Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to μ-LC-LIF determination

Abstract

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

Sequential injection lab-on-valve platform as a miniaturisation tool for solid phase extraction

“…This resin was able to retain the LAS that were subsequently eluted and quantied by capillary electrophoresis (CE). The same authors used this resin for the quantication of sphingoid percursors 18 and female steroid hormones 19 in human biouids. The quantication of sphingoid percursors 18 was based on the retention of the target analyte in the bead column and the resultant derivatised analyte was eluted towards the liquid chromatograph (LC) and quan-tied by laser induced uorescence (LIF).…”

“…The same authors used this resin for the quantication of sphingoid percursors 18 and female steroid hormones 19 in human biouids. The quantication of sphingoid percursors 18 was based on the retention of the target analyte in the bead column and the resultant derivatised analyte was eluted towards the liquid chromatograph (LC) and quan-tied by laser induced uorescence (LIF). In the case of the steroids hormones, 19 the bead column served as sample cleanup and preconcentration before the LC and the entire eluate was electrosprayed, ionised and monitored by tandem mass spectrometry (MS-MS) detection in multiple reaction monitoring (MRM) mode.…”

Rapid evaluation of 25 key sphingolipids and phosphosphingolipids in human plasma by LC-MS/MS

“…Even more challenging is to achieve their efficient separation in a short analysis time, ensuring at the same time robustness, reproducibility, and sensitivity. Several new sample preparation methods have also been established like dried blood spot extraction [28] and solid-phase extraction followed by in situ derivatization [29]. Despite these advances, the well-known Bligh-Dyer [30] total lipid extraction still remains very popular, thanks to its efficacy and ease.…”