Accurate LC-MS/MS Analysis of Diacylglycerols in Human Plasma with Eliminating Matrix Effect by Phospholipids Using Fluorous Biphasic Extraction.

We developed an accurate method for determining diacylglycerols (DAGs) in human plasma using a fluorous biphasic liquid-liquid extraction method, followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The lipid mixture in the plasma was first extracted with chloroform by using the Bligh-Dyer method. The resulting solution was subjected to fluorous biphasic liquid-liquid extraction to remove phospholipids, which are known to cause matrix effects during the LC-MS/MS analysis. In this method, phospholipids in a lipid mixture solution (nonfluorous solvent) were selectively extracted to tetradecafluorohexane (fluorous solvent) via the specificity of fluorous affinity by forming a complex with a perfluoropolyethercarboxylic acid-lanthanum(III) salt. The remaining DAGs in the nonfluorous solvent could be directly injected into the LC system through the positive electrospray ionization-MS/MS mode. The removal rate of the phospholipids through the fluorous biphasic extraction was more than 99.9%; thus, the matrix-effect-eliminating analysis of DAGs in human plasma with LC-MS/MS was enabled. Furthermore, the applicability of this method and the possibility of using DAGs as biomarkers were evaluated by applying this method to human plasma samples obtained from major depressive disorder as a related disease.

Liquid Chromatographic Determination of o-Phosphoethanolamine in Human Plasma Using Fluorescent Derivatization.

In this study, an HPLC analysis method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was developed for the determination of o-phosphoethanolamine (PEA), which is a potential biomarker for the diagnosis of major depressive disorder, in human plasma sample. After PEA was derivatized with AQC under mild conditions, the obtained derivative was subjected to purification with a titanium dioxide-modified monolithic silica spin column (MonoSpin® TiO). The eluate from the MonoSpin® TiO was directly injected into an amide-type hydrophilic interaction liquid chromatography (HILIC) column-equipped HPLC system, and the resulting derivative could be separated on the column under alkaline mobile phase conditions and subsequently detected fluorometrically at excitation and emission wavelengths of 250 and 395 nm, respectively. The limit of detection and limit of quantification for a 10 µL injection volume of PEA were 0.052 and 0.17 µM, respectively. The method was validated at 0.2, 1.0, and 5.0 nmol/mL levels in plasma sample, and the precision values were 2.0-6.6% as relative standard deviation and the correlation coefficient (r) of the calibration curve was 0.9995. Furthermore, applicability of this method was demonstrated by analyzing PEA levels in plasma samples from mental illness patients.

Discrimination of Malignant Pleural Mesothelioma Cell Lines Using Amino Acid Metabolomics with HPLC.

Malignant pleural mesothelioma (MPM) is a malignancy closely associated with asbestos exposure. Although early diagnosis provides a chance of effective treatment and better prognosis, invasive biopsy and cytological procedure are required for definitive diagnosis. In this study, we developed a method to differentiate between MPM and control cell lines, named "amino acid metabolomics," consisting in the assessment of the balance of their amino acid levels in the cell culture medium. Culture media of MESO-1 (MPM cell line) and Met-5A (control) cells were used in this study to evaluate amino acid levels using HPLC, following the fluorescence derivatization method. The time-dependent changes in amino acid levels were visualized on the score plot following principal component analysis, and the results revealed differential changes in amino acid levels between the two cell culture supernatants. A discriminative model based on linear discriminant analysis could distinguish MPM and control cells.

Deep eutectic solvent extraction of cortisol and cortisone from human saliva followed by LC-UV analysis.

A novel extraction method was developed for the determination of cortisol and cortisone. In this study, we prepared a hydrophobic deep eutectic solvent (DES) by mixing trioctylmethylammonium chloride and pentafluorophenol as a hydrogen bond acceptor and a hydrogen bond donor, respectively, for use as the extraction solvent. The extraction of cortisol and cortisone was performed by adding a small volume of the DES to the aqueous sample. After centrifugation, the resulting sedimented DES phase was injected into a reversed-phase liquid chroamtography column, and the analytes were detected with an ultraviolet detector at 254 nm. Under the optimized extraction conditions, the enrichment factors of cortisol and cortisone were 9.3 and 8.5, respectively. Furthermore, the linear dynamic ranges were established over a concentration range of 10-200 pmol mL-1 (r2 > 0.9992), and the limits of detection of cortisol and cortisone were found to be 2.1 and 1.8 pmol mL-1, respectively. The applicability of this method was evaluated by analyzing the cortisol and cortisone contents of human saliva samples.

Fluorous and Fluorogenic Derivatization for Selective Liquid Chromatographic Analysis of Cyanide in Human Plasma.

A liquid chromatographic (LC) method with fluorous derivatization for the determination of cyanide in human plasma is described. In this method, the cyanide was transformed to a fluorous and fluorogenic compound by derivatizing with 2,3-naphthalenedialdehyde and perfluoroalkylamine reagent under mild reaction conditions (a reaction time of 5 min at room temperature). The obtained derivative was successfully retained on the perfluoroalkyl-modified LC column with the use of a high concentration of organic solvent in the mobile phase, whereas non-fluorous derivative was hardly retained, followed by fluorometric detection at excitation and emission wavelengths of 420 and 490 nm, respectively. Under the optimized conditions, the limit of detection and the limit of quantification for cyanide in a 5-µL injection volume were 1.3 µg/L (S/N = 3) and 4.4 µg/L (S/N = 10), respectively. The recovery from spiked human plasma was achieved in the range of 54 - 90% within a relative standard deviation of 3.5%. The feasibility of this method was further evaluated by applying it to the analysis of human plasma samples.

Application of a fluorous derivatization method for characterization of glutathione-trapped reactive metabolites with liquid chromatography-tandem mass spectrometry analysis.

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.

Mechanism Underlying Linezolid-induced Thrombocytopenia in a Chronic Kidney Failure Mouse Model.

OBJECTIVE: To investigate the relationship between renal function and linezolid (LZD)-induced thrombocytopenia and elucidate the underlying mechanism using a chronic renal disease (CRD) mouse model. MATERIALS AND METHODS: CRD was induced in 5-week-old male Institute of Cancer Research (ICR) mice by 5/6 nephrectomy. After this procedure, LZD (25 and 100 mg/kg) was administered intraperitoneally once every day for 28 days. Platelet counts, white blood cell (WBC) counts, and hematocrit (HCT) levels were measured every 7 days. 2-14C-thymidine (0.185 MBq) was administrated intravenously to LZD-administered mice to evaluate the thymidine uptake ability of bone marrow. RESULTS: Platelet counts were significantly lower in the LZD-administered CRD group than in the LZD-nonadministered groups at 14, 21, and 28 days (P < 0.05); however, these changes were not observed in LZD-administered mice with normal renal function, regardless of the duration of LZD administration. No significant changes were observed in WBC counts or HCT levels in any LZD-administered CRD mouse. Moreover, radioactive levels in bone marrow were not significantly different in each group. CONCLUSIONS: These results indicate that LZD-induced decreases in platelet counts were enhanced by renal impairment in vivo, suggesting that LZD-induced thrombocytopenia is not caused by nonimmune-mediated bone marrow suppression.