Simultaneous quantification of sphingolipid metabolites in human plasma using UPLC-MS/MS

Sphingosine (SP), dihydro-sphingosine (DSP), and sphingosine 1-phosphate (S1P) are integral metabolites in the sphingolipid pathway. They exhibit intricate and diverse functions in the development of metabolic-associated fatty liver disease (MAFLD). In this study, we developed a highly sensitive and specific analytical approach for the concurrent quantification of these three sphingolipids in human plasma, utilizing ultra-high-performance liquid chromatography (UPLC), in conjunction with tandem mass spectrometry (MS/MS). A stripped biological matrix was generated through optimized activated‑carbon treatment to facilitate more accurate calibration. The sample preparation involved a straightforward protein precipitation method using methanol, followed by concentrating the supernatant and reconstituting it prior to injection. Detection was executed in the multiple reaction monitoring mode with a triple quadrupole mass spectrometer. The chromatographic separation was achieved on a Waters ACQUITY UPLC HSS C₁₈ column employing gradient elution. D-erythro-sphingosine-d₇ served as the internal standard. The newly established method conformed to recognized standards for selectivity, linearity, accuracy, precision, recovery, matrix effects, and stability. This technique was effectively applied to quantify the three sphingolipid metabolites in plasma samples collected from patients diagnosed with MAFLD (n = 30), and healthy controls (n = 30). Analysis of the data revealed significant differences between the groups, with the levels of SP, DSP, and S1P considerably elevated in MAFLD patients compared to healthy subjects.