Selective and rapid determination of raltegravir in human plasma by liquid chromatography-tandem mass spectrometry in the negative ionization mode.

A selective and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 µL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C18 (100 mm×4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1→316.1 for raltegravir and m/z 446.1→319.0 for IS. The linearity of the method was established in the concentration range of 2.0-6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

High-sensitivity simultaneous liquid chromatography-tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma.

A sensitive and simultaneous liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of ethinyl estradiol and levonorgestrel. The analytes were extracted with methyl-tert-butyl ether: n-hexane (50:50, v/v) solvent mixture, followed by dansyl derivatization. The chromatographic separation was performed on a Kinetex C18 (50 mm×4.6 mm, 2.6 µm) column with a mobile phase of 0.1% (v/v) formic acid in water and acetonitrile in gradient composition. The mass transitions were monitored in electrospray positive ionization mode. The assay exhibited a linear range of 0.100-20.0 ng/mL for levonorgestrel and 4.00-500 pg/mL for ethinyl estradiol in human plasma. A run time of 9.0 min for each sample made it possible to analyze a throughput of more than 100 samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic and bioequivalence studies.

Reliable LC-MS/MS assay for the estimation of rilpivirine in human plasma: application to a bioequivalence study and incurred sample reanalysis.

A simple, precise, and rapid stable isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of rilpivirine, a non-nucleoside reverse transcriptase inhibitor in human plasma. Rilpivirine and its deuterated analogue, rilpivirine-d6, used as an internal standard (IS) were quantitatively extracted by liquid-liquid extraction with methyl-tert-butyl ether and diethyl ether solvent mixture from 50 µL plasma. The chromatography was achieved on Gemini C18 (150 × 4.6 mm, 5 µm) analytical column in a run time of 2.2 min. The precursor → product ion transitions for rilpivirine (m/z 367.1 → 128.0) and IS (m/z 373.2 → 134.2) were monitored on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL. The mean extraction recovery for rilpivirine (94.9%) and IS (99.9%) from spiked plasma samples was consistent and reproducible. The IS-normalized matrix factors for rilpivirine ranged from 0.98 to 1.02 across three quality controls. Bench top, freeze-thaw, wet extract, and long-term stability of rilpivirine was examined in spiked plasma samples. The application of the method was demonstrated by a bioequivalence study with 25 mg rilpivirine tablet formulation in 40 healthy subjects. The assay reproducibility was shown by reanalysis of 200 study samples and the % change in the concentration of repeat values from the original values was within ±15%.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography-tandem mass spectrometry in the negative ionization mode / 药物分析学报

A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C18 (100 mm ? 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

Sensitive LC-MS/MS-ESI method for simultaneous determination of montelukast and fexofenadine in human plasma: application to a bioequivalence study.

A rapid, simple, sensitive and selective LC-MS/MS method was developed and validated for simultaneous quantification of montelukast (MT) and fexofenadine (FF) in human plasma (200 µL) using montelukast-d6 (MT-d6 ) and fexofenadine-d10 (FF-d10 ), respectively as an internal standard (IS) as per the US Food and Drug Administration guidelines. The chromatographic resolution was achieved on a Chromolith RP18e column using an isocratic mobile phase consisting of 20 mm ammonium formate-acetonitrile (20:80, v/v) at flow rate of 1.2 mL/min. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. The total run time of analysis was 4 min and elution of MT, FF, MT-d6 and FF-d10 occurred at 2.5, 1.2, 2.4 and 1.2 min, respectively. The standard curve found to be linear in the range 2.00-1000 ng/mL with a coefficient of correlation of ≥0.99 for both the drugs. The intra- and inter-day accuracy and precision values for MT and FF met the acceptance as per FDA guidelines. MT and FF were found to be stable in a battery of stability studies viz., bench-top, auto-sampler and repeated freeze-thaw cycles. The validated assay was applied to an oral bioequivalence study in humans.

Parallel achiral-chiral determination of oxybutynin, N-desethyl oxybutynin and their enantiomers in human plasma by LC-MS/MS to support a bioequivalence trial.

A parallel achiral and chiral determination of oxybutynin, its pharmacologically active metabolite N-desethyl oxybutynin and their enantiomers in human plasma is described using LC-MS/MS. Both the methods were developed and validated using deuterated analogues as internal standards. Achiral analysis of racemic oxybutynin and N-desethyl oxybutynin was carried out on Phenomenex Gemini C18 (150mm×4.6mm, 5µm) column under isocratic conditions using acetonitrile-5.0mM ammonium acetate, pH 4.0 (90:10, v/v) as the mobile phase. Separation of (S)- and (R)-enantiomers of the analytes was performed on Phenomenex Lux Amylose-2 (150mm×4.6mm, 3µm) chiral column using a mixture of solvent A [acetonitrile:10mM ammonium bicarbonate, 80:20 (v/v)] and solvent B [2-propanol:methanol, 50:50 (v/v)] in 20:80 (v/v) ratio as the mobile phase. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate-diethyl ether-n-hexane solvent mixture. A linear range was established from 0.025 to 10.0ng/mL and 0.25 to 100ng/mL for the enantiomers of oxybutynin and N-desethyl oxybutynin respectively. The extraction recovery varied from 96.0 to 105.1%, while the IS-normalized matrix factors ranged from 0.96 to 1.07 for all the enantiomers. The validated method was applied for a pilot bioequivalence study with 5mg oxybutynin tablet formulation in 8 healthy subjects. The pharmacokinetic profiles showed that the plasma concentration of (R)-oxybutynin was lower than that of (S)-oxybutynin, while a reverse trend was observed for the enantiomers of N-desethyl oxybutynin. The reproducibility in the measurement of study data was demonstrated by reanalysis of 20 incurred samples.

Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(+)-metoprolol in human plasma by chiral LC-ESI-MS/MS.

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 µm) column. Solid phase extraction of (S)-(-)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 µL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor→product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(t)-metoprolol in human plasma by chiral LC-ESI-MS/MS / 药物分析学报

A selective, sensitive and high throughput liquid chromatography-tandem mass spectro-metry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 mm) column. Solid phase extraction of (S)-(-)- and (R)-(t)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

Simultaneous analysis of oxybutynin and its active metabolite N-desethyl oxybutynin in human plasma by stable isotope dilution LC-MS/MS to support a bioequivalence study.

An isotope dilution high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of oxybutynin and its pharmacologically active metabolite N-desethyl oxybutynin in human plasma. Extraction of oxybutynin, its metabolite and their deuterated analogs as internal standards (ISs) from 300 µL human plasma was carried out by liquid-liquid extraction with methyl tert-butyl ether-ethyl acetate solvent mixture. Chromatographic separation of analytes was performed on Cosmosil C18 (150 mm × 4.6 mm, 5 µm) column under isocratic conditions with acetonitrile-1.0mM ammonium acetate (90:10, v/v) as the mobile phase. Six endogenous plasma phospholipids (496.3/184.0, 524.3/184.0, 758.5/184.0, 786.5/184.0, 806.5/184.0 and 810.5/184.0) were monitored to determine the extraction efficiency under different extraction conditions. The precursor→product ion transition for both the analytes and ISs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a concentration range of 0.050-10.0 ng/mL for oxybutynin and 0.500-100 ng/mL for N-desethyl oxybutynin. The mean extraction recovery for analytes (80.4%) and ISs (76.9%) was consistent across five QC levels. Bench top, wet and dry extract, freeze-thaw and long term stability was evaluated for both the analytes. The method was applied to support a bioequivalence study of 5mg tablet formulation in 74 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 344 incurred samples.

Reliable and sensitive determination of dutasteride in human plasma by liquid chromatography-tandem mass spectrometry.

An accurate and precise method was developed and validated using LC-MS/MS to quantify dutasteride in human plasma. The analyte and dutasteride-13C6 as internal standard (IS) were extracted from 300 µL plasma volume using methyl tert-butyl ether-n-hexane (80:20, v/v). Chromatographic analysis was performed on a Gemini C18 (150 × 4.6 mm, 5 µm) column using acetonitrile-5 mm ammonium formate, pH adjusted to 4.0 with formic acid (85:15, v/v) as the mobile phase. Tandem mass spectrometry in positive ionization mode was used to quantify dutasteride by multiple reaction monitoring. The entire data processing was done using Watson LIMS(TM) software, which provided excellent data integrity and high throughput with improved operational efficiency. The calibration curve was linear in the range of 0.1-25 ng/mL, with intra-and inter-batch values for accuracy and precision (coefficient of variation) ranging from 95.8 to 104.0 and from 0.7 to 5.3%, respectively. The mean overall recovery across quality controls was ≥95% for the analyte and IS, while the interference of matrix expressed as IS-normalized matrix factors ranged from 1.01 to 1.02. The method was successfully applied to support a bioequivalence study of 0.5 mg dutasteride capsules in 24 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 103 incurred samples.

Validation of an LC-MS/MS method for the quantitative determination of mavoglurant (AFQ056) in human plasma.

A simple, sensitive, and selective liquid chromatography/tandem mass spectrometry method was validated for the identification and quantification of mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150 × 4.6 mm, 5 µm) column at 40 ± 0.5 °C with a mobile phase consisting of acetic acid in water (0.1%, v/v)/methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometry, operating with electrospray ionization in positive ion mode and applying multiple reaction monitoring. The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant, and the anticoagulant effect was tested by lithium heparin and K(3)EDTA. The method was successfully cross-validated between two bioanalytical sites. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area at the retention time of mavoglurant in zero samples was less than 20% compared with the mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 and 2,500 ng/mL.

Simultaneous quantification of prodrug oseltamivir and its metabolite oseltamivir carboxylate in human plasma by LC-MS/MS to support a bioequivalence study.

A simple, precise and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of oseltamivir and oseltamivir carboxylate, a neuraminidase inhibitor, using their deuterated analogs as internal standards (ISs). The method involved solid phase extraction of the analytes and ISs from 200 µL human plasma with no reconstitution and drying steps. The chromatographic separation was achieved on a Symmetry C18 (100 mm×4.6 mm, 5 µm) column using 10 mM ammonium formate and acetonitrile (30:70, v/v) as the mobile phase in a run time of 2.0 min. Quantitation of analytes and ISs were done by multiple reaction monitoring on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL and 2.0-800 ng/mL for oseltamivir and oseltamivir carboxylate respectively. The mean extraction recovery for oseltamivir (94.4%) and oseltamivir carboxylate (92.7%) from spiked plasma samples was consistent and reproducible. The application of this method was demonstrated by a bioequivalence study in 42 healthy Indian subjects with 75 mg oseltamivir phosphate capsules. The assay reproducibility was established by reanalysis of 151 incurred subject samples.

Determination of (S)-(+)- and (R)-(-)-ibuprofen enantiomers in human plasma after chiral precolumn derivatization by reversed-phase LC-ESI-MS/MS.

BACKGROUND: A selective, sensitive and high-throughput LC-ESI-MS/MS method has been developed and validated for the chromatographic separation and quantitation of (S)-(+)-ibuprofen and (R)-(-)-ibuprofen after derivatization with (S)-(-)-1-(1-napthyl)ethylamine using 1-hydroxybenzotriazole as the activator of the carboxylic acid group and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the coupling reagent in human plasma. RESULTS: Both the analytes were chromatographically separated with a resolution factor of 1.27 on a Kinetex PFP (50 × 4.6 mm, 2.6 µm) analytical column. The method was validated over the concentration range of 0.10-32.0 µg/ml for both the enantiomers. The magnitude of matrix effect was assessed by post-column analyte infusion and also by precision (%CV) values for the calculated slopes of calibration curves. The mean extraction recovery was >91% for both the enantiomers. CONCLUSION: The method was successfully applied to a bioequivalence study in 34 healthy human subjects. The assay reproducibility was confirmed by reanalysis of 130 subject samples.

Enantiomeric separation of verapamil and its active metabolite, norverapamil, and simultaneous quantification in human plasma by LC-ESI-MS-MS.

A simple, selective and high-throughput liquid chromatography-tandem mass spectrometry method has been developed and validated for the chromatographic separation and quantification of (R)- and (S)-enantiomers of verapamil and its active metabolite, norverapamil, in human plasma. All four analytes along with deuterated internal standards (D(6)-verapamil and D(6)-norverapamil) were extracted from 50 µL human plasma by liquid-liquid extraction. Separation was achieved on a Chiralcel OD-RH (150 × 4.6 mm, 5 µm) analytical column with resolution factors of 1.4 and 1.9 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. A mobile phase consisting of 0.05% trifluoroacetic acid in water-acetonitrile (70:30, v/v) afforded capacity factors of 2.45, 3.05, 2.27 and 3.13 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. Detection was carried out on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion modes. The method was validated over the concentration range of 1.0-250.0 ng/mL for all four analytes. Absolute recovery for the analytes ranged from 91.1 to 108.1%. Matrix factors calculated at three quality control levels varied from 0.96-1.07. The method was successfully applied to a pharmacokinetic study in 18 healthy Indian males after oral administration of a 240-mg verapamil tablet formulation under fasting conditions.

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood.

A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) "Guidance for Industry, Bioanalytical Method Validation". Chromatographic separation was performed using an RP C18 (50 mm × 4.6 mm, 5 µm) column at 40±3.0 °C with a mobile phase consisted of 2 mM ammonium acetate in water (pH 4.5):methanol:acetonitrile (25:15:60, v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 µL, low inter-run bias and variability (for Sotrastaurin, -4.4 to 0.4% and 1.8 to 2.5% and for N-desmethyl-sotrastaurin, ranged from 1.6 to 2.3% and 2.7 to 3.9%, respectively) with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00 ng/mL and 1200 ng/mL.

Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC-ESI-MS/MS.

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid-liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500microL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50mmx4.6mm, 5microm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (alpha) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent-->product ion transitions for both the isomers (m/z 306.1-->233.0) and IS (m/z 237.3-->194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3-6076ng/mL and 23.8-5960ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200mg (E)-entacapone tablet formulation under fasting conditions.

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma.

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2 mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10 mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5 mL/min. The linear dynamic range was established over the concentration range 1.1-213.1 ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5 min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10 mg extended release methoxsalen formulation under fasting condition.

Determination of pitavastatin from human plasma using high performance liquid chromatography with fluorescence detection.

Pitavastatin belongs to the class of coenzyme A reductase inhibitors. Very few methods of assaying pitavastatin from human plasma are available in literature. An analytical method is presented for the determination of the drug from human plasma making use of the fluorescent property of the drug. The drug is extracted from plasma using ethyl acetate under neutral condition and then analyzed by reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection (lambda(Ex) 245 nm; lambda(Em) 420 nm). Analysis of pitavastatin was carried out on a C18 HPLC column using a gradient flow of mobile phase (0.01 mol/L monobasic potassium phosphate (pH 3.20) -acetonitrile, 63:37, v/v). Fluorescein isothiocyanate was used as internal standard. The dynamic range of assay was 3 to 50 ng/mL. The intraday precision was less than 10% and accuracy ranged from 95.2% to 112.6%. The same for interday check was less than 12% and 92.8% to 105.1%, respectively. The drug was found to be stable under the assay conditions. The developed method is simple, precise, accurate, and stable. This indicates that it can be applied to routine analysis of this drug in human subjects where there are large numbers of samples without the need of specialized instruments like column switching.

Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC-MS.

Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (-) and S (+) mianserin hydrochloride in a dose of 30-90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC-MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid-liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00-60.00 ng/ml for mianserin and 0.50-14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85-100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.