title: An improved UPLC-MS/MS platform for quantitative analysis of glycerophosphoinositol in mammalian cells.
abstract: The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.
authors: Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy.
authors: Laura
authors: L
authors: Grauso
authors: Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, via Pietro Castellino 111, 80131 Napoli, Italy.
authors: Stefania
authors: S
authors: Mariggiò
authors: Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, via Pietro Castellino 111, 80131 Napoli, Italy.
authors: Daniela
authors: D
authors: Corda
authors: Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy.
authors: Angelo
authors: A
authors: Fontana
authors: Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy.
authors: Adele
authors: A
authors: Cutignano
mesh_terms: D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry*; D051379:Mice; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D007295:Inositol Phosphates; C014575:glycerylphosphoinositol