Simple enrichment and analysis of plasma lysophosphatidic acids

Abstract

A simple and highly efficient technique for the analysis of lysophosphatidic acid (LPA) subspecies in human plasma is described. The streamlined sample preparation protocol furnishes the five major LPA subspecies with excellent recoveries. Extensive analysis of the enriched sample reveals only trace levels of other phospholipids. This level of purity not only improves MS analyses, but enables HPLC post-column detection in the visible region with a commercially available fluorescent phospholipids probe. Human plasma samples from different donors were analyzed using the above method and validated by LC-ESI/MS/MS.

Fig. 1

Structures of lysophosphatidic acids (LPAs).

Fig. 2

Structure of 4-(4-(Dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA), used in the post-column fluorescence detection of LPA subspecies.

Fig. 3

Absorption spectra (top) and fluorescence spectra (bottom) of 3 μM aqueous solutions of DiA alone (dashed lines) and in the presence of 10 μM LPA 18:0 (solid lines). Excitation/emisson wavelengths: 470/590 nm.

Fig. 4

HPLC trace of a LPA mixture (10 μM LPA 14:0, 16:0, 18:0, 18:1, 20:4 and 20 μM LPA 17:0). Chromatographic conditions: column: Luna^™ C-8, 3 μm, 50 × 2.0 mm; mobile phase: MeOH:phosphate buffer (50 mM, pH 2.5) 16:5; flow rate: 0.32 mL/min; injection volume: 20 μL; sample concentration: 10 μM in MeOH:H₂O 9:1; post-column reagent: 10 μM DiA in H₂O; reagent flow rate: 0.62 mL/min; excitation/emission wavelengths: 450/570 nm.

Fig. 5

Emission spectra and calibration curve (inset) of 2.67 μM DiA upon titration with LPA (18:0).

Fig. 6

Calibration curves of specific LPA subspecies obtained by HPLC-post column fluorescence detection. The area ratio is the peak area of individual LPAs divided by the peak area of the internal standard (LPA 17:0). Data points represent the average of 4 runs.

Fig. 7

Calibration curves of LPAs using the LC-ESI/MS/MS method. The area ratio is the peak area of individual LPAs divided by the peak area of the internal standard (LPA 17:0).

Fig. 8

Chromatograms of a mixture containing 10 μM of each LPA (LPA 14:0, 16:0, 17:0, 18:0, 18:1 and 20:4) and LPAs isolated from human plasma (donor A) using the post-column detection method.

Fig. 9

LC-ESI/MS/MS traces of a 10 μM standard mixture of LPAs. Column: Luna^™ C-8 (50 × 2 mm, 3 μm) at 40 °C. Injection volume: 10 μL. Mobile phase: 9:1 MeOH:aqueous HCOOH (pH 2.5) at a flow rate of 0.4 mL/min. Parent and daughter ions were detected in the negative ion mode, sprayer voltage; 3.0 kV, capillary temperature at 300 °C.

Fig. 10

LC-ESI/MS/MS traces of a plasma sample (donor A). Conditions are the same as in Fig. 9.