Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for quantification of rosuvastatin in small volume human plasma samples and its application to a clinical study

A high-throughput, simple, highly sensitive and specific LC-MS/MS method (liquid chromatography coupled with tandem mass spectrometry) has been developed for the estimation of rosuvastatin (CAS 287714-41-4, RST) with 100 microl human plasma using atorvastatin (CAS 134523-00-5) as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique. The assay procedure involved direct precipitation of RST and IS from plasma with acetonitrile. Sample preparation with this method yielded clean extracts and consistent recoveries: 91.39% for RST and 99.28% for IS. The total chromatographic run time was 3.5 min and the elution of RST and IS occurred at 2.5 and 3.1 min, respectively; this was achieved with a mobile phase consisting of 0.05 mol/L formic acid: acetonitrile (20:80, v/v) at a flow rate of 0.50 ml/min on an Inertsil ODS-3 column (4.6 x 100 mm, 3.0 microm). The developed method was validated in human plasma with a limit of quantitation of 0.05 ng/ml. A linear response function was established for the range of concentrations of 0.05 to 50.0 ng/ml with a correlation coefficient (r) of 0.999. The inter- and intra-day precision in the measurement of RST quality control (QC) samples at 0.05, 0.15, 25 and 40 ng/ml were in the range of 6.55 to 11.40% relative standard deviation (RSD) and 1.76 to 11.17% RSD, respectively. Accuracy in the measurement of QC samples for RST was in the range of 95.02 to 101.37% of the nominal values. RST was stable in the battery of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. The stability of RST was established for 1 month at -80 degrees C. The application of the assay to a clinical study confirmed the utility of the assay to derive human pharmacokinetic parameters.     

High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.     

Validation of a simple bioanalytical method for the determination of DRF-6196, a novel oxazolidinone, in mouse plasma: application to a single-dose pharmacokinetic study

An isocratic simple, specific, sensitive and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the estimation of DRF-6196, a novel oxazolidinone in mouse plasma. This method involves a simple liquid/liquid extraction of DRF-6196 and the internal standard (IS; chlorzoxazone, CAS 95-25-0) from plasma into dichloromethane/ethyl acetate mixture that was evaporated under nitrogen. The HPLC analysis was carried out on an Inertsil ODS 2 column using 0.01 mol/L potassium dihydorgen ortho phosphate (pH 3.2) and acetonitrile (65:35, v/v) as mobile phase. The eluate was monitored using an UV detector set at 266 nm. Ratio of peak area of analyte to IS was used for quantification of plasma samples. The retention time of DRF-6196 and IS were 8.2 and 11.1 min, respectively. The assay was linear (r2 > 0.999) in the concentration range 0.1-50 microg/ml. Absolute recovery for analyte and IS was > 94 % from mouse plasma. The lower limit of quantification (LLOQ) of DRF-6196 was 0.1 microg/ml. The inter- and intra-day precision in the measurement of quality control (QC) samples, 0.1, 0.3, 15.0 and 40.0 microg/ml, were in the range 3.64 to 9.51 % relative standard deviation (RSD) and 0.92 to 6.23 % RSD, respectively. Accuracy in the measurement of QC samples was in the range 88.15 to 106.05 % of the nominal values. The analyte and IS were stable in the stability studies viz., benchtop, autosampler and freeze/thaw cycles. The stability of DRF-6196 was established for 1 month at -80 degrees C. The assay method was successfully applied to a pharmacokinetic study of DRF-6196 in mice.     

Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry

High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.     

Development and validation of a sensitive LC-MS/MS method for the determination of adefovir in human serum and urine

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of adefovir (PMEA) in human serum and urine. The analyte was separated on a Diamonsil C(18) column (250 mm x 4.6 mm i.d., 5 microm particle size) by isocratic elution with methanol-water-formic acid (20:80:0.1, v/v/v) at a flow rate of 0.6 ml/min, and analyzed by mass spectrometry in multiple reaction-monitoring mode. The precursor-to-product ion transitions of m/z 274-->162 and m/z 226-->135 were used to measure and quantify the analyte and internal standard (I.S.), respectively. The weighted (1/x(2)) calibration curve was linear over serum concentration range 1.25-160.00 ng/ml and urine concentration range 0.05-8.00 microg/ml, with a correlation coefficient (r) of 0.9992 and 0.9978, respectively. The lower limit of quantification in human serum was 1.25 ng/ml. The inter- and intra-day precisions (R.S.D.%) in both serum and urine were lower than 8.64%, the mean method accuracies and recoveries from spiked serum samples at three concentrations ranged from 96.3 to 102.0% and 56.5 to 59.3%, respectively. The serum extract was stable when stored for 24h. The developed method was successfully applied to determine PMEA in human serum and urine, and proved suitable to clinical pharmacokinetic study.     

Liquid chromatography-mass spectrometry method for the determination of thiamphenicol in rabbit tears

A simple and sensitive liquid chromatography-mass spectrometry (LC-MS) method was described for the determination of thiamphenicol in rabbit tears. Chromatographic separation of the analyte was achieved on a C18 column using a mobile phase of acetonitrile and 10 mmol/l ammonium acetate solution (60:40, v/v). Selected ion monitoring (SIM) in negative mode was used for analyte quantification at m/z 354.4 for thiamphenicol and at m/z 137.1 for salylic acid. The run time was less than 6 min. Linearity over the concentration range of 0.032-32.0 ng/ml for thiamphenicol was obtained and the lower limit of quantification was 0.032 ng/ml. For each level of QC samples, inter- and intra-day precisions (R.S.D.) were 相似文献   

Simultaneous determination of albendazole and its major active metabolite in human plasma using a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A method for the simultaneous determination of albendazole (ABZ) and its major active metabolite albendazole sulfoxide (ABZ-SO) was developed and validated. The analytes were extracted from plasma samples by liquid-liquid extraction and analyzed using liquid chromatography-tandem mass spectrometry with an electrospray ionization interface. Estazolam was used as the internal standard. The assay was linear in the concentration range 0.4-200 ng/ml for ABZ and 4.0-2000 ng/ml for ABZ-SO. The intra- and inter-run precision (R.S.D.), calculated from quality control (QC) samples was less than 7.1 and 9.4% for ABZ and ABZ-SO, respectively. The accuracy as determined from QC samples was within +/- 3% for the analytes. Recoveries of ABZ and ABZ-SO were greater than 77 and 53%, respectively, over the calibration curve range. The method developed was successfully applied to pharmacokinetic studies of ABZ and ABZ-SO after an oral dose of 400 mg albendazole to healthy volunteers.     

Enantiospecific assay of citadiol--a key intermediate of escitalopram by liquid chromatography on Chiralpak AD-H column connected with UV and polarimetric detectors in series

A simple, rapid, selective and reproducible LC method for separation and quantitative determination of citadiol (CTD), a key intermediate of escitalopram has been developed. An optimum resolution > 3.0 was achieved on Chiralpak AD-H (250 mm x 4.6 mm); 5 microm column connected with UV and polarimetric detectors in series. The effects of organic modifiers, viz., methanol, ethanol, n-propanol and 2-propanol on enantioselectivity were evaluated. The limits of detection (LOD) and quantification (LOQ) were 0.02 microg/ml, 0.03 microg/ml and 0.07 microg/ml, 0.10 microg/ml for R-CTD and S-CTD enantiomers, respectively. The linearity of the method was studied in the range of 0.07-300 microg/ml and 0.1-300 microg/ml for R-CTD and S-CTD, respectively and the r2 was > or = 0.9999. The inter- and intra-day assay precision was less than 0.74% (%R.S.D.) and the recoveries were in the range 99.68-100.72% with %R.S.D. < 0.49%.     

Enantioselective liquid chromatography-mass spectrometry assay for the determination of ifosfamide and identification of the N-dechloroethylated metabolites of ifosfamide in human plasma

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the enantioselective determination of ifosfamide [(R)-IF and (S)-IF] in human plasma and for the detection of the N-dechloroethylated metabolites of IF, 2-N-dechloroethylifosfamide [(R)-2-DCl-IF and (S)-2-DCl-IF] and 3-N-dechloroethylifosfamide [(R)-3-DCl-IF and (S)-3-DCl-IF]. IF, 2-DCl-IF and 3-DCl-IF were extracted from plasma using solid-phase extraction and resolved by liquid chromatography on a column containing a Chirabiotic T chiral stationary phase. The enantioselective separations were achieved using a mobile phase composed of 2-propanol:methanol (60:40, v/v) and a flow rate of 0.5 ml/min. The observed enantioselectivities (alpha) for IF, 2-DCl-IF and 3-DCl-IF were 1.20, 1.17 and 1.20, respectively. The calibration curve was linear in the concentration range of 37.50-4800 ng/ml for each ifosfamide enantiomer (r(2)>0.997). The lower limit of detection (LLOD) was 5.00 ng/ml. The inter- and intra-day precision ranged from 3.63 to 15.8% relative standard deviation (R.S.D.) and 10.1 to 14.3% R.S.D., respectively, and the accuracy ranged from 89.2 to 101.5% of the nominal values. The method was applied to the analysis of plasma samples obtained from a cancer patient who received 3.75 g/m(2)/day dose of (R,S)-ifosfamide as a 96-h continuous infusion.     

Determination and pharmacokinetic study of amlodipine in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry

A novel, specific and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination and pharmacokinetic study of amlodipine in human plasma. The analysis was carried out on an ACQUITY UPLC BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with gradient elution at a flow-rate of 0.35 ml/min. The mobile phase was water and acetonitrile under gradient conditions (both containing 0.3% formic acid) and nimodipine was used as the internal standard. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via Turbo ion spray ionization (ESI). Linear calibration curves were obtained over the concentration range 0.15-16.0 ng/ml, with a lower limit of quantification of 0.15 ng/ml. The intra- and inter-day precision (R.S.D.) values were below 15% and the accuracy (R.E.) was -2.3% to 6.9% at all three QC levels. The method was used to support clinical pharmacokinetic studies of amlodipine in healthy volunteers following oral administration.     

Development and validation of a high-resolution capillary electrophoresis method for multi-analysis of ragaglitazar and arginine in active pharmaceutical ingredients and low-dose tablets

A selective, sensitive and robust capillary electrophoresis (CE) method has been developed and validated for multi analysis of ragaglitazar (also known as NNC 61-0029 or DRF 2725) and its counter ion arginine in Active Pharmaceutical Ingredients (API) and low-dose tablets (0.5, 1.0 and 2.0 mg). The method covers a total number of 12 analyses for the API and tablets: assay and identification of ragaglitazar and arginine, chiral purity of ragaglitazar and purity of ragaglitazar. After a simple extraction of samples with acetonitrile and 0.01 M sodium hydroxide (10:90), separation was performed using a combination of two cyclodextrins; sulfobutylether-beta-cyclodextrin (SB-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) in the electrolyte. The method showed good specificity and could separate and detect ragaglitazar, the distomer (the (+)-enantiomer) and arginine. The LOQ was found to be 0.10%, corresponding to 0.2 ng (0.5 microg/ml) for ragaglitazar and the distomer. Linearity was observed to be from 0.5 to 15 microg/ml (range 0.2-60 ng) and 400-600 microg/ml (range 1603-2404 ng) for ragaglitazar and 166-250 microg/ml (range 668-1000 ng) for arginine. The accuracy (as percent recovery) for ragaglitazar was found to be 101-106% (at 400-600 microg/ml), 101-125% (at 0.5-15 microg/ml) and for arginine 97-101% (at 166-250 microg/ml). The repeatability for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.05%, distomer: 1.01%, largest single impurity: 5.84%, total impurities: 0.90% and arginine: 2.00%. The intermediate precision for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.63%, distomer: 1.98%, largest single impurity: 5.22%, total impurities: 13.17% and arginine: 3.50.     

Simultaneous determinations of paeonol and palmatine hydrochloride in Shangshi Aerosols by HPLC method

A simple and rapid HPLC method was described for the simultaneous determination of paeonol and palmatine hydrochloride in Shangshi Aerosols. The optimum separation for these analytes was achieved using the mixture of 0.025 M sodium dihydrogen phosphate-acetonitrile-diethylamine (64:35:1, v/v/v) as the mobile phase and a Nova-Pak((R)) C8 column. The linear ranges of paeonol and palmatine hydrochloride were 0.2-80 and 0.06-60 microg/ml with the regression equations being Y=11716.4+2.96 x 10(6)X (R=0.99969), Y=-6388.8+1.89 x 10(5)X (R=0.99976), and limit of quantifications (LOQ) for paeonol and palmatine hydrochloride were 0.2 and 0.06 microg/ml, respectively (n=6). Other validation parameters: intra-day precision (R.S.D.: 0.71-1.65%) and inter-day precision (R.S.D.: 0.89-2.11%), and reproducibility (recoveries values: 94.6-98.2% for paeonol, 94.85-97.58% for palmatine hydrochloride) were found to be satisfactory. The proposed HPLC method had been applied for the determination of paeonol and palmatine hydrochloride in Shangshi Aerosols; R.S.D. values were 1.45 and 1.13%, respectively. In short, this method was rapid and convenient, which could be used for the routine control of paeonol and palmatine hydrochloride in Shangshi Aerosols.     

Simultaneous quantification of enalapril and enalaprilat 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 simultaneously determine enalapril and enalaprilat in human plasma. With benazepril as internal standard, sample pretreatment involved in a one-step protein precipitation (PPT) with methanol of 0.2 ml plasma. Analysis was performed on an Ultimate™ XB-C₁₈ column (50 mm × 2.1 mm, i.d., 3 μm) with mobile phase consisting of methanol–water–formic acid (62:38:0.2, v/v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction-monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for enalapril and enalaprilat were both obtained in the concentration range of 0.638–255 ng/ml (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.638 ng/ml. The intra-day precision (R.S.D.) was below 7.2% and inter-day R.S.D. was less than 14%, while accuracy (relative error R.E.) was within ±8.7 and ±5.5%, determined from QC samples for enalapril and enalaprilat which corresponded to requirement of the guidance of FDA. The HPLC–MS/MS method herein described was fully validated and successfully applied to the pharmacokinetic study of enalapril maleate capsules in 20 healthy male volunteers after oral administration.     

Development and validation of a liquid chromatographic method for determination of enantiomeric purity of citalopram in bulk drugs and pharmaceuticals

A simple, rapid and robust LC method for enantiospecific separation and determination of citalopram in drugs and pharmaceuticals was developed using UV and polarimetric detectors connected in series. Baseline separation with resolution > or = 3.0 was achieved within 20 min on Chiralcel OD-H (250 mm x 4.6 mm) 5 microm column using a mobile phase containing of n-hexane:2-propanol:triethylamine (TEA) (95:05:0.1 v/v/v) at a flow rate of 1.0 ml/min at 25 degrees C. Effects of 2-propanol, triethylamine and temperature on enantioselectivity and resolution of the enantiomers were evaluated. Clopidogrel hydrogen sulphate was used as an internal standard (IS) for quantitative determinations using UV detector at 240 nm. Polarimetric detector was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 1.3 microg/ml respectively for both the enantiomers. The linearity of the method was in the range of 50-600 microg/ml with r2 > 0.9999. The inter- and intra-day assay precision was less than 0.63% (%R.S.D.) and recoveries were in the range 99.38-100.41%. The method was validated and found to be suitable for determination enantiomeric purity of citalopram in bulk drugs and pharmaceutical formulations.     

Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with beta-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 degrees C. The standard curves were linear over the range of 0.0975-7.800 and 0.1744-13.95 microg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze-thaw cycles, storage at room temperature for 4 h, and at -20 degrees C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).     

Simultaneous determination of lansoprazole and its metabolites 5'-hydroxy lansoprazole and lansoprazole sulphone in human plasma by LC-MS/MS: application to a pharmacokinetic study in healthy volunteers

A highly sensitive and specific liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the simultaneous determination of lansoprazole and its metabolites 5'-hydroxy lansoprazole and lansoprazole sulphone. The detection was operated with multiple reaction-monitoring (MRM) using the electrospray ionization technique. The assay procedure involved precipitation of plasma samples with acetonitrile after indapamide was added as internal standard (IS). The chromatographic separation was achieved with a mixture of methanol-0.2% ammonium acetate and 0.1% methanoic acid in water (75:25, v/v) as mobile phase on an Inertsil ODS-3 column. The method was proved to be accurate and precise with linearity ranges of 10-4,000 ng/ml, 5.0-400 ng/ml, and 1.0-400 ng/ml for lansoprazole, 5'-hydroxy lansoprazole and lansoprazole sulphone, respectively, with the correlation coefficients (r) better than 0.999. The lower limits of quantification (LLOQ) were 2.0 ng/ml, 2.0 ng/ml, and 0.5 ng/ml for lansoprazole, 5'-hydroxy lansoprazole and lansoprazole sulphone, respectively. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits (R.S.D.% within +/-15) in accordance with FDA guidelines. The validated LC-MS/MS method has been successfully applied for the determination of lansoprazole and its metabolites in human plasma.     

Experimental design and capillary electrophoresis for simultaneous analysis of arbutin, kojic acid and hydroquinone in cosmetics

A statistical experimental design was used to optimize one micellar electrokinetic capillary electrophoresis (MEKC) for simultaneous analysis of arbutin (AR), kojic acid (KA) and hydroquinone (HQ). Untreated fused-silica capillaries were operated using a phosphate buffer (20mM, pH 6.5) under 20 kV and detection at 200 nm. Quantitative linear ranges were 20-200 microg/ml for AR, 20-100 microg/ml for KA and 8-80 microg/ml for HQ with correlation coefficients >or=0.9994. R.S.D. and R.E. were less than 3.0% for the intra-day and inter-day analysis, and all recoveries were greater than 99%. Our method was applied to assay commercial cosmetics. The results were within the labeled amount of 99.6-102.5%.     

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.     

Quantification of levetiracetam in human plasma by liquid chromatography-tandem mass spectrometry: application to therapeutic drug monitoring

A rapid, selective, reliable, precise, accurate, and reproducible tandem mass spectrometric (MS-MS) method for the quantification of levetiracetam (LEV) in human plasma using adenosine as an internal standard (IS) has been developed and validated. The drug and IS were extracted by solid phase extraction (SPE) technique and analyzed on Symmetry((R)) C(18) column (5 microm, 3.9 mm x 50 mm) using a mobile phase of methanol-water-formic acid (97:03:0.25, v/v/v) at a flow rate of 0.2 ml/min. Quantitation was achieved using a positive electrospray ionization (ESI+) interface employing multiple reaction monitoring (MRM) mode at MRM transitions m/z 171>126 and m/z 268>136 for LEV and IS, respectively. The method was validated over the concentration range of 1.0-40 microg/ml (r>0.99) with a limit of quantification of 1.0 microg/ml (R.S.D.%; 4.1 and Bias%; -9.0 to + 11.0%). Intra- and inter-run precision of LEV assay at three concentrations ranged from 0.6 to 8.9% with accuracy (bias) varied from -4.0 to 8.6% indicating good precision and accuracy. Analytical recoveries of LEV and IS from spiked human plasma were in the range of 91.7-93.4% and 80.2-84.1%, respectively. Stability of LEV in human plasma samples at different conditions showed that the drug was stable under the studied conditions. Matrix effect study showed a lack of matrix effect on mass ions of LEV and IS. The described method compared well with the commercial HPLC-UV method of Chromsystem (r(2)=0.99). The suitability of the developed method for therapeutic drug monitoring was demonstrated by measuring LEV in human plasma samples of epileptic patients treated with LEV.