril. The mean extraction recovery for analytes and ISs were >(86.0%), consistent across all four QC levels. The challenges to evaluate matrix effect and continuous reproducibility of method during long analytical run was studied and resolved. Processed samples, freeze-thaw, long-term and whole blood stability were evaluated for both the analytes. The method was applied to support a bioequivalence study of 25 mg of HCTZ and 5 mg of ramipril tablet formulation in nine healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 42 incurred samples.

Novel bio analytical method development, validation and application for simultaneous determination of nebivolol and S-amlodipine in human plasma using ultra performance liquid chromatography-tandem mass spectrometry.

A sensitive and specific ultra-performance liquid chromatography tandem mass spectrometric (UPLC-MS/MS) method has been developed, validated and applied for the assay of Nebivolol and S-amlodipine in human plasma. Sample extraction was carried out through hybrid extraction method from 250 µL of human plasma sample. Linearity of the method was (r ≥ 0.9996) was found to be dynamic for both the analytes over concentration range of 25.0-4000 pg/mL. Chromatographic separation was achieved on UPLC column {Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 micrometer)} with the mobile phase composition of 0.1% (v/v) formic acid in 5 mM Ammonium formate in water-acetonitrile (20:80, %v/v). Analytes Stability was assured under different requisite conditions in human plasma, reconstitution solution and diluents. Inter and intra-day assay precision and relative error (accuracy) were within ±5% for both analytes. The method was applied and reproduced to support a pharmacokinetic study of 5 mg Nebivolol (NEB) and 2.5 mg S-amlodipine (LAM) tablet on 9 healthy subjects.

The simultaneous UPLC-MS/MS determination of emerging drug combination; candesartan and chlorthalidone in human plasma and its application.

A novel, precise, sensitive and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of a novel drug combination, candesartan (CAN) and chlorthalidone (CHL), in human plasma. Chromatographic separation was achieved on Waters Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 µm). Mobile phase consisting of 1 mm ammonium acetate in water-acetonitrile (20:80 v/v) was used. The total chromatographic runtime was 1.9 min with retention times for CAN and CHL at 0.7 and 1.1 min respectively. Ionization and detection of analytes and internal standards was performed on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and negative ionization mode. Quantitation was done to monitor protonated precursor → product ion transition of m/z 439.2 → 309.0 for CAN, 337.0 → 189.8 for CHL and 443.2 → 312.1 for candesartan D4 and 341.0 → 189.8 for chlorthalidone D4. The method was validated over a wide dynamic concentration range of 2.0-540.0 ng/mL for candesartan and 1.0-180.0 ng/mL for chlorthalidone. The validated method was successfully applied for the assay of CAN and CHL in healthy volunteers.

Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography-tandem mass spectrometry method in human plasma.

The present study describes a simple, reliable and reproducible liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of allopurinol and its active metabolite, oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation (PPT) of 100 µL plasma sample with 1.0% formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold (150 mm×4.6 mm, 5 µm) column using 0.1% formic acid-acetonitrile (98:2, v/v) as the mobile phase. Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000 ng/mL for allopurinol and 80.0-8000 ng/mL for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300 mg allopurinol tablet formulation in healthy subjects.

An improved LC-MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study☆.

A highly sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of alverine (ALV) and its active metabolite, para hydroxy alverine (PHA), in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18 (150 mm×3.9 mm, 5 µm) column with a mobile phase consisting of acetonitrile and 10 mM ammonium formate (65:35, v/v). Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0-15,000 pg/mL for ALV and 30.0-15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision (% CV) ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect, expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Application of an LC-MS/MS method for reliable determination of amodiaquine, N-desethylamodiaquine, artesunate and dihydroartemisinin in human plasma for a bioequivalence study in healthy Indian subjects.

A sensitive and high throughput bioanalytical method has been developed for reliable determination of amodiaquine (AQ), N-desethylamodiaquine (DEAQ), artesunate (AS) and dihydroartemisinin (DHA) in human plasma by LC-MS/MS. The method employs a solid phase extraction procedure without an evaporation step and with optimum use of organic solvents to circumvent degradation of artemisinin derivatives. The analytes and their deuterated internal standards (ISs) were analyzed on Hypersil Gold (100 mm × 4.6mm, 5 µm) column using acetonitrile and 2.0mM ammonium formate (pH 2.50) in 80:20 (v/v) ratio as the mobile phase. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was used to detect and quantify the analytes. The method was established over the concentration range of 0.250-30.0 ng/mL, 1.50-180 ng/mL, 2.00-600 ng/mL and 5.00-1400 ng/mL for AQ, DEAQ, AS and DHA respectively using 250 µL human plasma. The intra-day and inter-day accuracy and precision (% CV) across quality controls varied from 93.3-105.0% and 1.7-8.3 respectively for all the analytes. The stability was assessed in whole blood as well as in plasma samples under different conditions. All four analytes were stable in whole blood up to 2h on melting ice. The long term stability in plasma was ascertained up to 90 days. IS-normalized matrix factors ranged from 0.988-1.023 for all the analytes. The method was successfully applied to a bioequivalence study using 50mg artesunate and 135 mg amodiaquine fixed dose formulation in 14 healthy subjects.

Systematic evaluation of matrix effect and cross-talk-free method for simultaneous determination of zolmitriptan and N-desmethyl zolmitriptan in human plasma: a sensitive LC-MS/MS method validation and its application to a clinical pharmacokinetic study.

The objective of the present work was to carry out systematic evaluation to eliminate matrix effect owing to plasma phospholipids as observed during sample preparation and to develop a cross-talk-free sensitive, selective and rapid bioanalytical method for the simultaneous determination of zolmitriptan (ZT) and N-desmethyl zolmitriptan (DZT) in human plasma by liquid chromatography-tandem mass spectrometry using naratriptan as internal standard (IS). The analytes and IS were quantitatively extracted from 200 µL human plasma by solid phase extraction. No cross-talk was found between ZT and DZT having identical product ions. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The total chromatographic run time was 2.5 min. The method was fully validated for sensitivity, selectivity, specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity and stability studies. The method was validated over a dynamic concentration range of 0.1-15 ng/mL for ZT and DZT. The method was successfully applied to a bioequivalence study of 2.5 mg ZT tablet formulation in 18 healthy Indian male subjects under fasting conditions. Assay reproducibility was assessed by reanalysis of 62 incurred samples.

Liquid chromatography--tandem mass spectrometry method for simultaneous determination of albendazole and albendazole sulfoxide in human plasma for bioequivalence studies.

An improved high performance liquid chromatography--tandem mass spectrometry (LC-MS/MS) method has been developed for sensitive and rapid determination of albendazole (ABZ) and its active metabolite, albendazole sulfoxide (ABZSO), in the positive ionization mode. The method utilized solid phase extraction (SPE) for sample preparation of the analytes and their deuterated internal standards (ISs) from 100 µL human plasma. The chromatography was carried out on Hypurity C18 column using acetonitrile-2.0 mM ammonium acetate, pH 5.0 (80:20, v/v) as the mobile phase. The assay exhibited a linear response over the concentration range of 0.200-50.0 ng/mL for ABZ and 3.00-600 ng/mL for ABZSO. The recoveries of the analytes and ISs ranged from 86.03%-89.66% and 89.85%-98.94%, respectively. Matrix effect, expressed as IS-normalized matrix factors, ranged from 0.985 to 1.042 for the both analytes. The method was successfully applied for two separate studies in healthy subjects using single dose of 400 mg conventional tablets and 400 mg chewable ABZ tablets, respectively.

Application of UPLC-MS/MS for separation and quantification of 3α-Hydroxy Tibolone and comparative bioavailability of two Tibolone formulations in healthy volunteers.

A novel, fast, sensitive and robust method based on ultra-performance liquid chromatography coupled to atmospheric pressure electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) has been developed to separate two Tibolone stereoisomers i.e., 3α-Hydroxy Tibolone and 3ß-Hydroxy Tibolone and to quantify 3α-Hydroxy Tibolone using p-toulenesulfonyl isocyanate (PTSI) as a derivatizing reagent in human plasma. 3α-Hydroxy Tibolone-13CD3 was used as an internal standard (IS). The analyte and IS were extracted from human plasma by liquid-liquid extraction using ethyl acetate. Extracted samples were analyzed by UPLC-ESI-MS/MS. Chromatography was performed using binary gradient on UPLC analytical column. A linear calibration curve over the range of 0.100-35.000 ng/mL was obtained and lower limit of quantification (LLOQ) was 0.100 ng/mL demonstrating acceptable accuracy and precision. This method was successfully applied to a pharmacokinetic study in order to compare a test Tibolone 2.5 mg formulation vs. a reference 2.5 mg Tibolone tablet formulation in 50 post-menopausal/surgical menopause female human volunteers under fasting conditions. It is concluded that test formulation of Tibolone is bioequivalent to reference formulation of Tibolone.

A single, selective and simple validated method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma by liquid chromatography-tandem mass spectrometry.

A single, simple and selective method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma was validated using triamterine and hydrochlorothiazide (13)C,d2 as internal standard. The compounds were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mm ammonium acetate pH 3.0 and acetonitrile (30:70, v/v) and detected by tandem mass spectrometry with positive/negative ion mode. The analytes and internal standards were extracted from plasma using simple solid phase extraction. The ion transitions recorded in multiple reaction monitoring mode were m/z 230.1 → 116.0 for amiloride, m/z 254.1 → 237.1 for internal standard, triamterine in positive mode and m/z 296.1 → 204.9 for hydrochlorothiazide, m/z 299.2 → 205.8 for internal standard, hydrochlorothiazide (13)C,d2 in negative ion mode. Linearity in plasma was observed over the concentration range 0.1-10 ng/mL for amiloride and 5.0-500.0 ng/mL for hydrochlorothiazide. The mean recovery was 41.1 and 81.5% for amiloride and hydrochlorothiazide respectively. The coefficient of variation of the assay was less than 11.2 and 5.2% for amiloride and hydrochlorothiazide, respectively, and the accuracy was 89.0-98.1 and 96.6-102.9% for amiloride and hydrochlorothiazide, respectively. The validated method can be applied to the pharmacokinetic study of amiloride and hydrochlorothiazide.

A simple, selective and rapid validated method for estimation of anastrazole in human plasma by liquid chromatography-tandem mass spectrometry and its application to bioequivalence study.

A rapid, simple and specific method for estimation of anastrazole in human plasma was validated using letrozole as internal standard. The analyte and internal standard were extracted from plasma using simple solid-phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12 : 88, v/v) and detected by tandem mass spectrometry in positive ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 294.1 --> 225.1 for anastrazole and m/z 286.1 --> 217.1 for internal standard. Linearity in plasma was observed over the concentration range 0.3-30 ng/mL for anastrazole. The mean recovery for anastrazole was 83.7% with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8% and the accuracy was 96.1-102.2%. The validated method was applied to a bioequivalence study of 1 mg anastrazole tablet in healthy human volunteers.

Highly sensitive and rapid LC-ESI-MS/MS method for the simultaneous quantification of uroselective alpha1-blocker, alfuzosin and an antimuscarinic agent, solifenacin in human plasma.

An accurate, selective and sensitive bioanalytical method has been developed and validated for the simultaneous quantification of alfuzosin and solifenacin in human plasma using propranolol as internal standard (IS). The analytes and IS were extracted in methyl tert-butyl ether, separated on Hypurity C8 column and detected by tandem mass spectrometry with a turbo ion spray interface. The method had a chromatographic run time of 3.0 min and linear calibration curves over the concentration range of 0.25-25 ng/mL for alfuzosin and 0.6-60 ng/mL for solifenacin. The intra- and inter-day accuracy and precision (%CV) evaluated at four quality control levels were within 88.2-106.4% and 0.9-7.7% respectively. The absolute recovery from spiked plasma samples was 71.8% for alfuzosin and 93.1% for solifenacin. Stability of alfuzosin and solifenacin was assessed under different storage conditions. The validated method was successfully employed for bioavailability study after oral administration of 10 mg of alfuzosin hydrochloride and 5mg of solifenacin succinate tablet formulations in eight healthy volunteers under fed condition.

Sensitive and rapid method to determine trimetazidine in human plasma by liquid chromatography/tandem mass spectrometry.

A simple, sensitive, and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method, using electrospray ionization, was developed and validated to quantify trimetazidine in human plasma using propranolol hydrochloride as an internal standard (IS). Samples were prepared by solid-phase extraction and analyzed without drying and reconstitution. The analyte and IS were chromatographed on a C18 reversed-phase column under isocratic conditions using 2 mM ammonium acetate (pH 3.5)-acetonitrile (40 + 60, v/v) as the mobile phase with a run time of 2.0 min. Quantitation was done on a triple-quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring mode to detect parent --> product ion (m/z 267.2 --> 181.4) transition. The method was validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, and stability. Linearity in plasma was observed over the concentration range of 1.5-300 ng/mL. Lower limit of quantification achieved was 1.5 ng/mL with precision < 10% using 10 microL injection volume. The mean relative recovery of analyte (97.36%) and IS (99.93%) was consistent and reproducible. Interbatch and intrabatch precision was < 8.0% and the accuracy determined was within +/- 8% in terms of relative error.

HPLC-ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma.

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.

Liquid chromatography tandem mass spectrometry method for simultaneous determination of antidiabetic drugs metformin and glyburide in human plasma.

A simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantitation of antidiabetic drugs metformin and glyburide in human plasma using glimepiride as internal standard (IS). After acidic acetonitrile-induced protein precipitation of the plasma samples, metformin, glyburide and IS were chromatographed on reverse phase C18 (50 mm x 4.6 mm i.d., 5 microm) analytical column. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 3.5 min and calibration curves were linear over the concentration range of 20-2500 ng/ml for metformin and 5-500 ng/ml for glyburide. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision, dilution integrity and stability studies. The recoveries obtained for the analytes and IS (>or=69%) were consistent and reproducible. Inter-batch and intra-batch coefficient of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 8%. The accuracy determined at these levels was within +/-8% in terms of relative error (RE). The method was applied to a bioequivalence study of 500 mg metformin and 5mg of glyburide tablet after oral administration to 28 healthy human subjects under condition of fasting.

High throughput LC-MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma.

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mmx3.9 mm i.d., 5 microm particle size) column using 5 microL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25-3000 ng/mL for 3TC, 20-2000 ng/mL for d4T and 50-5000 ng/mL for NVP. The absolute recoveries for analytes (>or=86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within +/-8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC)+12 (d4T)+100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.

Electrospray ionization LC-MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study.

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify griseofulvin in human plasma using propranolol hydrochloride as internal standard (IS). Samples were prepared using solid phase extraction and analysed without drying and reconstitution. The analytes were chromatographed on Hypersil, hypurity C18 reverse phase column under isocratic conditions using 0.05% formic acid in water:acetonitrile (30:70, v/v) as the mobile phase. Total chromatographic run time was 3.0 min. Quantitation was done on a triple quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring (MRM) mode to detect parent-->product ion transition for analyte and IS. The method was validated for sensitivity, matrix effect, accuracy and precision, linearity, recovery and stability studies. Linearity in plasma was observed over the concentration range 20-3000 ng/mL for griseofulvin. Lower limit of quantification (LLOQ) achieved was 20 ng/mL with precision (CV) less than 10% using 5 microL injection volume. The absolute recovery of analyte (87.36%) and IS (98.91%) from spiked plasma samples was consistent and reproducible. Inter-batch and intra-batch coefficients of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 7.5%. The accuracy determined at these levels was within +/-4.2% in terms of relative error. The method was applied to a pilot bioequivalence study of 500 mg griseofulvin tablet in six healthy human subjects under fed condition.

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the estimation of nicorandil in human plasma.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of nicorandil in human plasma. Nicorandil was extracted from human plasma using solid-phase extraction technique. Imipramine was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid 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 for a linear range of 1.0-500.0 ng/mL with a correlation coefficient of > or =0.9993. The intra-run and inter-run precision and accuracy was within 10.0%. The overall recovery for nicorandil was 63.81%. The total run time was just 3.0 min.

Rapid and sensitive liquid chromatography-mass spectrometry method for determination of ropinirole in human plasma.

A rapid and robust liquid chromatography-mass spectrometry (LC-MS/MS) method was developed for non-ergoline dopamine D(2)-receptor agonist, ropinirole in human plasma using Es-citalopram oxalate as an internal standard. The method involves solid phase extraction from plasma, reversed-phase simple isocratic chromatographic conditions and mass spectrometric detection that enables a detection limit at picogram levels. The proposed method was validated with linear range of 20-1,200 pg/ml. The extraction recoveries for ropinirole and internal standard were 90.45 and 65.42%, respectively. The R.S.D.% of intra-day and inter-day assay was lower than 15%. For its sensitivity and reliability, the proposed method is particularly suitable for pharmacokinetic studies.

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.