Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography–tandem mass spectrometry

doi:10.1016/j.ab.2004.12.006

Analytical Biochemistry

Volume 339, Issue 1, 1 April 2005, Pages 129-136

https://doi.org/10.1016/j.ab.2004.12.006 Get rights and content

Abstract

Sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) are important signaling sphingolipids. The presence of nanomolar levels of S1P and DHS1P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S1P and DHS1P. This article details a negative ion ESI LC–MS/MS technique following modification of the zwitterionic nature of S1P and DHS1P via derivatization. A highly selective and sensitive LC–MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S1P and DHS1P without significant sample carryover was developed. Standard curves for S1P and DHS1P are linear over wide ranges (0–300 pmol) of analyte concentrations with correlation coefficients (r²) greater than 0.995. The levels of S1P and DHS1P in human platelet poor plasma were 590.8 ± 42.1 and 130.7 ± 20.7 pmol/ml, respectively. The levels of S1P and DHS1P in fetal bovine serum were 141.7 ± 4.6 and 0.6 ± 0.2 pmol/ml, respectively. The addition of sphingosine (1 μM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S1P, whereas the level of DHS1P was unchanged.

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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Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography–tandem mass spectrometry

Abstract

Section snippets

Standards and reagents

Signal carryover in positive ion ESI LC–MS/MS analysis

Conclusions

Acknowledgments

Prostaglandins

Pharmacol. Res.

Biochim. Biophys. Acta

Prostaglandins

Blood

Prostaglandins

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Methods Enzymol.

Anal. Biochem.

Anal. Biochem.