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Quality Control Samples (quality + control_sample)
Selected AbstractsDevelopment and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasmaJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2005Paraskevi Valavani Abstract A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 × 3.0 mm i.d., particle size 5 µm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0,1000 ng ml,1 for all compounds analyzed. The limit of quantitation was 5 ng ml,1 for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml,1) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of ,9.1%, an inter-assay precision of ,6.0% and an overall accuracy (relative error) of <4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies. Copyright © 2005 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolitesBIOMEDICAL CHROMATOGRAPHY, Issue 1 2009Shuang Fu Abstract Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones,desmethoxyyangonin, methysticin and kavain,in rat liver microsomes using diazepam as an internal standard; liquid,liquid extraction was used for sample preparation and analysis was performed on a Shimadzu® 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna® C18 column (150 × 2.00 mm, 3 µm) at 40°C. The limit of quantitation was 0.1 µg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1,10 µg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites. Copyright © 2008 John Wiley & Sons, Ltd. [source] Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detectionBIOMEDICAL CHROMATOGRAPHY, Issue 3 2009Nobuo Mochizuki Abstract Therapeutic drug monitoring of sirolimus (rapamycin) is important for immunosuppressive therapy in solid organ transplantation. We have developed a simple and reliable method for determining blood concentrations of sirolimus using reversed-phase HPLC with electrochemical detection (ECD). The E2 potential was set at +900 mV. The potential of guard cell was set at +950 mV and that of the E1 cell at +400 mV. The method was linear for a concentration range of 1,50 ng/mL when 0.5 mL blood was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 0.5 ng/mL. The inter-assay precision ranged from 3.22 to 7.48%, and the coefficient of variation (CV) for a quality control sample at 10 ng/mL was 7.48% with a bias of 8.4% from the target value. The intra-assay precision ranged from 0.72 to 3.71%, and the CV for a quality control sample at 10 ng/mL was 0.72% with a bias of 6.8% from the target value. In a solid organ transplant recipient, trough concentrations of sirolimus were well within the analytic range of the HPLC/ECD procedure. The method described here is suitable for management of sirolimus therapy in solid organ transplantation. Copyright © 2008 John Wiley & Sons, Ltd. [source] Human thiopurine methyltransferase activity varies with red blood cell ageBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 5 2001L. Lennard Aims, Inherited differences in thiopurine methyltransferase (TPMT) activity are an important factor in the wide interindividual variations observed in the clinical response to thiopurine chemotherapy. The aim of this study was to establish a population range for red blood cell (RBC) TPMT activity in children with acute lymphoblastic leukaemia (ALL) at disease diagnosis. An additional aim was to investigate factors that can influence TPMT activity within the RBC. Methods,Blood samples were collected from children with ALL at disease diagnosis, prior to any blood transfusions, as part of the nationwide UK MRC ALL97 therapeutic trial. RBC TPMT activity was measured by h.p.l.c. RBCs were age-fractionated on Percoll density gradients. Results,Pretreatment blood samples were received from 570 children within 3 days of venepuncture. TPMT activities at disease diagnosis ranged from 1.6 to 23.6 units/ml RBCs (median 7.9) compared with 0.654,18.8 units (median 12.9), in 111 healthy control children (median difference 4.5 units, 95% CI 3.9, 5.1 units, P < 0.001). A TPMT quality control sample, aliquots of which were assayed in 60 analytical runs over a 12 month period, contained a median of 11.98 units with a CV of 11.6%. Seven children had their RBCs age-fractionated on density gradients. TPMT activities in the top gradient (young cells) ranged from 4.2 to 14.1 units (median 7.5) and in the bottom gradient (old cells) 1.5,12.6 units (median 4.7 units), median difference 2.3 units, 95% CI 0.7, 4.1, P = 0.035. Conclusions,Circulating RBCs do not constitute a homogeneous population. They have a life span of around 120 days and during that time undergo a progressive ageing process. The anaemia of ALL is due to deficient RBC production. The results of this study indicate that RBC TPMT activities are significantly lower in children with ALL at disease diagnosis. This may be due, at least in part, to a relative excess of older RBCs. [source] Measurement of HbA1c from stored whole blood samples in the Atherosclerosis Risk in Communities studyJOURNAL OF DIABETES, Issue 2 2010Elizabeth SELVIN Abstract Background:, The aims of the present study were to demonstrate the reliability of HbA1c measurements during two time periods and to compare these measurements with HbA1c distribution in the general US population. Methods:, HbA1c was measured in 14 069 whole blood samples in the Atherosclerosis Risk in Communities (ARIC) study using different HPLC instruments across two time periods, namely 2003,2004 and 2007,2008. At the time of measurement, samples had been in storage at ,70°C for up to 18 years. To assess differences in values, HbA1c measurements were repeated in 383 samples at both periods. Indirect comparisons were made by comparing our measurements against those from a nationally representative study. Results:, The coefficients of variation for quality control samples were 1.8% (n = 89) in 2003,2004 and 1.4% (n = 259) in 2007,2008. The correlation between measurements at the two time points was high (r = 0.99), but with a slight bias: 0.29% points higher in 2007,2008 vs 2003,2004 (n = 383; P < 0.0001). The comparison yielded the following Deming regression equation: y(2007,2008) = 0.073 + 1.034x(2003,2004). After alignment using this equation, the distribution of HbA1c in the ARIC study was similar to that in the national study using fresh samples. Conclusions:, Measurements of HbA1c from samples stored for up to 18 years are highly reliable when using state-of-the-art HPLC instruments, but with some bias introduced over time. The HbA1c data now available in the ARIC study should be invaluable for investigations into the clinical utility of HbA1c as a diagnostic test for diabetes. [source] Simultaneous quantification of CTN986 and its deglycosylation products in rat serum using liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2006Jifen Guo A quantitative method for the simultaneous determination of CTN986, a flavonol triglycoside, and its two deglycosylation products rutin and hirsutin in rat serum was developed and validated for the investigation of the pharmacokinetics of CTN986. Analytes were isolated from the serum samples (200,µL) prior to analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using C18 solid-phase extraction, and were separated on a Zorbax C8 reversed-phase column with an isocratic mobile phase consisting of methanol/isopropanol/water/formic acid (20:10:70:0.1, v/v/v/v). The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring in an eletrospray ionization source. Calibration was performed by internal standardization with CTN987, a flavonoid structurally similar to CTN986, and regression curves were constructed ranging from 2 to 1000,ng/mL in 200,µL serum samples. The intra- and inter-day precision values were below 11% and accuracy was between ,2.37 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of CTN986 in rats following oral and intravenous administration. Rutin and hirsutin were not detected in rat serum. Copyright © 2006 John Wiley & Sons, Ltd. [source] High-throughput determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteersRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2006Hui-chang Bi A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine carbocysteine in human plasma was developed and fully validated. After methanol-induced protein precipitation of the plasma samples, carbocysteine was subjected to LC/MS/MS analysis using electrospray ionization (ESI). The MS system was operated in the selected ion monitoring (SRM) mode. Chromatographic separation was performed on a Hypurity C18 column (i.d. 2.1,mm,×,50,mm, particle size 5,µm). The method had a chromatographic running time of 2.0,min and linear calibration curves over the concentration ranges of 0.1,20,µg/mL for carbocysteine. The lower limit of quantification (LLOQ) of the method was 0.1,µg/mL for carbocysteine. The intra- and inter-day precision was less than 7% for all quality control samples at concentrations of 0.5, 2.0, and 10.0,µg/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0,min) for carbocysteine compared with methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used to a bioequivalence study of two tablet formulations of carbocysteine in healthy volunteers. Copyright © 2006 John Wiley & Sons, Ltd. [source] Pharmacokinetic measurements of IDN 5390 using electrospray ionization tandem mass spectrometry: structure characterization and quantification in dog plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2005Liguo Song In this report, electrospray ionization tandem mass spectrometry (ESI-MS/MS) for a pharmacokinetic study of IDN 5390, a novel C- seco taxane derivative, which is under preclinical evaluation, has been investigated. Our results showed that IDN 5390 and other taxanes including paclitaxel and IDN 5109 could ionize well in not only positive-, but also in negative-ion mode. Under collision-induced dissociation (CID) conditions, these compounds could fragment into similar M- (molecular), T- (taxane ring) and S- (side chain) series ions. In positive-ion ESI, the formation of both T- and S-series ions involved the breaking of the C-13 ester bond. In negative-ion ESI, however, while the formation mechanism of S-series ions remained the same, the breaking of the C-1, carboxylic ester bond resulted in T-series ions. At optimum collision energy (CE) values, M-, T- and S-series ions of IDN 5390 in both positive- and negative-ion ESI-MS/MS spectra had good intensity. This phenomenon makes both positive- and negative-ion ESI-MS/MS good methods for IDN 5390 metabolite structural characterization, i.e. to reveal the location of modification groups in IDN 5390 metabolites versus IDN 5390 either on the side chain or the taxane ring. A liquid chromatography (LC)/ESI-MS/MS method using the multiple-reaction monitoring (MRM) technique was thereafter developed to quantify IDN 5390 in dog plasma using paclitaxel as internal standard. The method was validated using a concentration range between 5 and 1000,ng/mL and had a limit of detection of 1,ng/mL. The inter-day %CV (%coefficient of variation) of the calibration standards ranged between 4.36 and 9.64%, the intra-day %CV of the calibration standards between 0.61 and 13.44%, and the mean % accuracy of the quality control samples at the low, middle and high end of the concentration curves were 12.5, 6.8 and 9.6%, respectively. Copyright © 2005 John Wiley & Sons, Ltd. [source] Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of rabeprazole in human plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2005Jinchang Huang A simple and sensitive liquid chromatography/tandem mass spectrometry method, employing electrospray ionization, has been developed and validated to quantify rabeprazole in human plasma using omeprazole as the internal standard. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. Selected reaction monitoring was specific for rabeprazole and omeprazole (the internal standard, IS); no endogenous materials interfered with the analysis of rabeprazole and IS from blank plasma. The assay was linear over the concentration range 0.2,200,ng/mL using a 2,µL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9988,0.9994. The intra- and inter-day precision, calculated from quality control samples, were less than 6.65%. A mixture of methanol and water (50:50) was used as the isocratic mobile phase, with 0.1% of formic acid in water, that did not affect the stability of rabeprazole or IS. A simple sample preparation method of protein precipitation with methanol was chosen. The method was employed in a pharmacokinetic study after oral administration of 20,mg rabeprazole to 24 healthy volunteers. Copyright © 2005 John Wiley & Sons, Ltd. [source] Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry: practical approaches to their eliminationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2003Joachim Schuhmacher Some cases of occurrence of matrix effects (mostly ion suppression) in protein-precipitated plasma samples, and their influence on the validity of plasma concentrations and pharmacokinetic parameters, are discussed. The comparison of matrix effects using either electrospray (TurboIonspray, TISP) or atmospheric pressure chemical ionization (APCI) indicated that APCI is less prone to matrix effects. Nevertheless, TISP is usually the first choice of ionization technique since unknown thermally labile metabolites might be present in the plasma samples causing erroneous results. A high impact of ion suppression on the plasma concentrations after intravenous (i.v.) administration was found, depending on the drug formulation (vehicle). Since ion suppression caused significantly lower plasma concentrations (by a factor of up to 5.5) after i.v. dosing, the area under the curve (AUC) was underestimated and the plasma clearance was consequently erroneously high, with an impact on drug candidate selection. By simple stepwise dilution (e.g. 10-fold and 50-fold) of the supernatant of protein-precipitated plasma samples, including all calibration and quality control samples, the matrix effects were recognized and eliminated. Copyright © 2003 John Wiley & Sons, Ltd. [source] A simple and rapid high-performance liquid chromatography method for determination of alendronate sodium in beagle dog plasma with application to preclinical pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 2 2010Jian Meng Abstract A simple and rapid high performance liquid chromatographic (HPLC) method for quantifying alendronate in beagle dog plasma was developed, validated and applied to a pharmacokinetic study. The sample preparation involved coprecipitation with CaCl2 and derivatization with o -phthalaldehyde. Chromatographic separation was achieved on a DiamonsilÔ C18 (250 × 4.6,mm, 5,µm) using acetonitrile,0.4% EDTA-Na2 (16:84, v/v) containing 0.034% of NaOH as mobile phase. The fluorimetric detector was operated at 339,nm (excitation) and 447 nm (emission). The linearity over the concentration range of 5.00,600,ng/mL for alendronate was obtained and the lower limit of quantification was 5.00,ng/mL. For each level of quality control samples, inter-day and intra-day precisions were less than 8.52 and 7.42% and accuracies were less than 9.07%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70,mg alendronate sodium to beagle dogs, the maximum plasma concentration (Cmax) and elimination half-life were 152,±,27.3 and 1.75,± 0.267,h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd. [source] Solid-phase extraction and analysis of paroxetine in human plasma by ultra performance liquid chromatography,electrospray ionization mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 2 2010Mitesh Bhatt Abstract A rapid, sensitive and rugged solid-phase extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for determination of paroxetine in human plasma. The procedure for sample preparation includes simple SPE extraction procedure coupled with Hypersil Gold C18 column (100 mm , 2.1 mm, i.d., 1.9 ,m) with isocratic elution at a flow-rate of 0.350 mL/min and fluoxetine was used as the internal standard. The analysis was performed on a triple-quadrupole tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization. Using 500 ,L plasma, the methods were validated over the concentration range 0.050,16.710 ng/mL for paroxetine, with a lower limit of quantification of 0.050 ng/mL. The intra- and inter-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 69.2 and 74.4% for paroxetine and fluoxetine respectively. Total run time was only 1.9 min. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2009 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography method for determination of carnosic acid in rat plasma and its application to pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 7 2009Haixia Yan Abstract A sensitive and reproducible high-performance liquid chromatography method with ultraviolet detection (UV) was developed for the determination of carnosic acid (CA) in rat plasma. After simple acidification and liquid,liquid extraction of plasma samples using gemfibrozil as an internal standard, the supernatant was evaporated to dryness under a gentle stream of nitrogen. The residue was reconstituted in 200 µL before being injected into the chromatographic system. The analysis was performed on a C18 column protected by an ODS guard column using acetonitrile,0.1% phosphoric acid (55:45, v/v) as mobile phase, and the wavelength of the UV detector was set at 210 nm. The calibration curve was linear over the range of 0.265,265.0 µg/mL with a correlation coefficient of 0.9997. The recovery for plasma samples of 0.530, 13.25, 132.5 and 265.0 µg/mL was 72.2, 87.9, 90.4 and 94.7%, respectively. The intra-day and inter-day relative standard deviations for the measurements of quality control samples were less than 3.1%. The stability of the plasma samples was also validated. This method was successfully used to study the pharmacokinetics and bioavailability of CA in rats. Copyright © 2009 John Wiley & Sons, Ltd. [source] Rapid analysis of tetracaine for a tape stripping pharmacokinetic study using short-end capillary electrophoresisBIOMEDICAL CHROMATOGRAPHY, Issue 5 2009F. Al-Otaibi Abstract A rapid and simple short-end (reverse) capillary zone electrophoresis method was developed and validated for the separation and quantification of tetracaine in skin using tape samples. The separation was performed in a 485 mm (400 mm to window) × 50 µm internal diameter fused silica capillary using a background electrolyte of phosphoric acid,Tris pH2.5 at ,25 kV. The extraction of tetracaine from tape samples was achieved using methanol diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and tetracaine were 1.25 and 1.36 min, respectively. The limit of quantification for tetracaine was 50 µg, with a signal-to-noise ratio greater than 10. The calibration curve was linear from 50 to 1200 µg with r2 greater than 0.99. The CV for both within- and between-assay imprecision and the percentage inaccuracy for the quality control samples including lower and upper limits of quantitation were <12.1% and <11%, respectively. The absolute mean recovery of tetracaine was >97%. The accuracy and selectivity of this method allowed the rapid measurement of tetracaine in tape samples obtained from a skin tape stripping study of local anaesthetics in healthy subjects. Copyright © 2008 John Wiley & Sons, Ltd. [source] A rapid and specific approach for direct measurement of donepezil concentration in human plasma by LC-MS/MS employing solid-phase extractionBIOMEDICAL CHROMATOGRAPHY, Issue 2 2009Hiten J. Shah Abstract A selective, rapid and simple liquid chromatography,tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C8, 5 µm, 100 × 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15,50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were ,8.92 and 10.35% and accuracy (%RE) were ,7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps. Copyright © 2008 John Wiley & Sons, Ltd. [source] Simultaneous determination of mifepristone and monodemethyl-mifepristone in human plasma by liquid chromatography,tandem mass spectrometry method using levonorgestrel as an internal standard: application to a pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 1 2009Cheng Tang Abstract A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine mifepristone and monodemethyl-mifepristone in human plasma using levonorgestrel as the internal standard (IS). After solid-phase extraction of the plasma samples, mifepristone, monodemethyl-mifepristone and the IS were subjected to LC-MS/MS analysis using electro-spray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an XTERRA MS C18 column (150 × 2.1 mm i.d., 5 µm). The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration ranges of 5,2000 ng/mL for mifepristone and monodemethyl-mifepristone. The recoveries of the method were found to be 94.5,103.7% for mifepristone and 70.7,77.3% for monodemethyl-mifepristone. The method had a lower limit of quantification (LLOQ) of 5.0 ng/mL and a lower limit of detection (LOD) of 1.0 ng/mL for both mifepristone and monodemethyl-mifepristone. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 10, 100 and 1000 ng/mL. These results indicate that the method was efficient with a short run time (4.5 min) and acceptable accuracy, precision and sensitivity. The validated LC-MS/MS method was successfully used in a pharmacokinetic study in healthy female volunteers after oral administration of 25 mg mifepristone tablet. Copyright © 2008 John Wiley & Sons, Ltd. [source] LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparationBIOMEDICAL CHROMATOGRAPHY, Issue 11 2007Joseph Kim Abstract As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 × 150 mm, 5 µm, 120 Å) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 µL sample volume. The achieved r2 coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between ,4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between ,8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was ,4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies. Copyright © 2007 John Wiley & Sons, Ltd. [source] Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 9 2007Osama Y. Al-Dirbashi Abstract N -acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a ,dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d3 -NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C8 minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d3 -NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 µmol/L with limit of quantification at 1 µmol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9,102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of S -phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography-tandem mass spectrometric methodBIOMEDICAL CHROMATOGRAPHY, Issue 6-7 2006Yinghe Li Abstract S -phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S- phenylmercapturic acid in human urine. Isotope-labeled S- phenylmercapturic acid- d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S- phenylmercapturic acid (m/z 238 , 109) and S- phenylmercapturic acid - d5 (m/z 243 , 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400,200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved. Copyright © 2006 John Wiley & Sons, Ltd. [source] Determination of antiviral nucleoside analogues AM365 and AM188 in perfusate and bile of the isolated perfused rat liver using HPLCBIOMEDICAL CHROMATOGRAPHY, Issue 3 2006Jiping Wang Abstract Development, validation and application of an HPLC assay for new antiviral nucleoside analogues AM365 and AM188 in isolated perfused rat liver perfusate and bile were performed. An analytical column (Phenosphere-NEXT, 250 × 4.6 mm, C18, 4 µm, Phenomenex) was used in tandem with a guard column (4 × 3 mm, C18, Phenomenex) and operated at 25°C. The mobile phase [methanol:10 mmol/L sodium orthophosphate buffer (pH 7.0), 15:85, v/v] was pumped at 1 mL/min. The signal from a diode array detector was collected from 190 to 300 nm. The chromatogram was processed at 220 and 252 nm for AM365 and AM188, respectively. The HPLC method was validated by six intraday and seven interday runs. Standard curves were linear in the range 0.125,8.00 µg/mL for AM365 and AM188, and the lower limit of quantification for AM365 and AM188 was 0.125 µg/mL. Mean interday precision and accuracy of IPL perfusate quality control samples were within 8.8%, and mean intraday precision and accuracy were within 13.1%. The assay has been successfully used in the study of metabolism and disposition of AM365 in the isolated perfused rat liver. Copyright © 2005 John Wiley & Sons, Ltd. [source] HPLC method for determination of DRF-4367 in rat plasma: validation and its application to pharmacokinetics in Wistar rats,BIOMEDICAL CHROMATOGRAPHY, Issue 8 2004Ramesh Mullangi Abstract A speci,c, 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°C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 × 250 mm, 5 µm). 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 ,ow 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 quanti,cation 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 (r2 > 0.999) in the concentration range 0.1,20 µg/mL. Absolute recovery was >86% from rat plasma for both analyte and IS. The lower limit of quanti,cation of DRF-4367 was 0.1 µg/mL. The inter- and intra-day precisions in the measurement of quality control samples, 0.1, 0.3, 8.0 and 15.0 µg/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°C. The application of the assay to a pharmacokinetic study in rats is described. Copyright © 2004 John Wiley & Sons, Ltd. [source] SPE/RIA vs LC/MS for measurement of low levels of budesonide in plasmaBIOMEDICAL CHROMATOGRAPHY, Issue 1 2003H. Dimova Abstract A radioimmunoassay is described that measures budesonide in plasma after solid-phase extraction (SPE/RIA) of the analyte. The performance of the assay was compared with that of a selective LC/MS method. The limit of quantitation of budesonide determined for the LC/MS and SPE/RIA assay was 50,pg/mL and 120,pg/mL, respectively. Based on quality control samples, a higher variability was observed for the SPE/RIA (CV between 4.5 and 23.0%) than for the LC/MS method (CV between 7.5 and 12.5%). Plasma samples obtained from healthy volunteers after administration of budesonide rectal foam were assayed by both methods. In a subset of samples, these results were compared with those measured by direct RIA to evaluate the selectivity of two assays. About two times higher budesonide levels were measured with the direct RIA (lacking the extraction step), presumably because of cross-reactivity with budesonide metabolites, indicating that the extraction step in SPE/RIA is necessary for selectivity. Both SPE/RIA and LC/MS methods were found to be selective, sensitive and suitable for pharmacokinetic studies. Results obtained from the two methods were compared with a number of statistical methods. Ratios of results obtained for the clinical samples were close to 1 (ratio LC-MS/ SPE/RIA,=,0.98,±,0.27). Linear regression indicated a slope of 1.17,±,0.0378. The concordance correlation (r,=,0.91) indicated that the agreement between both methods was fair while the Bland,Altman plot indicated that the agreement was less pronounced at higher concentrations (1,3,ng/mL). In summary, the results confirm that the SPE/RIA is an alternative to HPLC/MS and that among the statistical methods tested the concordance correlation analysis was judged to be the most informative test to assess the comparability of two methods. Copyright © 2002 John Wiley & Sons, Ltd. [source] | |||||||||||||