Simultaneous Quantification of Ceramides and Sphingosine-1-Phosphate in Serum by LC-MS/MS with Application to Alcohol Intoxication Cases

Ceramides and sphingosine-1-phosphate (S1P) are bioactive sphingolipids critically involved in the regulation of cell survival and death, whose homeostasis is disrupted by alcohol exposure. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of nine ceramide species and S1P in serum. Following protein precipitation, analytes were separated on a CSH C18 column within an 11-min chromatographic run. Calibration curves were linear over 5 to 500 ng/mL with R2 greater than 0.99 for all analytes. Intra- and inter-day accuracy and precision met acceptance criteria across all quality control levels. Extraction recoveries ranged from 79.51% to 109.23%, matrix effects were adequately compensated by stable isotope-labeled internal standards, and dilution integrity was confirmed across factors of 1:5, 1:10, and 1:20. The method was applied to a large cohort of serum samples from suspected alcohol-impaired driving cases stratified by blood alcohol concentration (BAC) into negative control, mild, moderate, and severe intoxication groups. Very long-chain ceramides including Cer 22:0, Cer 24:0, and Cer 24:1, as well as S1P, were consistently elevated in alcohol-exposed groups relative to the negative control. These findings indicate that circulating sphingolipid profiles reflect lipid metabolism alterations induced by ethanol exposure and may provide complementary information to blood alcohol concentration in the assessment of alcohol-associated cases.