Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography–tandem mass spectrometry
Section snippets
Standards and reagents
Methanol, water, and acetonitrile (HPLC grade) were purchased from Burdick and Jackson (Muskegon, MI, USA). 1,3-Dihydroxy-2-amino-4-octadecene-1-phosphate (S1P), 1,3-dihydroxy-2-amino-4-heptadecene-1-phosphate (a 17-carbon analog of S1P, C17-S1P), and 1,3-dihydroxy-2-amino-octadecane-1-phosphate (sphinganine-1-phosphate, dihydro-S1P, DHS1P) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The S1P, C17-S1P, and DHS1P standards were dissolved in methanol with a trace amount of
Signal carryover in positive ion ESI LC–MS/MS analysis
In our initial studies toward development of a simple and reliable method for measurement of S1P levels (and subsequently of DHS1P levels) in biological samples, minor modifications of positive ion ESI LC–MS/MS techniques described in the literature [20], [21] were employed. The characteristic MRM transitions monitored for measurements of S1P in our initial studies and of C17-S1P employed as the internal standard were m/z 380/264 and m/z 366/250, respectively. In these studies, it was
Conclusions
Modification of the zwitterionic properties of sphingoid base-1-phosphates via derivatization was exploited for obtaining a negative ion LC–MS/MS technique devoid of significant sample carryover from a previous injection. The assay method possesses capabilities for detection of less than 50 fmol of the analytes injected on column. The analytical method displays linearity over a wide range of analyte concentrations with correlation coefficients (r2) greater than 0.995. The novel LC–MS/MS
Acknowledgments
The authors thank Viktor Laiko for helpful discussion of the data. This work was supported in part by National Institutes of Health Grants RO1 HL71152 and P50 HL073994 to Viswanathan Natarajan. Proceeds of NIH1 S10 RR16798 grant awarded to Walter Hubbard was used for the purchase of the LC–MS/MS system.
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