In vitro metabolism study of Strychnos alkaloids using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry

In this report, the in vitro metabolism of Strychnos alkaloids was investigated using liquid chromatography/high‐resolution mass spectrometry for the first time. Strychnine and brucine were selected as model compounds to determine the universal biotransformations of the Strychnos alkaloids in rat liver microsomes. The incubation mixtures were separated by a bidentate‐C₁₈ column, and then analyzed by on‐line ion trap/time‐of‐flight mass spectrometry. With the assistance of mass defect filtering technique, full‐scan accurate mass datasets were processed for the discovery of the related metabolites. The structural elucidations of these metabolites were achieved by comparing the changes in accurate molecular masses, calculating chemical component using Formula Predictor software and defining sites of biotransformation based upon accurate MSⁿ spectral information. As a result, 31 metabolites were identified, of which 26 metabolites were reported for the first time. These biotransformations included hydroxylation, N‐oxidation, epoxidation, methylation, dehydrogenation, de‐methoxylation, O‐demethylation, as well as hydrolysis reactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Urinary d‐lactate levels reflect renal function in aristolochic acid‐induced nephropathy in mice

Urinary d ‐lactate is highly correlated to diabetic nephropathy – a progressive kidney disease in renal glomeruli. In this study, we used a C3H/3e mouse model to investigate the relationship between urinary d ‐lactate and aristolochic acid nephropathy where the glomerular structure is not affected. The nephropathy was induced using intravenous injections of aristolochic acid at a dosage of 10 mg/kg per day for 5 days and was characterized biochemically and histologically. The urinary excretions of proteins, N‐acetyl‐β‐d ‐glucosaminidase and serum creatinine were determined and connected to histological conventional findings. Urinary d ‐lactate was analyzed using column‐switching high‐performance liquid chromatography with fluorescence detection. The results showed a remarkable increase of urinary markers, including of urinary proteins and N‐acetyl‐β‐d ‐glucosaminidase, and the histological examination confirmed a diagnosis of acute tubule necrosis. The ratio of d ‐lactate to creatinine in the urine of aristolochic acid‐treated mice was approximately 36 times greater than that of the mice in the control group (p < 0.05). The ratios for the two groups of mice were 311.00 ± 71.70 and 8.60 ± 1.80 µmol/mmol creatinine, respectively. These data confirm in vivo that urinary d ‐lactate reflects renal injury conditions in aristolochic acid‐treated mice and may be a marker for the assessment of nephropathy. Copyright © 2013 John Wiley & Sons, Ltd.     

Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry

This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C₈‐cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS‐LC‐MS/MS is reported. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of morniflumate and its major active metabolite,niflumic acid,in human plasma by high‐performance liquid chromatography in stability and pharmacokinetic studies

A rapid, sensitive and stable high‐performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of morniflumate and its major active metabolite, niflumic acid, in human plasma. HPLC analysis was carried out using a 5 µm particle size, C₁₈‐bonded silica column with a mixture of acetonitrile and 0.005 m potassium phosphate monobasic in water (60:40, v/v) as the mobile phase and UV detection at 287 nm. The method involved the treatment with 50 μL of 0.4 m hydrochloric acid for the stability of morniflumate, extraction with diethylether and evaporation to dryness under a nitrogen stream. The lower limit of quantitation for morniflumate and niflumic acid was 50 and 500 ng/mL, respectively. The calibration curves for morniflumate and niflumic acid were linear over the concentration range of 50–20,000 ng/mL and 500–50,000 ng/mL, respectively, with correlation coefficients greater than 0.9995 and inter‐ or intra‐batch coefficients of variation not exceeding 13.79%. The variability (percentage difference) of incurred sample re‐analysis did not exceed 11.72% and all of the repeat samples fell within 20% of the mean value. This assay procedure was applied successfully to an examination of the pharmacokinetics of morniflumate and its metabolite, niflumic acid, in human subjects. Copyright © 2013 John Wiley & Sons, Ltd.