Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4°C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C₁₈ column with a mobile phase of ammmonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 ± 3.2% and 81 ± 4.8% following dosage at concentrations of 10 μg/ml and 30 μ/ml, respectively. Using 25- μl of rat plasma the limit of quantitation was 1 μg/ml sinefungin, and the assay was linear from 1 to 30 μg/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models.     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

Chiral high-performance liquid chromatographic analysis of enantiomers of losigamone,a new candidate antiepileptic drug

An assay based on a single-step liquid–liquid extraction from human plasma followed by high-performance liquid chromatography on a chiral column was developed for the measurement of enantiomers of a racemic new candidate antiepileptic drug. Excellent intra- and inter-assay accuracy and precision and recovery were demonstrated in the desired concentration range of 0.031 to 5.00 μg/ml. The method is free from interferences by other anticonvulsant drugs and their metabolites. The method is being used in a clinical trial of losigamone.     

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

Determination of caffeic acid in rabbit plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method involving UV detection was developed for determination of caffeic acid in rabbit plasma. A Lichrosphere CN column (250 mm × 4 mm I.D., 5 μm) was used as the stationary phase and the mobile phase consisted of 2% acetic acid solution at a flow-rate of 1.0 ml/min. The UV absorbance was monitored at 320 nm. The plasma sample was acidified by the addition of 0.01 parts of concentrated phosphoric acid (85%) to maintain caffeic acid stability. After a simple clean-up procedure, the limit of quantitation achieved was 0.1 μg/ml, and the standard curve was found to be linear over the concentration ranges of 0.1–2.0 μg/ml and 0.1–40 μg/ml. The coefficient of variation for within- and between-run precision and accuracy was less than 10%, and the recovery was 82.3%.     

Determination of a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse,rat, dog,and monkey plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C₁₈ Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 μg/ml to 50 μg/ml with an intra-sassay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 μg/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 μg/ml in dog and monkey plasma and 0.1 μg/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at −70°C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

High-performance liquid chromatographic assay of cefotaxime,desacetylcefotaxime and ceftriaxone in rat plasma

A simple and selective high-performance liquid chromatographic method is described for the analysis of the cephalosporins cefotexime (CXM), desacetylcefotaxime (DACXM) and ceftriaxone (CFX) in rat plasma. Plasma was deproteinized with methanol, and the supernatant was directly injected into the chromatograph and monitored at 254 nm. For determination of the unbound drugs, a centrifugal ultrafiltration method was employed. The calibration curves were linear (r=0.999) from 2.5 to 500 μg/ml; the detection limits were 100 ng/ml for DACXM and 250 ng/ml for CXM and CFX. The method was not interfered with by other plasma components, nor by barbital sodium or caffeine, and has been applied to study the pharmacokinetics of the cephalosporins in rats.     

High-performance liquid chromatography of flufenamic acid in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for the determination of flufenamic acid in rat plasma is described. After liquid-liquid extraction, the drug is separated by HPLC on a 5-μm octadecylsilica column (Nucleosil C₁₈) with ultraviolet detection at 280 nm. Linear calibration graphs for flufenamic acid were constructed from 0.5 to 15 μg/ml. The method has been applied to a pharmacokinetic study in animals.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid—liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5–2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Determination of protein binding for novel 2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid enantiomers to rats,dogs, and humans plasma by UPLC-MS/MS

R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA) is a novel COX inhibitor with remarkable anti-inflammatory and antiplatelet aggregation activities, but no gastrointestinal toxicity. In our previous study, the different pharmacokinetic profiles of the two enantiomers in rats were observed after administration of R-HFBA and S-HFBA. Stereoselective protein binding of the two enantiomers may be a reason for the different pharmacokinetic behaviors. In this study, we developed and validated an UPLC-MS/MS method for determining stereoselective binding of HFBA enantiomers to rat, dog, and human plasma in vitro. Chromatographic separation was achieved by gradient elution with a flow rate of 0.4 mL/min. MS/MS detection was operated in positive electrospray using multiple reaction monitoring (MRM) mode. The method was proved to be linear over the concentration range of 0.005 to 10 μg/mL with a lower limit of quantification of 0.005 μg/mL. The developed method was successfully employed to the plasma protein binding study of HFBA enantiomers. Equilibrium dialysis method was applied to assess drug-plasma protein interactions. The results showed that the enantiomers were both extensively bound to three species plasma and protein binding of R-/S-HFBA was concentration dependent. R-HFBA and S-HFBA showed significant species difference among rat, dog, and human plasma and stereoselective plasma protein binding.     

High-performance liquid chromatographic analysis of the anti-tumor agent SCH 66336 in cynomolgus monkey plasma and evaluation of its chiral inversion in animals

SCH 66336 is a novel non-cytotoxic anti-tumor agent that is in phase I/II clinical trials for the treatment of solid tumors. This compound is a single enantiomer with one chiral center. Prior to evaluation of this drug candidate in man, it was necessary to evaluate its pharmacokinetics and possible chiral inversion in animals. Thus, high-performance liquid chromatographic (HPLC) methods have been developed for its determination in cynomolgus monkey plasma and for the evaluation of its chiral inversion in rats and cynomolgus monkeys. The achiral HPLC analysis involved extraction with 30% methylene chloride in hexane followed by separation on a CN column and quantitation by UV absorbance at 280 nm. The method was linear over a concentration range of 0.1 to 20 μg/ml in monkey plasma. The chiral HPLC analysis involved the use of a Chiralpak AD column set at 39°C with a mobile phase of hexane–ethanol–diethylamine mixture and a UV detector set at 280 nm. Plasma samples were subjected to solid-phase extraction on a C₂ cartridge prior to HPLC analysis. The method was linear over a concentration range of 0.25 to 10 μg/ml in rat and cynomolgus monkey plasma for both enantiomers. Both methods showed good linearity (r²>0.99), accuracy (bias<13%) and precision (CV<12%). Chiral HPLC analysis indicated that SCH 66336 was not subjected to chiral inversion in rats and cynomolgus monkeys     

Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

In this study, a high-performance liquid chromatographic method was developed for the quantitative determination of erythromycin (EM), roxithromycin (RXM), and azithromycin (AZM) in rat plasma with amperometric detection under a standardized common condition using clarithromycin (CAM) as an internal standard. This method was also proved to be applicable for the determination of CAM by employing RXM as an internal standard. Each drug was extracted from 150 μl of plasma sample spiked with internal standard under an alkaline condition with tert.-butyl methyl ether. The detector cell potential for the oxidation of the drugs was set at +950 mV. The linearity of the calibration curves were preserved over the concentration ranges of 0.1–10 μg/ml for EM and RXM, and 0.03–3.0 μg/ml for CAM and AZM. Coefficients of variation and relative error were less than 9% and ±7%, respectively. The analytical method presented here was proved to be useful for the investigation of the pharmacokinetic characteristics of EM, CAM, RXM, and AZM in rats.     

Liquid chromatographic determination of sodium mercaptoundecahydrododecaborate in rat urine and plasma after precolumn derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of disodium mercaptoundecahydrododecaborate (BSH) in biological fluids. Monobromobimane was used as a precolumn derivatizing agent. A stable derivative was obtained. The derivative was separated on a C₁₈ column using reversed-phase ion-pairing chromatography and detected by a spectrophotometric detector at 373 nm. The detection limit was 200 ng/ml (0.1 ppm boron). Calibration curves were prepared for rat urine and plasma samples. The calibration curves were linear in the range of 1 μg/ml to 100 μg/ml for urine samples and 0.2 μg/ml to 50 μg/ml for plasma samples.