Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.     

Simultaneous determination of gemcitabine and its main metabolite, dFdU, in plasma of patients with advanced non-small-cell lung cancer by high-performance liquid chromatography-tandem mass spectrometry

Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC) is a pyrimidine antimetabolite employed against several human malignancies. It undergoes intracellular activation to the pharmacologically active triphosphate form (dFdCTP) and metabolic inactivation to the metabolite 2',2'-difluorodeoxyuridine (dFdU). In order to investigate the human plasma pharmacokinetics of dFdC and dFdU, we developed and validated an HPLC-MS/MS method, adding 2'-deoxycytidine as internal standard and simply precipitating the protein with acetonitrile. The method requires a small sample (125 microl), and it is rapid and selective, allowing good resolution of peaks from the plasma matrix in only 7 min. It is sensitive, precise and accurate, with overall precision, expressed as CV%, always less than 10.0% for both analytes and high recovery: > or = 80%. The limits of detection for dFdC and dFdU were 0.1 and 1.1 ng/ml, but considering the high concentrations in the plasma of patients investigated, we set the limit of quantitation at 20 ng/ml (0.08 microM) for dFdC and 250 ng/ml for dFdU, and validated the assay up to the dFdC concentration of 6.0 microg/ml (22.8 microM). The method was successfully used to study the drug pharmacokinetics in patients with advanced non-small-cell lung cancer in a phase II trial with gemcitabine administered as a fixed dose-rate infusion.     

LC-MS/MS method for the quantification of myo- and chiro-inositol as the urinary biomarkers of insulin resistance in human urine

A simple LC‐MS/MS method has been developed and validated for the quantification of endogenous myo‐ and chiro‐inositol in human urine. myo‐ and chiro‐Inositol were completely resolved from other carbohydrates and there were no interference peaks in human urine. The correlation coefficient (n = 3) was greater than 0.9991 over the range 0.05–25.0 µg/mL with the weighted (1/C²) least square method. Precision (%RSD) and accuracy (%RE) were 0–10.0% and 0–6.0% for the intra‐day assay (n = 5) and 0–14.3% and 0–10.0% for the inter‐day assay (n = 5). myo‐ and chiro‐Inositol have been shown to be stable in human urine stored at room temperature and for three freeze–thaw cycles. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantitation of enalapril and enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.     

Quantification of pseudoephedrine in human plasma by LC-MS/MS using mosapride as internal standard

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of pseudoephedrine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple-reaction monitoring mode using the respective [M + H](+) ions, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 2-1000 ng/mL pseudoephedrine in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 9% for pseudoephedrine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze 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.     

Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Gemcitabine triphosphate (dFdCTP) is a highly active metabolite of gemcitabine. It is formed intra-cellularly via the phosphorylation of gemcitabine by deoxycytidine kinase. The monitoring of dFdCTP in human peripheral blood mononuclear cells (PBMCs), in addition to plasma concentrations of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine, is considered very useful in determining pharmacokinetic-pharmacodynamic relationships.We describe a novel sensitive assay for the quantification of dFdCTP in human PBMCs. The method is based on weak anion-exchange liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS). The assay has been validated from 1 ng/ml (lower limit of quantification, LLOQ) to 25 ng/ml (upper limit of quantification, ULOQ) using 180 microl aliquots of PBMC extracts containing approximately 0.648 mg protein or 3.8 x 10(6) lysed PBMCs. The LLOQ is equivalent to 94 fmol/10(6) cells (1 ng/ml = 0.18 ng/180 microl or 0.18 ng/0.648 mg protein = 0.047 ng/10(6) cells or 94 fmol/10(6) cells). This highly sensitive assay is capable of quantifying about 200-fold lower concentrations of dFdCTP in human PBMCs than currently available methods.     

Determination of L-carnitine in food supplement formulations using ion-pair chromatography with indirect conductimetric detection

A novel method for the determination of L-carnitine in food supplement formulations was developed and validated, using ion-pair chromatography with indirect conductimetric detection. The chromatographic method was based on a non-polar (C18) column and an aqueous octanesulfonate (0.64 mM) eluent, acidified with trifluoroacetic acid (5.2 mM). The retention time was 5.4 min and the asymmetry factor 0.65. A linear calibration curve from 10 to 1000 microg/ml (r= 0.99998), with a detection limit of 2.7 microg/ml (25 microl injection volume), a repeatability %RSD of 0.8 (40 microg/ml, n = 5) and reproducibility %RSD of 2.6 were achieved. The proposed method was applied for the determination of carnitine in oral solutions and capsules. No interference from excipients was found and the only pretreatment step required was the appropriate dilution with the mobile phase. Recovery from spiked samples was ranged from 97.7 to 99.7% with a precision (%RSD, n = 3) of 0.01-2.1%.     

Development and validation of a high‐performance liquid chromatography–tandem mass spectrometry method for the determination of the novel proteasome inhibitor CEP‐18770 in human plasma and its application in a clinical pharmacokinetic study

CEP‐18770, [(1R)‐1‐{[(2S,3R)‐3‐hydroxy‐2‐{[(6‐phenyl‐2‐pyridinyl)carbonyl]amino}butanoyl]amino}‐3‐methylbutyl]boronic acid, is a novel proteasome inhibitor, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to measure the drug in human plasma, based on simple protein precipitation with acetonitrile after the addition of irbesartan as internal standard. The method requires a small volume of sample (100 µl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise and accurate, with overall precision, expressed as coefficient of variation (CV%), always < 10.0%, accuracy in the range 93.8–107.7% and high recovery, close to 100%. The limit of detection is 0.01 ng/ml and the lower limit of quantitation (LLOQ) is 0.20 ng/ml. The assay was validated in the range from the LLOQ up to 50.00 ng/ml. This is the first method developed and validated for analyzing a proteasome inhibitor with a boronic‐acid‐based structure in human plasma. The method was successfully applied to study the pharmacokinetics of CEP‐18770 in cancer patients with solid tumors or multiple myeloma who had received the drug as a short intravenous bolus during the initial Phase I trial. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

Simultaneous determination of Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta9-tetrahydrocannabinol in human plasma by high-performance liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the simultaneous confirmatory analysis of three forensic most relevant cannabinoids, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), by means of high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in human plasma was developed and fully validated. Sample clean-up was performed by automated silica-based solid-phase extraction and the separation was carried out using a PhenylHexyl column (50 x 2 mm i.d., 3 micro m) and acetonitrile-5 mM ammonium acetate gradient elution. Data were acquired with an API 3000 LC/MS/MS system equipped with a turboionspray interface and triple quadrupole mass analyzer using positive electrospray ionization and multiple reaction monitoring. Two MS/MS transitions for each substance were monitored and deuterated analogues of analytes were used as internal standards for quantitation. The limit of quantitation was 0.8 ng ml(-1) for THC, 0.8 ng ml(-1) for 11-OH-THC and 4.3 ng ml(-1) for THC-COOH and linearity with a correlation coefficient r(2) = 0.999 was achieved up to 100 ng ml(-1) for THC and 11-OH-THC and 500 ng ml(-1) for THC-COOH. The limits of detection were 0.2 ng ml(-1) for THC, 0.2 ng ml(-1) for 11-OH-THC and 1.6 ng ml(-1) for THC-COOH. The developed LC/MS/MS method was also successfully used for the determination of THC-COOH-glucuronide, the phase II metabolite of THC-COOH.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma using liquid-liquid extraction

A sensitive high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 408-235 for sitagliptin and m/z 310-148 for the internal standard. The assay exhibited a linear dynamic range of 0.1-250 ng/mL for sitagliptin in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

Detection and validated quantification of toxic alkaloids in human blood plasma--comparison of LC-APCI-MS with LC-ESI-MS/MS

Poisonings with toxic plants may occur after abuse, intentional or accidental ingestion of plants. For diagnosis of such poisonings, multianalyte procedures were developed for detection and validated quantification of the toxic alkaloids aconitine, atropine, colchicine, coniine, cytisine, nicotine and its metabolite cotinine, physostigmine, and scopolamine in plasma using LC-APCI-MS and LC-ESI-MS/MS. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a C8 base select separation column and gradient elution (acetonitrile/ammonium formate, pH 3.5). Calibration curves were used for quantification with cotinine-d(3), benzoylecgonine-d(3), and trimipramine-d(3) as internal standards. The method was validated according to international guidelines. Both assays were selective for the tested compounds. No instability was observed after repeated freezing and thawing or in processed samples. The assays were linear for coniine, cytisine, nicotine and its metabolite cotinine, from 50 to 1000 ng/ml using LC-APCI-MS and 1 to 1000 ng/ml using LC-ESI-MS/MS, respectively, and for aconitine, atropine, colchicine, physostigmine, and scopolamine from 5 to 100 ng/ml for LC-APCI-MS and 0.1 to 100 ng/ml for LC-ESI-MS/MS, respectively. Accuracy ranged from -38.6 to 14.0%, repeatability from 2.5 to 13.5%, and intermediate precision from 4.8 to 13.5% using LC-APCI-MS and from -38.3 to 8.3% for accuracy, from 3.5 to 13.8%, for repeatability, and from 4.3 to 14.7% for intermediate precision using LC-ESI-MS/MS. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. With the exception of the greater sensitivity and higher identification power, LC-ESI-MS/MS had no major advantages over LC-APCI-MS. Both presented assays were applicable for sensitive detection of all studied analytes and for accurate and precise quantification, with the exception of the rather volatile nicotine. The applicability of the assays was demonstrated by analysis of plasma samples from suspected poisoning cases.     

Determination of 3-hydroxy pterocarpan, a novel osteogenic compound in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study

A rapid, sensitive and selective LC‐MS/MS method for the quantitative analysis of 3‐hydroxy pterocarpan (S006‐1709) in female rat plasma has been developed and validated. A Discovery RP₁₈ column was used for the chromatographic elution using acetonitrile and 0.1% acetic acid in water as mobile phase (80:20 v/v) at the flow rate of 0.5 mL/min. MS/MS analysis was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative ion mode using biochanin as an internal standard (IS). Extraction of S006‐1709 and IS from rat plasma was done by liquid–liquid extraction method using diethyl ether. The LC‐MS/MS method was sensitive with 1.95 ng/mL as the limit of detection and 3.9 ng/mL as the lower limit of quantification. The method was linear in the concentration range of 3.9–1000 ng/mL. The percentage bias for intraday and interday accuracy was not greater than 4.2 and the %RSD for intraday and interday precision was not greater than 13.2. The recoveries of S006‐1709 and IS were 73.9–79.3 and 85.7%, respectively. S006‐1709 was found to be stable in various stability studies. The validated LC‐MS/MS method was successfully applied for the oral pharmacokinetics study of S006‐1709 at 10 mg/kg in female Sprague–Dawley rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

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 x 3.0 mm i.d., particle size 5 microm). 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 < or = 9.1%, an inter-assay precision of < or = 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.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of metformin and gliclazide in human plasma by liquid chromatography-tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine gliclazide and metformin in human plasma using huperzine A as the internal standard (IS). After acetonitrile-induced protein precipitation of the plasma samples, gliclazide, metformin and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 10-10,000 ng ml(-1) for gliclazide and 7.8-4678.9 ng ml(-1) for metformin. The recoveries of the method were found to be 71-104%. The lower limits of quantification (LOQ) of the method were 10.0 and 7.8 ng ml(-1) for gliclazide and metformin, respectively. The intra- and interday precision was less than 15% for all quality control samples at concentrations of 100, 500, and 2000 ng ml(-1). The validated LC/MS/MS method has been used to study bioequivalence in healthy volunteers. These results indicate that the method was efficient with a very short running time (2.0 min) for metformin and gliclazide compared to the methods reported in the literature. The presented method had acceptable accuracy, precision and sensitivity and was used in clinical bioequivalence study.     

Separation, determination and identification of the diastereoisomers of podophyllotoxin and its esters by high-performance liquid chromatography/tandem mass spectrometry

A rapid and stable high-performance liquid chromatography-diode array detection (HPLC-DAD) and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) method were developed and validated for the separation, determination, and identification of eight pairs of diastereoisomers of podophyllotoxin and its esters at C-2 position. The separation was carried out on BDS Hypersil C18 column with CH3OH-CH3CN-H2O as the mobile phase in a gradient program. Interestingly, every 2alpha-H compound migrated before its corresponding 2beta-H epimer under optimum conditions. Also, the [M+NH(4)](+) of all eight pairs of compounds was observed in the HPLC-ESI/MS spectra. The characteristic elimination from the precursor protonated ions and the product ions at m/z 397, 313, 282, and 229 were the common diagnostic masses. The ion ratios of relative abundance [M-ROH+H](+) (ion 397) to [M+NH(4)](+), [A+H](+) (ion 313) to [M-ROH+H](+), and [M-ROH-ArH+H](+) (ion 229) to [M-ROH+H](+) in the ESI/MS/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. Thus, by using identical sample solutions and chromatographic conditions (including the same columns and gradient programs), the combination of DAD and MS/MS data permitted the separation and identification of the eight pairs of diastereoisomers of the podophyllotoxin and its esters in the mixture. The method could be used in rapidly identifying the purity and monitoring of the epimerization of 2-H of podophyllotoxin and its analogues from natural products, chemical reactions, and pharmaceutical metabolism.     

Development and validation of an LC-MS/MS analysis for simultaneous determination of delphinidin-3-glucoside, cyanidin-3-glucoside and cyanidin-3-(6-malonylglucoside) in human plasma and urine after blood orange juice administration

Blood orange juice has a high content in anthocyanins, especially represented by delphinidin-3-glucoside (D3G), cyanidin-3-glucoside (C3G) and cyanidin-3-(6-malonylglucoside) (CMG). An LC-MS/MS method for the simultaneous determination of D3G and C3G in human plasma and urine was developed and validated. After sample preparation by SPE, chromatographic separation was performed with an RP-C(18) column, using a water/methanol linear gradient. The quantitation of target compounds was determined by multiple reaction monitoring (MRM) mode, using ESI. The method showed good selectivity, sensitivity (LOD = 0.05 and 0.10 ng/mL for C3G in plasma and urine, respectively; LOD = 0.10 ng/mL for D3G in plasma and urine), linearity (0.20-200 ng/mL; r >or= 0.998), intra- and interday precision and accuracy (相似文献   

Application of 1-alkylamines to a liquid chromatographic/turbo ionspray tandem mass spectrometric method for quantifying metabolites of a new bone anabolic agent,TAK-778, in human serum

We investigated the application of alkylamines, as additives to the mobile phase, to a quantification method for the metabolites, M-III and M-IV, of TAK-778, which is a new bone anabolic agent, in human serum using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Prior to setting up the analytical method, we found that 1-alkylamines co-existing with M-III and M-IV in the turbo ionsprayed solution formed 1-alkylammonium adduct molecules of these metabolites during the ionization process, and the abundance of the adduct ions was considerably higher than that of protonated molecules ([M + H](+)s) of these metabolites. Based on these findings, we investigated a variety of 1-alkylamines and their spiked concentrations in the mobile phase for LC/MS/MS analysis to obtain higher sensitivities for the quantification of these metabolites. After these examinations, we found that 1-hexylamine at a final concentration of 0.05 mmol l(-1) was the most suitable additive for the mobile phase, and set the selected reaction monitoring (SRM) ions for the 1-hexylammonium adduct molecule and [M + H](+), allowing about a fivefold gain in the SRM chromatographic peak compared with that without 1-hexylamine. The adduct ion was considered to be formed by interaction between the amino group of 1-hexylamine and the phosphoryl group of M-III and M-IV. The internal standard (I.S.) used was deuterated M-III for each metabolite. The analytes and I.S. were extracted with diethyl ether from serum samples at neutral pH and injected into the LC/MS/MS system with a turbo ionspray interface. The limit of quantification for both analytes was 0.5 ng ml(-1) when 0.1 ml of serum was used, and the calibration curves were linear in the range 0.5-100 ng ml(-1). The method was precise; the intra- and inter-day precisions of the method were not more than 5.6%. The accuracy of the method was good, with deviations between added and calculated concentrations of M-III and M-IV being typically within 16.6%. This method provided reliable pharmacokinetic data for M-III and M-IV after the intramuscular administration of TAK-778 sustained-release formulation in humans.     

Determination of Abacavir in maternal plasma, amniotic fluid, fetal and placental tissues by a polarity switching liquid chromatography/tandem mass spectrometry method

A rapid and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of concentrations of the carbocyclic nucleoside antiviral Abacavir in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. All tissue samples were homogenized in water prior to analysis and all samples were prepared by acetonitrile precipitation followed by dilution with HPLC-grade water. Separation of the analyte and internal standard from the matrices was achieved on a C(8) analytical column (2.1 x 150 mm, 5 microm particle size). The mobile phase consisted of 10 mM ammonium acetate/acetonitrile using a gradient method at a flow rate of 0.25 mL/min for all matrices. The method yields retention times of approximately 3.4 and 5.1 min for the internal standard (Azidouridine) and Abacavir, respectively. For all matrices the limit of detection was approximately 1 ng/ml. Recoveries from the different matrices ranged from 53-87% for Abacavir and from 69-84% for Azidouridine. Within- and between-run precision (%RSD) and accuracy (%Error) were under 15% for all matrices.