Journal of Chromatography B: Biomedical Sciences and Applications最新文献

Modern atmospheric pressure ionization (API) ion-trap mass spectrometry in connection with fast chromatographic separations using a short narrow-bore C₈ column was developed to determine 5-phenyl-3-thioureido-1,2,4-thiadiazole (301029), a novel virus inhibitor in serum. Both 301029 and an internal standard (I.S.) were separated from serum samples by acetonitrile deproteinization and extraction without time-consuming reconstitution. The chromatographic separation was achieved on a C₈ reversed-phase narrow-bore column using acetonitrile–water–acetic acid (90:10:0.01, v/v/v) as a mobile phase. The mass spectrometric analysis was performed by atmospheric pressure chemical ionization (APCI) mode with positive ion detection. Single ion monitoring (SIM) scan mode of m/z 237 and 158 was used to quantitatively determine 301029 and I.S., respectively. The low limit of quantitation was 25 ng/ml. The assay exhibited a linear range of 25–2500 ng/ml. Recovery from serum proved to be 100–113%. The precision (C.V.) and accuracy (RE) of the method were 2–12% and 94–112%, respectively. The present method was applied to determine the pharmacokinetic parameters of 301029 following oral administration of the agent to mice at 5 g/kg. The results revealed that the elimination half-life of 301029 was 413 min and the area under serum concentration–time curve was 354 μg/ml/min.

A chiral separation using carboxymethyl-β-cyclodextrin and methyl-β-cyclodextrin for the direct assay of tramadol in human urine by capillary electrophoresis (CE) with laser-induced native fluorescence detection was developed. Furthermore, the phase II metabolite O-demethyl tramadol glucuronide was determined from the urine samples and the ratio of the diasteromers was determined. The chiral method was validated. Correlation coefficients were higher than 0.999. Within day variation showed accuracy in the range 96.1–105.8% with a RSD less than 6.00%. Day to day variation present an accuracy ranging from 100.2 to 103.5% with a RSD less than 5.4%. After oral administration of 150 mg tramadol hydrochloride to a healthy volunteer, the urinary excretion was monitored during 24 h. About 11.4% of the dose was excreted as 1S,2S-tramadol, 16.4% as 1R,2R-tramadol and 23.7% as O-demethyl tramadol glucuronide. The amount of 1S,2S O-demethyl tramadol glucuronide was more than three fold higher as 1R,2R-O-demethyl tramadol glucuronide. The enantiomeric ratio of tramadol and the diastereomeric ratio of O-demethyl tramadol glucuronide was deviated from 1.0 showing that a stereoselective metabolism of tramadol occurs.

Hyperforin, hypericin and pseudohypericin are the main ingredients of St. John’s wort extract, which is available over the counter for treatment of mild to moderate depression. To facilitate clinical studies we developed two sensitive HPLC methods for determination of hypericin/pseudohypericin and hyperforin, respectively, in human plasma samples. The achieved limits of quantitation of 0.25 ng/ml for hypericin and pseudohypericin and 10 ng/ml for hyperforin were low enough to allow determination of pharmacokinetic parameters of the substances. Following liquid–liquid extraction of human plasma the samples were separated by isocratic reversed-phase HLPC and analyzed using fluorimetric detection for hypericin/pseudohypericin and UV detection for hyperforin.

The phase I and phase II metabolism of the anabolic steroid methandrostenolone was investigated following oral administration to a standardbred gelding. In the phase I study, metabolites were isolated from the urine by solid-phase extraction, deconjugated by acid catalysed methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. GC–MS analysis indicated the major metabolic processes to be sequential reduction of the A-ring and hydroxylation at C6 and C16. In the phase II study, unconjugated, β-glucuronidated and sulfated metabolites were fractionated and deconjugated using a combination of liquid–liquid extraction, enzyme hydrolysis, solid-phase extraction and acid catalysed methanolysis. Derivatization followed by GC–MS analysis revealed extensive conjugation to both glucuronic and sulfuric acids, with only a small proportion of metabolites occurring in unconjugated form.

A rapid high-performance liquid chromatography method for the determination of phosphoarginine (PArg) in invertebrate tissue has been redeveloped and validated. The method employs a reversed-phase amino column and a KH₂PO₄–acetonitrile mobile phase. PArg peak identity was confirmed by comparison with a known standard and via enzymatic conversion. Additionally linear calibration data, low intra-assay variability (<4%), and a detection limit of 5 pmol were determined. The method was demonstrated using PArg extracted from red abalone (Haliotis rufescens) adductor muscle. Validation of the extraction procedure was also completed, including the measurement of a 100.2±0.9% extraction efficiency.