Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column.

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.

Umezawa H ，Lee XP ，Arima Y ，Hasegawa C ，Marumo A ，Kumazawa T ，Sato K ... -  《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》

Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry.

Umezawa H ，Lee XP ，Arima Y ，Hasegawa C ，Izawa H ，Kumazawa T ，Sato K ... -  《-》

Extending the detection window of diazepam by directly analyzing its glucuronide metabolites in human urine using liquid chromatography-tandem mass spectrometry.

A method for the simultaneous direct analysis of diazepam oxazepam glucuronide, temazepam glucuronide, oxazepam, nordiazepam, and temazepam in human urine was developed and validated. Urine sample was purified by solid phase extraction (SPE), and the analysis was achieved using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system equipped with an electrospray ionization source (ESI). Multiple reaction monitoring (MRM) mode was used to analyze the target compounds. Extraction recoveries were 65-122% for all the analytes. The method showed acceptable intra-assay and inter-assay precision (both relative standard deviation (RSD)≤11.2%) for quality control (QC) samples. The limits of detections (LODs) were in the range of 0.1-2 ng/mL. The present assay was applied to analyze the urine obtained from three volunteers after oral administration of a single dose 5mg of diazepam. The results showed that, the detection periods of oxazepam glucuronide and temazepam glucuronide were much longer than diazepam and other metabolites.

Wang X ，Wang R ，Zhang Y ，Liang C ，Ye H ，Cao F ，Rao Y ... -  《-》

A sensitive and selective method for the detection of diazepam and its main metabolites in urine by gas chromatography-tandem mass spectrometry.

Kinani S ，Bouchonnet S ，Milan N ，Ricordel I ... -  《-》

LC-MS/TOF and UHPLC-MS/MS study of in vivo fate of rifamycin isonicotinyl hydrazone formed on oral co-administration of rifampicin and isoniazid.

The formation and fate of 3-formylrifamycin isonicotinyl hydrazone (HYD) was investigated following oral co-administration of rifampicin (RIF) and isoniazid (INH) in Sprague-Dawley (SD) rats (n=5) using advanced analytical modalities. The study was carried out with 20 and 5mg/kg doses of RIF and INH, respectively. The plasma, urine and faeces samples were collected at different time points up to 48h, which were qualitatively and quantitatively evaluated for the presence of HYD after proper sample preparation. For the same, initially liquid chromatography-mass spectrometry/time-of-flight (LC-MS/TOF) method was developed in electrospray ionization (ESI) positive mode, wherein separation was achieved on a C18 column (4.6mmx250mm, 5microm), using a volatile mobile phase in a gradient mode. The presence of HYD was confirmed by accurate mass study, spiking with the standard and UV-visible spectra matching. For quantitative evaluation of HYD, a selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for all the three matrices. In this case, elution of HYD was achieved on a small C18 column (4.6mmx50mm, 1.8microm) using a short gradient method. The quantitation was done by selective reaction monitoring (SRM) in ESI positive mode. The validation parameters like linearity, accuracy, precision, selectivity, matrix effect, recovery and stability were assessed as per regulatory guidelines. The calibration range was established between 1 and 200ng/ml, with r(2)>0.99 in all the cases. The back calculated values for three quality control (QC) samples, and at lower limit of quantitation (LLOQ) were within 15 and 20%, respectively, of the nominal values. Similarly, the intra- and inter-day precisions were found within 15% at the four tested levels. The HYD was found to be stable for the duration of sample preparation and analysis in the controlled experimental conditions. The analysis of in vivo samples showed a significant extent of HYD in faeces, however, the interaction product was not found in plasma and urine. To verify the results, 5mg/kg oral dose of HYD standard was given to rats separately, and its presence was studied in all the three matrices. Further, in vitro plasma stability of HYD was also carried out to explain its absence in plasma and urine, which showed approximately 55% disappearance of HYD in 2h.

Prasad B ，Singh S 《-》