Mallika Sanyal

An accurate, rapid and sensitive determination of tramadol and its active metabolite O-desmethyltramadol in human plasma by LC-MS/MS.

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous determination of tramadol and its active metabolite, O-desmethyltramadol in human plasma is developed using propranolol as internal standard (IS). The analytes and IS were extracted from 200 microL aliquots of human plasma via protein precipitation using acetonitrile. Chromatographic separation was achieved in a run time of 2.0 min on an Aquasil C18 (100mm x 2.1mm, 5microm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method was fully validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. A linear dynamic range was established from 1.0 to 600.0ng/mL for tramadol and 0.5-300.0ng/mL for O-desmethyltramadol. The method was successfully applied to a bioequivalence study of 200mg tramadol tablet formulation in 27 healthy Indian male subjects under fasting condition.

Liquid chromatography tandem mass spectrometry assay for the simultaneous determination of venlafaxine and O-desmethylvenlafaxine in human plasma and its application to a bioequivalence study

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous determination of venlafaxine (VEN) and its active metabolite, O-desmethylvenlafaxine (ODV) in human plasma was developed using nadolol as internal standard (IS). The analytes and IS were extracted from 200 microl aliquots of human plasma via protein precipitation using 0.43% formic acid in acetonitrile and separated on a Hypurity cyano (50 mm x 4.6 mm, 5 microm) column. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM) and positive ion mode. The precursor to product ion transitions monitored for VEN, ODV and IS were m/z 278.3-->58.1, 264.3-->58.1 and 310.4-->254.1, respectively. The total chromatographic runtime was 3 min with retention time for VEN, ODV and IS at 1.93, 1.50 and 1.29 min, respectively. The method was fully validated for its sensitivity, accuracy and precision, linearity, recovery, matrix effect, dilution integrity and stability studies. The linear dynamic range of 2.0-500 ng/ml was established for both VEN and ODV with mean correlation coefficient (r), 0.9994 and 0.9990, respectively. The intra-batch and inter-batch precision (%CV) in three validation batches across five concentration levels (LLOQ, LQC, MQC, HQC and ULOQ) was less than 12.6% for both the analytes. The accuracy determined at these levels was within -9.8 to +3.9% in terms of %bias. The method was successfully applied to a bioequivalence study of 150 mg venlafaxine extended release capsule formulation in 22 healthy Indian male subjects under fed condition.

High throughput and sensitive determination of trazodone and its primary metabolite, m-chlorophenylpiperazine, in human plasma by liquid chromatography–tandem mass spectrometry

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine (mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm x 2.1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/z 372.2-->176.2, 197.2-->118.1 and 470.5-->274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0-3000.0 ng/mL for TRZ and 0.2-60.0 ng/mL for mCPP was evaluated with mean correlation coefficient (r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was < or =8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.

A high throughput and selective method for the estimation of valproic acid an antiepileptic drug in human plasma by tandem LC-MS/MS.

A high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of valproic acid, an antiepileptic drug, in human plasma is described. It is a rapid and sensitive isocratic reversed-phase liquid chromatography-tandem mass spectrometric method equipped with turbo ion spray (TIS) source, operating in the negative ion and pseudo selective reaction monitoring (SRM) acquisition mode to quantify valproic acid. The extraction of valproic acid and hydrochlorothiazide (IS) from the plasma involved sample treatment with phosphoric acid followed by solid-phase extraction using Waters hydrophilic-lipophilic balance (HLB) cartridge giving extracts free from endogenous interferences. Sample preparation by this method yielded very good and consistent mean recoveries of 99.73 and 74.47% for valproic acid and IS, respectively. The method was linear over the dynamic range of 2.0-200.0mug/ml (covering entire therapeutic range) with a correlation coefficient r>/=0.9989. The coefficient of variance (CV, %) was 7.03% at 2.0mug/ml (LLOQ). This method was fully validated for its accuracy, precision, recovery and matrix effect especially because the pattern of elution of all the analytes may appear as flow injection type. The analyte stability was examined under conditions mimicking the sample storage, handling and analysis procedures. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 500mg formulations.

Analysis of second-generation antidepressant drug, sertraline and its active metabolite, N-desmethyl sertraline in human plasma by a sensitive and selective liquid chromatography–tandem mass spectrometry method

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of sertraline (SER) and its primary metabolite, N-desmethyl sertraline (NDS) in human plasma is developed and validated. The analytes and the internal standard-fluoxetine were extracted from 300 microL aliquots of human plasma via liquid-liquid extraction in methyl tert-butyl ether. Chromatographic separation was achieved in a run time of 2.5 min on a Betasil C8 column (100 mm x 2 .1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for SER, NDS and IS were m/z 306.2-->159.0, 292.1-->159.0 and 310.6-->148.4, respectively. The method was fully validated for its sensitivity, selectivity, linearity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5-150 ng/mL was established for both the analytes with mean correlation coefficient (r) of 0.9993 and 0.9980, respectively. The intra-batch and inter-batch precision (%CV) across five quality control levels was <or=10.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100mg sertraline tablet formulation in 32 healthy Indian male subjects under fasting condition.

Simultaneous determination of simvastatin and simvastatin acid in human plasma by LC-MS/MS without polarity switch: application to a bioequivalence study.

A simple, specific and sensitive LC-MS/MS assay for simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite, simvastatin acid (SVA) in human plasma was developed using a statin analog as internal standard (IS). The method was validated over a dynamic linear range of 0.20-100.00 ng/mL for SV and 0.10-50.00 ng/mL for SVA with correlation coefficient r > or = 0.9987 and 0.9989, respectively. The analytes and IS were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction using methyl tert-butyl ether and separated through an Aquasil C18 column (100 mm x 2.1 mm, 5 microm). Detection of analytes and IS was done by MS/MS with a turbo ion spray interface operating in positive ion and selective reaction monitoring acquisition mode. The total chromatographic run time was 3.0 min. Flash freezing of the aqueous phase was an added advantage during liquid-liquid extraction, which considerably reduced time and labour. The method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect. The method was successfully used for bioequivalence study of 40 mg SV tablet formulation in 12 human subjects under fasting condition.

Challenges in the simultaneous quantitation of sumatriptan and naproxen in human plasma: application to a bioequivalence study.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of sumatriptan and naproxen in human plasma using naratriptan and indomethacin as the internal standards (ISs). The plasma samples were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample. Chromatography was carried out on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column under isocratic conditions using a mobile phase consisting of methanol-acetonitrile-4.0mM ammonium acetate (70:10:20, v/v/v). The precursor→product ion transition for both the analytes and ISs was monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a wide dynamic concentration range of 0.050-100 ng/mL for sumatriptan and 0.050-100 μg/mL for naproxen. Matrix effect was assessed by post-column analyte infusion and the extraction recovery was >95.0% across four quality control levels for both the analytes. Stability was evaluated under different conditions including bench top, processed sample, freeze and thaw and long term. The method was applied to support a bioequivalence study of 85 mg sumatriptan+500 mg naproxen sodium fixed dose formulation in 28 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 123 incurred samples.

Quantitation of donepezil and its active metabolite 6-O-desmethyl donepezil in human plasma by a selective and sensitive liquid chromatography–tandem mass spectrometric method

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous determination of donepezil (D) and its pharmacologically active metabolite, 6-O-desmethyl donepezil (6-ODD) in human plasma is developed using galantamine as internal standard (IS). The analytes and IS were extracted from 500 microL aliquots of human plasma via solid-phase extraction (SPE) on Waters Oasis HLB cartridges. Chromatographic separation was achieved in a run time of 6.0 min on a Waters Novapak C18 (150 mm x 3.9 mm, 4 microm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for D, 6-ODD and IS were at m/z 380.1-->91.2, 366.3-->91.3 and 288.2-->213.2, respectively. The method was fully validated for its selectivity, interference check, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, cross-specificity, stability and dilution integrity. A linear dynamic range of 0.10-50.0 ng mL(-1) for D and 0.02-10.0 ng mL(-1) for 6-ODD was evaluated with mean correlation coefficient (r) of 0.9975 and 0.9985, respectively. The intra-batch and inter-batch precision (%CV, coefficient of variation) across five quality control levels was less than 7.5% for both the analytes. The method was successfully applied to a bioequivalence study of 10mg donepezil tablet formulation in 24 healthy Indian male subjects under fasting condition.

Application of a validated ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of darunavir in human plasma for a bioequivalence study in Indian subjects.

A simple, precise and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the quantification of darunavir, a protease inhibitor, using darunavir-d9 as internal standard (IS). The method involved liquid-liquid extraction of darunavir and IS in methyl-tert-butyl ether from 50 μL human plasma. The chromatographic separation was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1mm, 1.7 μm particle size) analytical column under gradient conditions, in a run time of 1.6 min. The precursor → product ion transitions for darunavir (m/z 548.1 → 392.0) and IS (m/z 557.1 → 401.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was extensively validated for its selectivity, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The linearity of the method was established in the concentration range of 1.0-5000 ng/mL. The mean relative recovery for darunavir (100.8%) and IS (89.8%) from spiked plasma samples was consistent and reproducible. The application of this method for routine measurement of plasma darunavir concentration was demonstrated by a bioequivalence study conducted in 40 healthy Indian subjects for a 600 mg tablet formulation along with 100mg ritonavir as booster under fast and fed conditions. To demonstrate the reproducibility in the measurement of study data, an incurred sample reanalysis was done with 400 subject samples and the % change in concentration was within ± 12%.

Development and validation of a selective and sensitive LC–MS/MS method for determination of cycloserine in human plasma: Application to bioequivalence study

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20-30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.