Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry

A specific LC-MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mmx2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mmx4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5-200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 microL plasma aliquot. The LC-MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

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.     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of 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.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

Liquid chromatographic assay for a butenolide endothelin antagonist (PD 156707) in plasma

A sensitive and selective liquid chromatographic assay for determining the non-peptide endothelin A receptor antagonist PD 156707 (I) in rat plasma has been developed and validated. The analyte was isolated from matrix by solid-phase extraction. Liquid chromatographic separation was achieved isocratically ona 3.2 mm I.D., ODS column with a mobile hase of acetonitrile-ammonium phosphate (50 mM, pH 3.5) (44:56, v/v). Column effluent was monitored fluorometrically. Peak-height ratios (analyte/IS) were proportional to I concentrations in rat plasma from 25 to 1000 ng/ml. Assay precision and accuracy for I, based on quality controls, was 9.5% relative standard deviation, with relative error of ±6.5%. The quantitation limit was 25 ng/ml for a 200-μl sample aliquot.     

Liquid chromatography-tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150mmx2mm, 5microm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3-->118.9 and m/z 226.4-->123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.     

High-performance liquid-chromatographic determination of warfarin enantiomers in plasma with automated on-line sample enrichment

A sensitive and selective chiral high performance liquid chromatographic method was developed for the direct determination of R- and S-warfarin enantiomers in human plasma. The method involved direct injection of human plasma onto a semipermeable surface (SPS) guard column, washing the proteins from the column with aqueous acetonitrile and back flushing the analytes onto a reversed phase ovomucoid silica HPLC column using switching valves. After separation, the analytes were simultaneously detected and quantitated with a fluorometer. The recoveries of R-warfarin from human plasma at 25 and 2500 ng/ml were 98.9% and 88.1%, respectively. The recoveries of S-warfarin at 25 and 2500 ng/ml were 105.4% and 93.9%, respectively. Using 100 microl of human plasma, the lower limit of quantification for both R- and S-warfarins was 25 ng/ml. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 25 to 2500 ng/ml for both enantiomers. Fluorescence chromatograms of drug-free human plasma showed no interfering peaks with retention times similar to those for R- and S-warfarins and the internal standard. Results from a 3-day validation study for both enantiomers demonstrated excellent precision (1.7-9.0%) and accuracy (97-109%) across the calibration range.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm x 4.6 mm i.d., 5 microm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50-2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.     

Improved and simplified liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized glucosamine in human plasma

Glucosamine is an amino monosaccharide reagent. It is difficult to assay using typical reversed-phase column due to the early elution, by optimizing the chromatographic conditions, especially the analytical column and the mobile phase composition, an improved analytical method was developed and validated, which offers rapid, sensitive and specific determination of glucosamine in human plasma. Following protein precipitation, the analyte and internal standard (valibose) were separated using an isocratic mobile phase on an Inertsil CN-3 column and detected by mass spectrometry in the multiple reaction monitoring mode using the respective precursor to product ion combinations of m/z 180/72 for glucosamine and m/z 252/198 for valibose. The chromatographic time was just 4.2 min for each sample, which made it possible to analyze more than 120 human plasma samples per day. The method exhibited a linear dynamic range of 4.00-4000 ng/mL for glucosamine in human plasma. The lower limit of quantification (LLOQ) was 4.00 ng/mL with a relative standard deviation of less than 10.9%. Acceptable precision and accuracy were obtained for the plasma concentrations over the standard curve range. By monitoring the two different MRM transitions, it was proved that no endogenous glucosamine was found in human plasma. The validated method has been successfully used to analyze human plasma samples for application in a bioequivalence study.     

Liquid chromatography-negative ion electrospray tandem mass spectrometry method for the quantification of tacrolimus in human plasma and its bioanalytical applications

A simple, rapid, novel and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tacrolimus (I) in human plasma, a narrow therapeutic index, potent macrolide immunosuppressive drug. The analyte and internal standard (tamsulosin (II)) were extracted by liquid-liquid extraction with t-butylmethylether using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of 99% methanol and 1% 10mM ammonium acetate buffer. The deprotonate of analyte was quantitated in negative ionization by multiple reaction monitoring (MRM) with a mass spectrometer. The mass transitions m/z 802.5-->560.3 and m/z 407.2-->151.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.05-25ng/ml for tacrolimus in human plasma. The lower limit of quantitation was 50pg/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 2min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in comparative bioavailability studies. The tacrolimus plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (C(max)) of tacrolimus (5mg oral dose) is 440pg/ml, time to observed maximum plasma concentration (T(max)) is 2.5h and elimination half-life (T(1/2)) is 21h.     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma by liquid chromatography-tandem mass spectrometry following automated solid-phase extraction

A selective, accurate, and reproducible LC-MS-MS assay was developed for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma samples. The method involved automated solid-phase extraction of atazanavir and a stable isotope analog internal standard (I.S.) using Oasis HLB 10 mg 96-well SPE plates. A portion of the reconstituted sample residue was injected onto a C(18) HDO analytical column which was configured with a triple quad mass spectrometer for analyte determination by positive ion electrospray. The assay was linear from 1.00 to 1,000 ng/ml with a lower limit of quantitation of 1.00 ng/ml. The inter- and intra-day coefficients of variation (C.V.) for the assay were <4%, and the accuracy was 99-102%. Atazanavir was stable in human plasma for at least 109 h at room temperature and for at least 1 year at -20 degrees C.     

Simultaneous determination of methylephedrine and noscapine in human plasma by liquid chromatography-tandem mass spectrometry

A selective and sensitive method has been developed and validated for simultaneous quantification of methylephedrine and noscapine in human plasma. Analytes were extracted from human plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface. Diphenhydramine was used as the internal standard (I.S.). The method was found to be precise and accurate within the linear range 0.1-100 ng/ml for each analyte. The intra- and inter-day relative standard deviations (R.S.D.s) were below 5.2% for methylephedrine and 6.7% for noscapine. The inter-day relative error (RE) as determined from quality control samples (QCs) was less than 3.0% for each analyte. The assay was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation containing 20 mg DL-methylephedrine hydrochloride, 16 mg noscapine, 300 mg paracetamol and 1mg of chlorpheniramine maleate.     

Determination of famotidine in human plasma and urine by high-performance liquid chromatography

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C₁₈ reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 μg/ml, respectively; intra- and inter-day coefficients of variation were ≤10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.     

Determination of nefazodone and its pharmacologically active metabolites in human blood plasma and breast milk by high-performance liquid chromatography

A method is described for the determination of nefazodone and its active metabolites hydroxynefazodone, the dione BMS-180492 and m-chlorophenylpiperazine in blood plasma and expressed human milk based on reversed-phase high-performance liquid chromatography. Measurements were performed on drug-free plasma and expressed human milk spiked with nefazodone and metabolites to prepare and validate standard curves and specimens collected from nursing mothers. Parent drug and metabolites were separated from the biological matrices by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved with a C₁₈ column and compounds were detected by their absorbance at 205 nm. Trazodone was used as an internal standard. The assay was validated for each analyte in the concentration range 200 to 1200 ng/ml.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.