ABSTRACT
A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of valsartan in human plasma was developed and validated. A 0.5 ml aliquot was extracted using solid-phase extraction in an Empore high performance extraction disk plate, universal resin 96-well format. The estimated calibration range of the method was 2-2000 ng/ml. The method was fully validated with intra-day mean accuracy and precision of 94.8-107% and 2.19-5.40% and inter-day mean accuracy and precision of 93.5-105% and 1.87-5.67%, respectively. No significant loss of valsartan in processed samples was confirmed in processed samples for up to 24 h at 10 degrees C. Sample dilution up to 50-fold with blank human plasma provided acceptable analyses. No interference peaks or matrix effects were observed. No effect of QC sample location results was observed in a 96-well plate. This LC-MS/MS technique was found to improve quantitative determination of valsartan allowing its pharmacokinetic evaluation with clinically relevant doses.
Subject(s)
Antihypertensive Agents/blood , Antihypertensive Agents/pharmacokinetics , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Tetrazoles/blood , Tetrazoles/pharmacokinetics , Valine/analogs & derivatives , Antihypertensive Agents/chemistry , Drug Stability , Humans , Molecular Structure , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Temperature , Tetrazoles/chemistry , Valine/blood , Valine/chemistry , Valine/pharmacokinetics , ValsartanABSTRACT
An assay based on cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed for the quantitative determination of metformin in human plasma. The analytical method consists of cation exchange solid-phase extraction (VersaPlate CBA) without any further evaporation/dissolution steps and cation exchange-based HPLC separation (Capcell Pak SCX column) with a normal-phase gradient system followed by semi-micro LC/MS/MS in positive ion selected reaction monitoring mode using electrospray ionization. The method exhibited excellent performance in terms of selectivity, robustness, short run time (7 min/sample) and simplicity of sample preparation. The calibration range was 10-1000 ng/ml with 0.2 ml of plasma. Intra- and inter-day mean accuracies were within the ranges of 100.3-105.0% and 101.2-105.3%, respectively. Intra- and inter-day precisions were within the ranges of 0.8-1.9% and 1.5-8.6%, respectively. Mean absolute recovery was 67.0% for metformin. No apparent loss of metformin after extraction was observed in an autosampler at 10 degrees C for 24 h. Dilution of metformin by blank human plasma up to 20-fold was tested and revealed no impact on the results of determination. Furthermore, the method exhibited high selectivity, since no effect on metformin analysis was observed on comparison of samples with or without nateglinide and other agents in plasma. Results obtained with the method were also comparable to a published LC-UV method on cross-validation. This method can be applied to various clinical pharmacokinetic studies of metformin.
Subject(s)
Cation Exchange Resins/analysis , Metformin/blood , Chromatography, Liquid/methods , Humans , Mass Spectrometry/methods , Metformin/chemistryABSTRACT
A marked increase in the Na+ , K+ -ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na+ , K+ -ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na+ , K+ -ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na+ , K+ -ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8-16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li+ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na+ , K+ -ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.
