HPLC method for determination of DRF-4367 in rat plasma: validation and its application to pharmacokinetics in Wistar rats

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of DRF-4367, a novel cyclooxygenase-2 inhibitor in rat plasma. The assay procedure involved simple liquid/liquid extraction of DRF-4367 and internal standard (IS, celecoxib) from plasma into dichloromethane. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C(18) column (4.6 x 250 mm, 5 microm). The mobile phase consisting of 0.01 M potassium dihydrogen ortho-phosphate (pH 3.2) and acetonitrile (40:60, v/v) was used at a flow rate of 1.0 mL/min. The eluate was monitored using an UV detector set at 247 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of DRF-4367 and IS were 6.6 and 11.2 min, respectively. The standard curve for DRF-4367 was linear (r(2) > 0.999) in the concentration range 0.1-20 micro g/mL. Absolute recovery was >86% from rat plasma for both analyte and IS. The lower limit of quantification of DRF-4367 was 0.1 micro g/mL. The inter- and intra-day precisions in the measurement of quality control samples, 0.1, 0.3, 8.0 and 15.0 microg/mL, were in the range 6.93-9.34% relative standard deviation (RSD) and 0.48-6.59% RSD, respectively. Accuracy in the measurement of QC samples was in the range 91.24-109.36% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. Stability of DRF-4367 was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats is described.     

Determination of rosuvastatin in rat plasma by HPLC: Validation and its application to pharmacokinetic studies

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of rosuvastatin (RST), a novel, synthetic and potent HMG-CoA inhibitor in rat plasma. The assay procedure involved simple liquid-liquid extraction of RST and internal standard (IS, ketoprofen) from a small plasma volume directly into acetonitrile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). Mobile phase consisting of 0.05 m formic acid and acetonitrile (55:45, v/v) was used at a flow rate of 1.0 mL/min for the effective separation of RST and IS. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 240 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of RST and IS were 8.6 and 12.5 min, respectively. The standard curve for RST was linear (r2 > 0.999) in the concentration range 0.02-10 microg/mL. Absolute recoveries of RST and IS were 85-110 and >100%, respectively, from rat plasma. The lower limit of quantification (LLOQ) of RST was 0.02 microg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.02, 0.06, 1.6 and 8.0 microg/mL, were in the range 7.24-12.43% relative standard deviation (RSD) and 2.28-10.23% RSD, respectively. Accuracy in the measurement of QC samples was in the range 93.05-112.17% of the spiked nominal values. Both analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. RST was found to be stable for a period of 30 days on storage at -80 degrees C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.     

Stereospecific determination of cis- and trans-resveratrol in rat plasma by HPLC: application to pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for simultaneous determination of resveratrol isomers in rat plasma. Cis-resveratrol was made by exposure of a trans-resveratrol solution to sunlight for 5 days followed by separation by HPLC and identification by mass spectrometry (MS). The assay procedure involved simple liquid-liquid extraction of resveratrol isomers and internal standard (IS, caffeine) from a small plasma volume directly into acetonitrile. The supernatant liquid was added an equal volume of water and injected onto a Hypersil ODS(2) C(18) column (5 microm, 4.6 x 250 mm). Mobile phase consisting of methanol and distilled water was used at a flow rate of 1.0 mL/min for the effective separation of cis-, trans-resveratrol and caffeine (IS). The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of cis-, trans-resveratrol and IS were 3.2, 4.3 and 6.1 min, respectively. The calibration curve was linear ranging from 0.066 to 6.64 and 0.134 to 13.4 microg/mL with correlation coefficients of 0.9998 and 0.9997 for trans and cis isomers, respectively. The absolute recovery of both isomers was more than 85%. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.066, 0.664 and 6.64 microg/mL of trans-resveratrol, were in the range 2.37-6.95% relative standard deviation (RSD) and 0.77-6.97% RSD, respectively. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.134, 1.34 and 13.4 microg/mL of cis-resveratrol, were in the range 1.93-3.72% relative standard deviation (RSD) and 1.13-6.57% RSD, respectively. Both analytes and IS were stable in the battery of stability studies and freeze-thaw cycles. Resveratrol isomers were found to be stable for a period of 30 days on storage at -20 degrees C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.     

Validation of a simple HPLC method for DRF-4848, a novel COX-2 inhibitor suitable for pharmacokinetic application in rats

For pharmacokinetic and toxicokinetic purpose a simple HPLC-UV method has been developed and validated for the estimation of DRF-4848, a novel COX-2 inhibitor in rat plasma. A liquid-liquid extraction was used to extract DRF-4848 and internal standard (IS, DRF-4367) from rat plasma. The analysis was performed on a C(18) column with UV detection at 285 nm. The isocratic mobile phase, 0.01 M potassium dihydrogen ortho phosphate (pH 3.2) and acetonitrile (50:50, v/v) was run at a flow rate of 1 mL/min. The retention times of DRF-4848 and IS were 6.8 and 11.2 min, respectively. Absolute recovery for analyte and IS was >80% from rat plasma. A linear response was observed over a concentration range 0.1-20 microg/mL. The lower limit of quantification (LLOQ) of DRF-4848 was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.1, 0.3, 8.0 and 15.0 microg/mL, were in the range 1.74-8.70% relative standard deviation (RSD) and 0.75-8.43% RSD, respectively. Accuracy in the measurement of QC samples was in the range 93.29-116.51% of the nominal values. Analyte and IS were stable in the battery of stability studies viz., benchtop, autosampler, long-term and freeze/thaw cycles.     

Simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in plasma by high-performance liquid chromatography with UV detection

A specific, accurate, precise and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in human plasma. The method employed a simple liquid-liquid extraction of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib and internal standard (IS, DRF-4367) from human plasma (500 microL) into acetonitirile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid (pH 3)-acetonitrile-methanol-water at a flow rate of 1.0 mL/min. The eluate was monitored using an ultraviolet (UV) detector set at 235 nm. The ratio of peak area of each analyte to IS was used for quantification of plasma samples. Nominal retention times of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide, IS and celecoxib were 15.63, 17.20, 21.66, 24.95, 26.27, 30.24 and 32.22 min, respectively. The standard curve for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib was linear (r2 > 0.999) in the concentration range 0.1-50 microg/mL and for nimesulide (r2 > 0.999) in the concentration range 0.5-50 microg/mL. Absolute recovery was >83% from human plasma for all the analytes and IS. The lower limit of quantification (LLOQ) of nimesulide was 0.5 microg/mL and for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib the LLOQ was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of QC samples, 0.1, 0.3, 15.0 and 40.0 microg/mL (for all analytes except nimesulide), were in the range 2.29-9.37% relative standard deviation (RSD) and 0.69-10.28% RSD, respectively. For nimesulide the inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.5, 1.5, 15.0 and 40.0 microg/mL, were in the range 3.21-7.37% RSD and 0.97-7.06% RSD, respectively. Accuracy in the measurement of QC samples for all analytes was in the range 91.03-106.38% of the nominal values. All analytes including IS were stable in the battery of stability studies, viz. bench top, autosampler and freeze-thaw cycles. Stability of all analytes was established for 21 days at -20 degrees C. The application of the assay in an oral pharmacokinetic study in rats co-administered with celecoxib and valdecoxib is described.     

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

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

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

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for determining rosuvastatin in human plasma, a new synthetic hydroxymethylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (IS; cilostazol) were extracted by simple one-step liquid/liquid extraction with ether. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The chromatographic separation was performed on an Atlantis C18 column (2.1 mm x 150 mm, 5.0 microm) with a mobile phase consisting of 0.2% formic acid/methanol (30:70, v/v) at a flow rate of 0.20 mL/min. The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 482 --> 258 and m/z 370 --> 288. The areas of peaks from the analyte and the IS were used for quantification of rosuvastatin. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification (LLOQ) was 0.2 ng/mL and the assay exhibited a linear range of 0.2-50.0 ng/mL and gave a correlation coefficient (r) of 0.9991 or better. Quality control samples (0.4, 8, 25 and 40 ng/mL) in six replicates from three different runs of analysis demonstrated an intra-assay precision (RSD) 7.97-15.94%, an inter-assay precision 3.19-15.27%, and an overall accuracy (relative error) of < 3.7%. The method can be applied to pharmacokinetic or bioequivalence studies of rosuvastatin.     

Determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine carbocysteine in human plasma using 2-pyridylacetic acid as the internal standard (IS). The method employed derivatization with 10 M hydrochloric acid/methanol, which significantly improved the ionization efficiency of carbocysteine. After methanol-induced protein precipitation of plasma samples, carbocysteine and the IS were derivatized and subjected to LC/MS/MS analysis using atmospheric pressure chemical ionization. The method has a lower limit of quantitation of 20 ng/mL for a 0.2-mL plasma aliquot. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was less than 7%. The accuracy, determined using QC samples, was within +/- 1%. The method offered increased sensitivity, selectivity and speed of analysis over existing methods. The method was utilized to support clinical pharmacokinetic studies of carbocysteine in volunteers following oral administration.     

Pharmacokinetic study of salvianolic acid A in rat after intravenous administration of Danshen injection

In order to research the pharmacokinetics of salvianolic acid A (SalA), a herbal ingredient isolated from Salvia miltiorrhiza Bunge, after intravenous administration to rats, a specific and accurate high-performance liquid chromatography (HPLC) was developed. The assay procedure involved simple liquid-liquid extraction of SalA and internal standard (IS, ethyl-p-hydroxybenzoate) from plasma into ethyl acetate. The organic layer was separated and evaporated under reduced pressure at 40 degrees C. The residue was reconstituted in the mobile phase and analyzed on an Inertsil C8 column, monitored at 285 nm. The mobile phase, which consisted of methanol-acetonitrile-water-formic acid (10:20:70:0.4, by vol), was used at a flow rate of 1.0 mL/min. The ratio of the peak area of the analyte to IS was applied to quantify the plasma samples. The standard curve for SalA was linear (r2 = 0.9999) in the concentration range of 0.75-150 microg/mL. The limit of quantitation (LOQ) of SalA was 0.75 microg/mL. The intra- and inter-day precisions (RSD) of the quality control (QC) samples were in the ranges of 2.17-3.29 and 1.24-5.28%, respectively. Accuracy in the measurement of QC samples ranged from 94.7 to 101.1%. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of SalA in rat plasma after intravenous administration of Danshen injection.     

Determination of lipoic acid in rat plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a pharamcokinetic study

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.     

Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study

A simple, sensitive and specific high-performance liquid chromatography method is described for simultaneous determination of rosuvastatin (RST) and gemfibrozil (GFZ) in human plasma using celecoxib as an internal standard (IS). The assay procedure involved extraction of RST, GFZ and IS from plasma into acetonitrile. Following separation and evaporation of the organic layer the residue was reconstituted in the mobile phase and injected onto an X-Terra C(18) column (4.6 x 150 mm, 5.0 microm). The chromatographic run time was less than 20 min using flow gradient (0.0-1.60 mL/min) with a mobile phase consisting of 0.01 M ammonium acetate:acetonitrile:methanol (50:40:10, v/v/v) and UV detection at 275 nm. Nominal retention times of RST, GFZ and IS were 6.7, 13.9 and 16.4 min, respectively. Absolute recovery of both analytes and IS was greater than 90%. The lower limit of quantification (LLOQ) of RST and GFZ was 0.03 and 0.30 microg/mL, respectively. Linearity was excellent (r(2) = 0.999) in the 0.03-10 microg/mL and 0.3-100 microg/mL ranges for RST and GFZ, respectively. The inter- and intra-day precisions in the measurement of RST quality control (QC) samples 0.03, 0.09, 2.50 and 8.00 microg/mL were in the range 2.37-9.78% relative standard deviation (RSD) and 0.92-10.08% RSD, respectively. Similarly, the inter- and intra-day precisions in the measurement of GFZ quality control (QC) samples 0.30, 0.90, 25.0 and 80.0 microg/mL were in the ranges 2.79-6.27 and 0.96-9.69% RSD, respectively. Accuracies in the measurement of QC samples for RST and GFZ were in the range 85.43-107.23 and 84.98-102.35% respectively, of the nominal values. RST and GFZ were stable in the array of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. Stability of RST and GFZ was established for 1 month at -80C. The application of the assay in an oral pharmacokinetic study in rats co-administered with RST and GFZ is described.     

A sensitive and rapid liquid chromatography tandem mass spectrometry method for quantitative determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in human plasma containing liposome-based SN-38 (LE-SN38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of Irinotecan (CPT-11), an anticancer pro-drug. To support clinical pharmacokinetic studies for liposome based formulation of SN-38 (LE-SN38) in cancer patients, a rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of total SN-38 in human plasma. Sample preparation was carried out by one-step protein precipitation using cold acetonitrile with 0.5% acetic acid (v/v). Camptothecin was used as an internal standard (IS). Chromatographic separation of SN-38 and IS was achieved using a Synergi Hydro-RP column (C(18), 50 x 2 mm, 4 micro m), with a gradient elution of acetonitrile and 0.1% acetic acid. After ionization in electrospray source (positive ions), the acquisition was performed in the multiple reactions monitoring mode. Quantitation was accomplished using the precursor-->product ion combinations of m/z 393.1-->349.2 for SN-38 and 349.1-->305.1 for IS. The quantification limit of 0.05 ng/mL was achieved by using much lower volume (0.2 mL) of plasma and in the presence of LE-SN38. The method was validated over the concentration range of 0.05-400 ng/mL. Accuracy was within +/-12% of nominal at all concentration levels. Inter-day and intra-day precisions expressed as percentage coefficient of variation (%CVs) for quality control (QC) samples were less than 14 and 5%, respectively.     

LC–ESI–MS Determination of Lovastatin in Human Plasma

A convenient, selective and sensitive liquid chromatographic-electrospary ionization mass spectrometry (LC–ESI–MS) method was developed and validated to determine lovastatin in human plasma. The analyte was extracted from human plasma samples by typical liquid–liquid extraction, separated on a C₁₈ column by using the mobile phase consisting of water–methanol (13:87, v/v). Simvastatin was used as the internal standard (IS). The method was linear within the range of 0.1–20 ng mL⁻¹. The lower limit of quantification (LLOQ) was 0.1 ng mL⁻¹. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 10.2%. The accuracy as determined from QC samples was in the range of 99.3–102.9% for the analyte. The mean recoveries for lovastatin and IS were 84.8 and 88.0%, respectively. The method was successfully applied for evaluation of the pharmacokinetic of lovastatin in healthy volunteers.     

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of isoniazid and ethambutol in human plasma

Isoniazid and ethambutol are commonly used in various combination treatments for tuberculosis, and for this reason a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of these two drugs in human plasma. After a simple protein precipitation using methanol, the analytes and the internal standard metformin were chromatographed on a C18 column and detected by MS/MS. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The LC retention times for isoniazid and ethambutol were 2.46 and 2.27 min, respectively. The method was linear in the concentration range of 10.0-5000 ng/mL for each analyte using 100 microL plasma. The intra- and inter-day precisions, expressed as the relative standard deviation (RSD), were less than 5.7 and 6.4%, determined from QC samples for isoniazid and ethambutol, and the accuracies were within +/-2.1% and +/-4.5% in terms of relative error, respectively. The method was successfully employed in a pharmacokinetic study after oral administration of a multicomponent formulation containing 150 mg isoniazid, 500 mg ethambutol, 150 mg rifampicin and 250 mg pyrazinamide.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.     

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of paroxetine in human plasma

A sensitive, simple, fast and rugged hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of paroxetine was developed and validated over curve range 0.050-50 ng/mL using only 0.4 mL plasma. This is the first published LC-MS/MS method and the low limit of quantitation of this method is 10-fold lower than previously published methods. A simple liquid-liquid extraction method using methyl-tert butyl ether (MTBE) as the extraction solvent was used to extract paroxetine and the internal standard (IS) fentanyl-d(5) from plasma. The extract was evaporated to dryness, reconstituted and injected onto a silica column using a low aqueous-high organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.1 and 1.2 min for paroxetine and IS, respectively. The detection was by monitoring paroxetine at m/z 330 --> 192 and IS at m/z 342 --> 188, respectively. The inter-day precision and accuracy of the quality control (QC) samples were <5.0% relative standard deviation (RSD) and <2.9% relative error (RE). This method can be used for supporting therapeutical drug monitoring and pharmacokinetic or drug-drug interaction studies.     

Quantitative determination of isorhamnetin, quercetin and kaempferol in rat plasma by liquid chromatography with electrospray ionization tandem mass spectrometry and its application to the pharmacokinetic study of isorhamnetin

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of quercetin, kaempferol and isorhamnetin in rat plasma. After being treated with beta-glucuronidase and sulfatase, the analytes were extracted by liquid/liquid extraction with the internal standard (IS; baicalein). The chromatographic separation was performed on a Diamonsil C(18) column with a mobile phase consisting of 2% formic acid/methanol (10:90, v/v) at a flow rate of 1.00 mL/min, with a split of 200 microL to the mass spectrometer. Validation results indicated that the lower limit of quantification (LLOQ) was 1 ng . mL(-1). The assay exhibited a linear range of 1-200 ng . mL(-1) and gave a correlation coefficient of 0.9980 or better for each analyte. Quality control samples (1, 5, 20 and 100 ng . mL(-1)) in six replicates from each of three different runs demonstrated an intra-assay precision (RSD) of 1.1-8.9%, an inter-assay precision of 1.6-10.8%, and an overall accuracy (bias) of <13.4%. The extraction recovery of each analyte and internal standard was 70-80%. In the present study, we have investigated the pharmacokinetic profiles of isorhamnetin after oral application in rats equipped with a jugular catheter. After oral dosing of isorhamnetin, the mean values (n = 10) of C(max) were 57.8, 64.8 and 75.2 ng . mL(-1) which were achieved at a T(max) of 8.0, 6.4 and 7.2 h for oral doses of 0.25, 0.5 and 1.0 mg . kg(-1) body weight, respectively. The corresponding mean values for isorhamnetin area under the curver (AUC) from 0 to 60 h were 838.2, 1262.8, 1623.4 ng . h . mL(-1). Our results further demonstrated that the samples analyzed showed isorhamnetin could not be transformed into quercetin or kaempferol in rats, indicating that the demethylation of the 3'-oxymethyl group of isorhamnetin does not occur in Wistar rats.     

A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.