Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers

A simple, sensitive and selective LC-MS-MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C(18) column with a mobile phase consisting of 0.2% formic acid-methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508-5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC(0-24), AUC(0-infinity), C(max), T(max) and t(1/2) of huperzine A were 16.35 +/- 3.42 vs 16.38 +/- 3.61 ng h/mL, 17.53 +/- 3.80 vs 17.70 +/- 3.97 ng h/mL, 2.47 +/- 0.49 vs 2.51 +/- 0.51 ng/mL, 1.3 +/- 0.4 vs 1.2 +/- 0.3 h and 5.92 +/- 0.75 vs 6.18 +/- 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent.     

Determination of ligustilide in rat blood and tissues by capillary gas chromatography/mass spectrometry

A gas chromatography/mass spectrometry (GC/MS) method was developed to study the pharmacokinetics of ligustilide following oral administration to rats. The method was used for the analysis of samples taken from rats. Biological samples were prepared by liquid-liquid extraction (LLE) using an n-hexane-ether (2:1) solvent mixture for a sample clean-up step and analyzed by GC/MS with a quadrupole MS detector in selected ion monitoring mode (m/z 190). The calibration curves were linear over the concentration range 0.172-8.60 microg/mL (r > 0.99) for blood samples and a different range (r > 0.99) for different tissue samples. The limit of detection (LOD) was 1.0 ng/mL or 1.0 ng/g (three times the signal-noise ratio). Within- and between-day precision expressed as the relative standard deviation (RSD) for the method was 1.58-3.88 and 2.99-4.91%, respectively. The recovery for all samples was >80%, except for liver samples (>70%). The main pharmacokinetic parameters obtained were: T(max) = 0.65 +/- 0.07 h, C(max) = 1.5 +/- 0.2 microg/mL, AUC = 34 +/- 6 h microg/mL and K(a) = 3.5 +/- 0.6/h. The experimental results showed that ligustilide was easily absorbed, but its elimination was slow, from 3 to 12 h after oral administration. The concentrations of ligustilide in rat cerebellum, cerebrum, spleen and kidney were higher than those in other organs.     

Determination of trimebutine maleate in rat plasma and tissues by using capillary zone electrophoresis.

A simple and rapid capillary zone electrophoresis method was developed for the determination of trimebutine maleate in rat plasma and tissues. Rat plasma and tissue homogenates were mixed with acetonitrile containing internal standard, ephedrine hydrochloride, and then centrifuged. The supernatant was dried under a stream of nitrogen, and the residue was reconstituted in methanol-water (1:1). The electrophoresis was performed in uncoated capillary with 30 mmol/L phosphate buffer of pH 6.0 as the separation electrolyte. The applied voltage was 10 kV and the UV detection was set at 214 nm. The peak height ratio vs concentration in plasma or homogenates was linear over the range of 5-500 ng/mL and the limit of quantitation was 5 ng/mL. The intra- and inter-day precision was RSD < 14% and <15%. The accuracy was relative error (RE) within +/- 14%. This method was applied to studying the pharmacokinetics and tissue distribution after a single dose of trimebutine maleate was administrated to the rats. The T(max), AUC, C(max) and t(1/2) were 30 min, 7.8 x 10(2) (ng/mL) min, 39 ng/mL and 1.7 x 10(2) min. The drug distribution was found in a decreasing order of liver, kidney, spleen, lung and heart.     

A simple HPLC method for the quantification of mizoribine in human serum: pharmacokinetic applications

A simple high-performance liquid chromatographic (HPLC) method was developed and validated for the quantification of mizoribine in human serum. After the addition of 70% perchloric acid and 3-methylxanthine (50 microg/mL, internal standard) to human serum, the samples were mixed and centrifuged at 12,000 rpm (1432 g) for 10 min. The supernatant was injected onto a C(18) column eluted with a mobile phase of 20 mm Na2HPO4 and methanol (93:7, v/v, pH 3) containing 0.04% octanesulfonic acid and detected utilizing an ultraviolet detector at 275 nm. The linear calibration curve was obtained in the concentration range of 0.1-4.0 microg/mL and the lower limit of quantification was 0.1 microg/mL. This method was validated with selectivity, linearity, precision and accuracy. In addition, the method was successfully applied to estimate the pharmacokinetic parameters of mizoribine in Korean subjects following an oral administration of 100 mg mizoribine (two Bredinine 50 mg tablets). The maximum serum concentration (C(max)) of 2.30 +/- 0.83 microg/mL was reached 2.27 +/- 0.66 h after an oral dose. The mean AUC(0-12 h) and the elimination half-life (t(1/2)) were 13.2 +/- 4.79 microg h/mL and 3.10 +/- 0.74 h, respectively.     

Pharmacokinetic study of puerarin in rat serum by liquid chromatography tandem mass spectrometry

A highly sensitive and specific atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry method was developed for serum pharmacokinetic studies of puerarin in rats. Chromatography was carried out on a reversed-phase Phenomenex Synergi 4 microm Fusion-RP80 column (150 x 2.0 mm i.d.) using a mobile phase consisting of acetonitrile-water (10:90, v/v) in 10 mm NH(4)OAc with a flow rate of 0.2 mL/min. Puerarin was analyzed in the multiple reaction monitoring mode with a precursor/product ion transition of m/z 415/267. The method was demonstrated to be specific and sensitive, and a linear response was observed over a range of 2-5000 ng/mL in rat serum. The validated method was successfully applied to the characterization of the pharmacokinetics of puerarin in rat serum after oral administration to spontaneously hypertensive rats. The blood concentration-time profile of puerarin showed a rapid initial increase, reaching a maximum and then declining within 1 h. Puerarin could not be detected after 24 h. The main pharmacokinetic parameters for puerarin after oral administration were as follows: C(max) (3.54 +/- 2.03 mg/L), T(max) (0.68 +/- 0.37 h), AUC(0-t) (7.29 +/- 3.79 mg h/L), AUC(0-infinity) (9.17 +/- 4.87 mg h/L), T(1/2) (1.7 +/- 0.6 h), CL/F (7.24 +/- 4.27 L/h/kg) and V/F (17.88 +/- 13.55 L/h/kg).     

Determination of donepezil hydrochloride (E2020) in plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies in healthy, young, Chinese subjects

A sensitive, simple, and specific liquid chromatographic method coupled with electrospray ionization-mass spectrometry for the determination of donepezil in plasma is developed, and its pharmacokinetics in healthy, male, Chinese is studied. Using loratadine as the internal standard, after extraction of the alkalized plasma by isopropyl alcohol-n-hexane (3:97, v/v), solutes are separated on a C(18) column with a mobile phase of methanol-acetate buffer (pH 4.0) (80:20, v/v). Detection is performed with a time-of-flight mass spectrometer equipped with an electrospray ionization source operated in the positive-ionization mode. Quantitation of E2020 is accomplished by computing the peak area ratio (donepezil [M+H](+) m/z 380-loratadine [M+H](+) m/z 383) and comparing them with the calibration curve (r = 0.9998). The linear calibration curve is obtained in the concentration range 0.1-15 ng/mL. The limit of quantitation is 0.1 ng/mL. The mean recovery of E2020 from human plasma is 99.4% +/- 6.3% (ranging 93.4-102.6%). The inter- and intraday relative standard deviation is less than 15%. After an oral administration of 5 mg E2020 to 20 healthy Chinese volunteers, the main pharmacokinetic parameters of E2020 are as follow: T(max), 3.10 +/- 0.55 h; t((1/2)), 65.7 +/- 12.8 h; C(max), 10.1 +/- 2.02 ng/mL; MRT, 89.4 +/- 13.4 h; and CL/F, 9.9 +/- 4.3 L/h.     

Sensitive assay for triazolam in plasma following low oral doses.

At low doses of triazolam currently recommended increased assay sensitivity is required for measurement of low plasma concentrations. A highly sensitive capillary gas chromatographic analytical method with a limit of detection of 0.02 ng/ml was developed and used to describe the pharmacokinetics of triazolam following the oral intake of 0.125, 0.250 and 0.375 mg. Six male subjects were studied with blood sampling at the following times: 0, 15, 30 and 45 min and 1, 1.5, 2.0, 2.5, 3, 4, 5, 6 and 8 h. The mean pharmacokinetic parameters for the three doses, respectively, were as follows: half-life, 2.7 +/- 0.4, 3.2 +/- 0.5 and 3.2 +/- 0.6 h; apparent oral clearance, 302.3 +/- 59.0, 260.2 +/- 67.9 and 328.6 +/- 77.8 ml/min; apparent volume of distribution, 64.3 +/- 9.6, 62.0 +/- 12.6 and 73.3 +/- 7.7 l; time to maximum concentration, 0.7 +/- 0.2, 0.6 +/- 0.1 and 0.8 +/- 0.3 h; maximum concentration, 2.2 +/- 0.3, 4.3 +/- 0.6 and 5.0 +/- 0.5 ng/ml; and the area under the concentration-time curve (AUC) up to 8 h, 6.8 +/- 1.2, 16.8 +/- 2.9 and 19.6 +/- 3.5 ng/ml h; and AUC extrapolated to infinity, 8.5 +/- 1.7, 21.4 +/- 4.4 and 26.3 +/- 7.2 ng/ml h. There were no significant differences in the half-life, clearance, volume of distribution and time to maximum concentration among the three doses. The AUC was significantly different on the three occasions and was linearly correlated with dose: r = 0.64 (p less than 0.005).     

Determination of stavudine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of stavudine in human serum, using didanosine as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode. The method was linear over the studied range (10-2000 ng/mL), with r(2) > 0.99, and the run time was 4 min. The intra- and inter-assay precisions (%) were in the ranges 0.1-13.6 and 2.6-9.9, respectively, and the intra- and inter-assay accuracies were >92%. The absolute recoveries were approximately 100% (10 ng/mL), 98% (30 ng/mL), 105% (750 ng/mL) and 105% (1500 ng/mL). The limits of detection and quantitation were 4 and 10 ng/mL, respectively. The analytical method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (40 mg) of reference and two test stavudine formulations, in an open, three-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration), AUC(0-10) and AUC(0-inf) (areas under the serum concentration vs. time curve from time zero to 10 h and to infinity, respectively), were in the range 80-125%, which supports the conclusion that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of stavudine absorption.     

Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry

Anastrozole (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-1.3-phenylene]bis(2-methylpropionitrile)) and exemestane (6-methylenandrostan-1,4-diene-3,17-dione) are therapeutically used to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes they may be used to counteract adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase plasma testosterone concentrations. Excretion study urine samples and spot urine samples from women suffering from metastatic breast cancer, being treated with anastrozole or exemestane, were collected and analyzed to develop/optimize a detection system for anastrozole and exemestane to allow the identification of athletes who do not comply with the internationally prohibited use of these cancer drugs. The assay was based on liquid-liquid extraction after enzymatic hydrolysis following liquid chromatography/tandem mass spectrometry (LC/MS/MS). Anastrozole, exemestane and its main metabolite (17-dihydroexemestane) were identified in urine by comparison of mass spectra and retention times with respective reference substances. An assay validation for the analysis of anastrozole and exemestane was performed regarding lower limits of detection (anastrozole: 0.02 ng/mL; exemestane: 3.1 ng/mL; dihydroexemestane: 0.5 ng/mL), interday precisions (6.6-11.1%, 4.9-9.1% and 5.6-8.3% for low [10 ng/mL], medium [50 ng/mL] and high [100 ng/mL] concentration) and recoveries (ranged from 85-97%).     

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.     

Hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric analysis of anastrozole in human plasma and its application to a pharmacokinetic study

A hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric method was developed for determination of anastrozole in human plasma. Anastrozole and irbesartan (internal standard) were extracted from human plasma with a mixture of dichloromethane and methyl tert-butyl ether (30:70, v/v). Analysis of the analytes was performed on a Luna HILIC column with the mobile phase of acetonitrile-10 m m ammonium formate (95:5, v/v) and detected by electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r(2) = 0.9992) over the concentration range of 0.10-50.0 ng/mL using 200 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.2-10.0% and -7.2-3.2%, respectively. The present method was applied successfully to the pharmacokinetic study of anastrozole after oral administration of 1 mg anastrozole tablet to healthy male volunteers.     

Evaluation of propolis polyphenols absorption in humans by liquid chromatography/tandem mass spectrometry

Propolis has various biological activities such as antibacterial, antiviral, antioxidant, immunostimulating and antiinflammatory, which are generally ascribed to the polyphenolic fraction. The aim of this study was to evaluate the absorption of the main polyphenols [caffeic acid (CA), pinobanksin-5methyl ether (P-5ME), pinobanksin (Pb), chrysin (C), pinocembrin (P), galangin (G), pinobanksin-3-acetate, pinobanksin esters and caffeic acid phenylethyl ester (CAPE)] from a dewaxed and standardized extract of propolis (EPID). Fifteen healthy volunteers consumed 5 mL EPID in water, corresponding to 125 mg of flavonoids. Blood samples were collected before, each hour for 8 h and 24 h after EPID intake. After deconjugation by beta-glucuronidase/sulfatase the plasma samples were analyzed by a selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method using morin as internal standard (I.S.). A kinetic profile characterized by two t(max), respectively at 1 h and about 5 h post-ingestion, was observed in all the subjects. The two peaks may be due to enterohepatic cycling. Among the various polyphenols ingested, only P-5ME, Pb, C, P and G were detected in plasma and C(max)t(1h) were 65.7 +/- 13.3, 46.5 +/- 12.7, 79.5 +/- 18.6, 168.1 +/- 16.3 and 113.7 +/- 16.8 ng/mL, respectively. These levels decreased significantly after 8 h and were no longer detectable 24 h after EPID intake. The recovery of the extraction for CA, Pb, C, P, G and I.S. from spiked plasma was 95.2 +/- 3.1, 93.1 +/- 3.6, 91 +/- 2.5, 96.4 +/- 4.2, 93.4 +/- 2.4 and 85.5 +/- 2.4%, respectively. The results of this study evidence that flavonoids from EPID are absorbed, metabolized and Pb-5ME and G seem to have apparent absorption, measured as (AUC/dose), higher than C, P and Pb.     

Enantioselective pharmacokinetics of mabuterol in rats studied using sequential achiral and chiral HPLC

The enantioselective pharmacokinetics of mabuterol was studied in six rats after single oral dose administration of mabuterol racemate. Serial plasma samples were collected and the pharmacokinetic behavior of each enantiomer in rats was characterized using a sequential achiral and chiral liquid chromatographic method. This method involved the separation of mabuterol racemate from endogenous substances on an achiral ODS column and enantiomeric separation on a Chirobiotic V column. The plasma-concentration data were analyzed for individual mabuterol enantiomer using 3P97 software. After i.g. administration of mabuterol racemate at a dose of 10 mg/kg, both enantiomers were slowly absorbed, reaching mean C(max) of 266.8 and 277.9 ng/mL at t(max) of 5.3 and 5.7 h for R- and S-mabuterol, respectively. The AUC(0-infinity) (5,938.9 ng h/mL) of R-mabuterol was significantly higher than that (4,446.1 ng h/mL) of S-mabuterol, and the half-life (14.5 h) was longer than that (9.6 h) of S-mabuterol (p < 0.001 and p < 0.01, respectively), showing that enantioselective pharmacokinetics between mabuterol enantiomers occur during the metabolism phase.     

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 --> 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 --> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1-20.0 ng/mL. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between -2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 +/- 2.73 ng/mL which occurred at 3.50 +/- 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 +/- 12.05 h and 609.3 +/- 122.2 ng . h/mL. LC/MS/MS is a rapid, sensitive and specific method for determining donepezil in human plasma samples.     

Physico-chemical effects of the major quassinoids in a standardized Eurycoma longifolia extract (Fr 2) on the bioavailability and pharmacokinetic properties, and their implications for oral antimalarial activity

A simple validated LC-UV method for the phytochemical analysis of four bioactive quassinoids, 13alpha(21)-epoxyeurycomanone (EP), eurycomanone (EN), 13alpha,21-dihydroeurycomanone (ED) and eurycomanol (EL) in rat plasma following oral (200 mg/kg) and intravenous administration (10 mg/kg) of a standardized extract Fr 2 of Eurycoma longifolia Jack was developed for pharmacokinetic and bioavailability studies. The extract Fr 2 contained 4.0%, 18.5%, 0.7% and 9.5% of EP, EN, ED and EL, respectively. Following intravenous administration, EP displayed a relatively longer biological half-life (t1/2 = 0.75 +/- 0.25 h) due primarily to its lower elimination rate constant (k(e)) of 0.84 +/- 0.26 h(-1)) when compared with the t1/2 of 0.35 +/- 0.04 h and k(e) of 2.14 +/- 0.27 h(-1), respectively of EN. Following oral administration, EP showed a higher C(max) of 1.61 +/- 0.41 microg/mL over that of EN (C(max) = 0.53 +/- 0.10 microg/mL). The absolute bioavailability of EP was 9.5-fold higher than that of EN, not because of chemical degradation since both quassinoids were stable at the simulated gastric pH of 1. Instead, the higher log K(ow) value of EP (-0.43) contributed to greater membrane permeability over that of EN (log K(ow) = -1.46) at pH 1. In contrast, EL, being in higher concentration in the extract than EP, was not detected in the plasma after oral administration because of substantial degradation by the gastric juices after 2 h. Similarly, ED, being unstable at the acidic pH and together with its low concentration in Fr 2, was not detectable in the rat plasma. In conclusion, upon oral administration of the bioactive standardized extract Fr 2, EP and EN may be the only quassinoids contributing to the overall antimalarial activity; this is worthy of further investigation.     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

Liquid chromatography-mass spectrometry method for the determination of venlafaxine in human plasma and application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-mass spectrometric (LC-MS) method for the determination of venlafaxine in human plasma has been developed. Samples were prepared using liquid-liquid extraction and analyzed on a C(18) column interfaced with a triple quadrupole mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase was methanol-water containing 10 mmol/L ammonium acetate, pH 7.9 adjusted with aqueous ammonia (80:20, v/v) at the flow rate of 1.0 mL/min. The analyte and internal standard clozapine were both detected by use of selected ion monitoring mode. The method was linear in the concentration range of 1.0-200.0 ng/mL. The lower limit of quantification (LLOQ) was 1.0 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.1%. The accuracy determined at three concentrations (5.0, 50.0 and 150.0 ng/mL for venlafaxine) was within +/-10.0% in terms of relative error (RE). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsule in 20 healthy volunteers. The results show AUC, T(max), C(max) and T(1/2) between the testing formulation and reference formulation have no significant difference (p > 0.05). Relative bioavailability was 103.4 +/- 14.1%.     

A rapid and sensitive method for simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry detection

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lamivudine (3TC) and zidovudine (AZT) in human serum, using didanosine (ddI) as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 230.0 --> 111.8 for 3TC, m/z 268.1 --> 126.8 for AZT, and m/z 237.2 --> 136.8 for ddI. The limits of detection and quantitation were 3 and 10 ng/mL for 3TC, and 5 and 15 ng/mL for AZT. The method was linear in the studied ranges (10-1500 ng/mL for 3TC and 15-3000 ng/mL for AZT), with r(2) > 0.99 for each drug, and the run time was 4 min. The intra-assay precisions (%) were in the ranges 1.9-8.7 (3TC) and 2.2-8.9 (AZT), the inter-assay precisions were in the ranges 2.6-9.0 (3TC) and 4.2-8.1 (AZT), and the intra- and inter-assay accuracies were >97% for both drugs. The absolute recoveries were 95-99% for 3TC (45, 600 and 1200 ng/mL) and 104-112% for AZT (45, 1000 and 2400 ng/mL). The analytical method was applied to a bioequivalence study in which 24 healthy adult volunteers received single oral doses of the reference formulation and two test combined AZT/3TC tablets, in an open, three-period, balanced, randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (extrapolated area under the serum concentration vs. time curve from time zero to infinity), it was concluded that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of absorption of both 3TC and AZT.     

An ultraperformance liquid chromatography–tandem mass spectrometry method for determination of anastrozole in human plasma and its application to a pharmacokinetic study

A fast, selective and sensitive ultraperformance liquid chromatography–tandem mass spectrometry method was developed for determination and pharmacokinetic study of anastrozole in human plasma. Plasma sample pretreatment involved a one‐step extraction with diethyl ether of 500 µL plasma. The chromatographic separation was carried out on an Acquity UPLC^TM BEH C₁₈ column with a mobile phase consisting of methanol–10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.30 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive mode. A high throughput was achieved with a run time of 1.5 min per sample. The standard curve for anastrozole was linear (r² ≥ 0.99) over the concentration range of 0.0550–27.5 ng/mL with a lower limit of quantification of 0.0550 ng/mL. The intra‐ and inter‐day precision (relative standard deviation) values were not higher than 14% and the accuracy (relative error) was within ±3.2% at three quality control levels. This simple, fast and highly sensitive method was fully validated and successfully applied to a clinical pharmacokinetic study of anastrozole in healthy volunteers after oral administration. Copyright © 2010 John Wiley & Sons, Ltd.