The goal of this study is to find an experimental condition which enables us to perform enzymatic studies on the cellularbehavior of PTEN (phosphatase and tensine homolog) through identification of molecular species of phosphatidylinositol 3,4,5-trisphosphates and their quantitative analysis in a mammalian cell line using mass spectrometry. We initially exployed a two-stepextraction process using HCl for extraction of phosphatidylinositol 3,4,5-trisphosphates from two mammalian cell lines and furtheranalyzed the extracted phosphatidylinositol 3,4,5-trisphosphates using tandem mass spectrometry for the identification of them. Wefinally quantified the concentration of phosphatidylinositol 3,4,5-trisphosphates using internal standard calibration. From theseobservation, we found that HEK 293-T cells is a good model to examine the enzymatic behavior of PTEN in a cell, and theminimum amount of phosphatidylinositol 3,4,5-trisphosphates is more than 50 pmol for quantification in a mass spectrometer. These results suggest that the well-optimized experimental conditions are required for the investigation of the cellular PTEN interms of the catalytic mechanism and further for the detailed identification of cellular substrates.
An ultra-fast generic LC-MS/MS method was developed for high-throughput quantification of discovery pharmacokinetic(PK) samples and its reliability was verified. The method involves a simple protein precipitation for sample preparationand the analysis by ultra-fast generic LC-MS/MS with the ballistic gradient program and selected reaction monitoring (SRM)mode. Approximately 290 new chemical entities (NCEs) (over 10,000 samples) from 5 therapeutic programs were analyzed. The calibration curves showed good linearity in the concentration range of 1, 2 or 5 to 2000 ng/mL. No significant ion suppressionwas observed in the elution region of all the NCEs. When approximately 300 plasma samples were continuously analyzed,the peak area of internal standard was constant and reproducible. In the repeated analysis of samples, the plasma concentrationsand the area under the curve (AUC) were consistent with the results from the first analysis. These results showed that the presentultra-fast generic LC-MS/MS method is reliable in terms of selectivity, sensitivity, and reproducibility and could be useful forhigh-throughput quantification and other bioanalysis in drug discovery.
Isotopic analysis using thermal ionization mass spectrometry coupled with scanning electron microscopy (SEMTIMS)was performed to determine the isotopic ratios of uranium contained in micro-particles in the 6th Nuclear SignaturesInterlaboratory Measurement Evaluation Programme (NUSIMEP-6) sample. Elemental analysis by energy dispersive X-rayspectroscopy (EDS) was conducted on uranium-bearing mirco-particles, which were transferred to rhenium filaments for TIMSloading using a micromanipulation system in a SEM. A multi-ion-counter system was utilized to detect the ion signals of thefour isotopes of uranium simultaneously. The isotope ratios of uranium corrected by bracketing using a reference materialshowed excellent agreement with the certified values. The measurement accuracy for n(234U)/n(238U) and (b) n(235U)/n(238U) was10% and 1%, respectively, which met the requirements for qualification for the NetWork of Analytical Laboratories (NWAL).
Dioscin is a biologically active steroidal saponin with anticancer and hepatoprotective effects. A rapid, selective, andsensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantificationof dioscin in rat plasma. Dioscin was extracted from rat plasma using ethyl acetate at acidic pH. The analytes were separatedon a Halo C18 column using gradient elution of acetonitrile and 0.1% formic acid and detected by tandem massspectrometry in selected reaction monitoring mode. The standard curve was linear (r2 = 0.998) over the concentration range of1−100 ng/mL. The lower limit of quantification was 1.0 ng/mL using 50 μL of plasma sample. The coefficient of variation andrelative error for intra- and inter-assay at four QC levels were 1.3 to 8.0% and −5.4 to 10.0%, respectively. This method wasapplied successfully to the pharmacokinetic study of dioscin after oral administration of dioscin at a dose of 29.2 mg/kg in maleSprague-Dawley rats.