Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method for the determination of 25-hydroxyvitamin D₃, the major metabolite of vitamin D₃ in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D₃ 3-sulfate, a major conjugated metabolite of 25-hyroxyvitamin D₃, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.     

Determination of amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether—dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Determination of Z-butylidenephthalide in plasma by high-performance liquid chromatography

An HPLC assay is described for the determination of Z-butylidenephthalide (Z-Bdph) in plasma. Plasma samples were cleaned up by extraction with 2% chloroform in n-hexane. Z-Bdph was separated on a normal-phase silica column with a mobile phase of chloroform-n-hexane (1:1). The limit of quantitation with UV detection at 254 nm for Z-Bdph in plasma was 0.01 μg/ml. The recovery of Z-Bdph by organic solvent extraction of plasma was 99.5%. The intra-day and inter-day coefficients of variation and relative errors for Z-Bdph determination in plasma were both less than 10%. The present method was applied to pharmacokinetic studies of Z-Bdph in plasma after intravenous administration to rabbits.     

Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 μl each). A column packed with 5 μm octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol—water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30°C. The results were linear from 0.05 to 100 μg/ml (r = 0.999), and the detection limit was 0.01 μg/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at −20°C.     

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with β-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C₁₈ column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100–3000 ng/ml and 4–400 μg/ml in plasma and urine, respectively. The intra-assay variability was 5.1% and the inter-assay variability was 8.2% for each compound in each matrix. The method presented is applicable to pharmacokinetic and pharmacogenetic studies utilizing chlorzoxazone.     

Determination of indinavir, an HIV-protease inhibitor, in human plasma by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay has been developed to determine the concentrations of the HIV-protease inhibitor indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 μl of human plasma and 400 μl of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile–water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of indinavir in several clinical pharmacokinetic studies in HIV-infected patients.     

Determination of eltanolone in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of eltanolone in plasma has been developed. Plasma samples containing eltanolone were diluted with acetonitrile to precipitate plasma proteins, and derivatized with 2,4-dinitrophenylhydrazine before direct injection onto a C₁₈ column. The mobile phase was acetonitrile–water (70:30, v/v) containing 0.1% trifluoroacetic acid and detection was by UV absorbance at 367 nm. The quantitation limit was 0.020 μg/ml. The method has proven to be rapid, precise and sensitive in the range of concentrations found during and following intravenous anaesthesia.     

Determination by high-performance liquid chromatography of hydroxyurea in human plasma

An assay using reversed-phase high-performance liquid chromatography with electrochemical detection was developed to measure hydroxyurea in plasma at concentrations suitable for pharmacokinetic studies. The sample preparation is simple, the analysis rapid and assays can be batched. The between-run precision is excellent (coefficient of VARIATION = 2.8–4.5%) and the limit of detection is 0.02 mmol/l. Preliminary studies have shown that the method is suitable for pharmacokinetic studies.