Liquid chromatographic tandem mass spectrometric determination of trandolapril in human plasma

A liquid chromatographic tandem mass spectrometric method for the determination of trandolapril in human plasma has been developed and fully validated. The article describes, in detail, the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using HLB Oasis solid-phase extraction cartridges. The chromatographic separation was performed on X-Terra C8 MS column (150 mm × 4.6 mm i.d., 5 μm particle size) using a mobile phase consisting of acetic acid 20 mM and triethylamine 4.3 mM/acetonitrile (40:60 (v/v)), pumped isocratically at 0.35 ml/min.

The analytes were detected using a micromass quattro micro triple quadrupole mass spectrometer with positive electrospray ionization in multiple reaction-monitoring (MRM) mode. Tandem mass spectrometric detection enabled the quantitation of trandolapril down to 2.0 ng/ml. Calibration graphs were linear (r better than 0.996, n = 9) in the concentration ranges 2.0–750 ng/ml and the intra- and inter-day R.S.D. values were less than 3.83 and 3.86% for trandolapril.     

Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.     

A validated liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma: application to a pharmacokinetic study

A new liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma has been developed and fully validated. The article describes in detail the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using liquid-liquid extraction with a mixture of 1-chlorobutane/isopropanol/ethyl acetate (88:2:10, (v/v/v)). The chromatographic separation was performed on a reversed-phase XTerrra MS C₈ column ( i.d.; 3.5 μm particle size) using a mobile phase consisting of 0.010 M ammonium formate (pH 7.8) and acetonitrile (35:65, (v/v)), pumped at a flow rate of 0.80 ml min⁻¹. The analytes were detected using a Finnigan LCQ advantage ion-trap mass spectrometer with positive electrospray ionization in selected reaction monitoring (SRM) mode. Tandem mass spectrometric detection enabled the quantitation of both compounds down to 0.10 ng ml⁻¹. Calibration graphs were linear (r better than 0.990, n=11), in concentration ranges 0.10 to 200 ng ml⁻¹ for mirtazapine demethylmirtazapine. The intra- and inter-day R.S.D. values were less than 14.8 and 16.6% for mirtazapine and demethylmirtazapine, respectively. The method was successfully applied to a kinetic study in order to assess the main pharmacokinetic parameters of mirtazapine and demethylmirtazapine.     

A rapid and highly sensitive method for the determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a pre-clinical pharmacokinetic study

A sensitive and specific liquid chromatography-positive electrospray ionization-tandem mass spectrometry method has been developed and validated for the determination of glimepiride (GPD) in human plasma. GPD and the internal standard (IS, glibenclamide) were extracted from a small aliquot of human plasma (200 microL) by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. The compounds were separated on a YMC Propack, C18, 4.6x50 mm column using a mixture of ammonium acetate buffer, acetonitrile and methanol (30:60:10, v/v) as mobile phase at 0.5 mL/min on an API 4000 Sciex mass spectrometer connected to an Agilent HPLC system. Method validation and pre-clinical sample analysis was performed as per FDA guidelines and the results met the acceptance criteria. GPD and IS were detected without any interference from human plasma matrix. The method was proved to be accurate and precise at linearity range of 0.02-100.00 ng/mL with a correlation coefficient of 0.999. The method was robust with a lower limit of quantitation of 0.02 ng/mL. Intra- and inter-day accuracies for GPD were 88.60-113.50 and 96.82-103.93%, respectively. The inter-day precision was better than 12.21%. This method enabled faster and reliable determination of GPD in a pre-clinical study.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantification of fexofenadine in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry using mosapride as internal standard

A rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of fexofenadine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 1-500 ng/mL for fexofenadine in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5% for fexofenadine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of penciclovir in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the determination of penciclovir in human plasma. The method involved simple, one‐step SPE procedure coupled with a C₁₈, 75 × 4.mm, 3µm column with a flow‐rate of 0.5 mL/min, and acyclovir was used as the internal standard. The Quattro Micro mass spectrometry was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Using 250 µL plasma, the methods were validated over the concentration range 52.555–6626.181 ng/mL, with a lower limit of quantification of 52.55 ng/mL. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of lisinopril in human plasma by high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine lisinopril in human plasma. Sample pretreatment involved a one-step protein precipitation with methanol of 0.1 mL plasma. Analysis was performed on an Inertsil ODS-3 column (2.1 × 50 mm i.d., 3 μm) with mobile phase consisting of methanol-water (containing 0.2% formic acid; 55:45, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via an electrospray ionization source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for lisinopril were obtained in the concentration range of 1.03-206 ng/mL (r(2) ≥ 0.99) with a lower limit of quantification of 1.03 ng/mL. The intra- and inter-day precisions (relative standard deviation) were not higher than 11%, and accuracy (relative error) was within ±6.8%, determined from quality control samples for lisinopril, which corresponded to the guidance of the Food and Drug Administration. The method described herein was fully validated and successfully applied to the pharmacokinetic study of lisinopril tablets in healthy male volunteers after oral administration.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of benazepril, benazeprilat and hydrochlorothiazide in human plasma

A new method was developed and fully validated for the quantitation of benazepril, benazeprilat and hydrochlorothiazide in human plasma. Sample pretreatment was achieved by solid-phase extraction (SPE) using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled to a single-quadrupole mass spectrometer (MS) with an electrospray ionization interface. The MS system was operated in selected ion monitoring (SIM) modes. HPLC was performed isocratically on a reversed-phase porous graphitized carbon (PGC) analytical column (2.1 x 125.0 mm i.d., particle size 5 microm). The mobile phase consisted of 55% acetonitrile in water containing 0.3% v/v formic acid and pumped at a flow rate of 0.15 ml min(-1). Chlorthalidone was used as the internal standard (IS) for quantitation. The assay was linear over a concentration range of 5.0-500 ng ml(-1) for all the compounds analysed, with a limit of quantitation of 5 ng ml(-1) for all the compounds. Quality control (QC) samples (5, 10, 100 and 500 ng ml(-1)) in five replicates from three different runs of analyses demonstrated intra-assay precision (coefficient of variation (CV) < or =14.6%), inter-assay precision (CV < or = 5.6%) and overall accuracy (relative error less than -8.0%). The method can be used to quantify benazepril, benazeprilat and hydrochlorothiazide in human plasma, covering a variety of pharmacokinetic or bioequivalence studies.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP(18) (4.6 x 50 mm, 3 microm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 --> m/z 158.2 for CLA and m/z 837.7 --> m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r(2)) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and -10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method for the quantification of lacidipine in human plasma

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantification of lacidipine in human plasma using its structural analogue, amlodipine, as internal standard (IS). The method involves a simple single-step liquid-liquid extraction with tert-butyl methyl ether. The analyte was chromatographed on an Xterra MS C(18) reversed-phase chromatographic column by isocratic elution with 20 mM ammonium acetate buffer-acetonitrile (10:90, v/v; pH 6) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 456.4 --> 354.4 and m/z 409.3 --> 238.3 were used to measure the analyte and the I.S., respectively. The chromatographic run time was 1.5 min and the weighted (1/x(2)) calibration curves were linear over the range 0.1-25 ng ml(-1). Lacidipine was sensitive to temperature in addition to light. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 50 and 100 pg ml(-1), respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<15%). The analyte was stable after three freeze-thaw cycles (deviation <15%). The average absolute recoveries of lacidipine and amlodipine (IS) from spiked plasma samples were 51.1 +/- 1.3 and 50.3 +/- 4.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of lacidipine.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of febuxostat in human plasma

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d₇‐febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C₁₈ column (4.6 × 100 mm, 5 µm) using acetonitrile–5 mm ammonium acetate–formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor‐to‐product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d₇‐febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0–5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra‐ and inter‐day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at ?20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

A high‐performance liquid chromatography–tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study

A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre‐purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar‐RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1–10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter‐ and intra‐batch precision was <6.1% and the accuracy was within 95.6–100.0%. The mean extraction recovery was 96.3%. Selectivity, matrix effect and stability were also validated. The method was applied to the comparative pharmacokinetic study of granisetron in Chinese healthy subjects. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric analysis of anastrozole in human plasma and its application to a pharmacokinetic study

A hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric method was developed for determination of anastrozole in human plasma. Anastrozole and irbesartan (internal standard) were extracted from human plasma with a mixture of dichloromethane and methyl tert-butyl ether (30:70, v/v). Analysis of the analytes was performed on a Luna HILIC column with the mobile phase of acetonitrile-10 m m ammonium formate (95:5, v/v) and detected by electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r(2) = 0.9992) over the concentration range of 0.10-50.0 ng/mL using 200 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.2-10.0% and -7.2-3.2%, respectively. The present method was applied successfully to the pharmacokinetic study of anastrozole after oral administration of 1 mg anastrozole tablet to healthy male volunteers.     

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5 > 166.1 for itopride and m/z 342.3 > 111.6 for IS, respectively. Analytes were chromatographed on an YMC C₁₈ reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r² = 0.9999) over the studied range (0.5-1000 ng mL⁻¹) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma using liquid-liquid extraction

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 408-235 for sitagliptin and m/z 310-148 for the internal standard. The assay exhibited a linear dynamic range of 0.1-250 ng/mL for sitagliptin in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 x 3.0 mm i.d., particle size 5 microm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml(-1) for all compounds analyzed. The limit of quantitation was 5 ng ml(-1) for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml(-1)) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of < or = 9.1%, an inter-assay precision of < or = 6.0% and an overall accuracy (relative error) of < 4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies.