2022 Volume 43 Issue 3 Pages 143-147
A hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS)-based highly sensitive method utilizing limited sample volumes has been developed to analyze 5-aminolevulinic acid (5-ALA) in cell samples. Under the constructed HILIC conditions, 5-ALA is completely separated from biomolecules with similar molecular weights (leucine, isoleucine, hydroxyproline, and creatine), allowing for selective quantification through MS/MS. To analyze 5-ALA using limited amounts of 5-aminolevulinic acid synthase (ALAS) reaction solution, we established a pretreatment method to handle smaller sample volumes. A calibration curve of 5-ALA standard solutions in the range of 7.5–375 nM shows good quantitation with R2 = 0.999 and satisfactory precision in the range of 0.7–5.3%. The mentioned concentration range corresponds to 1–50 nM in 5-ALA synthase ALAS reaction solution and is below the quantification limit (3.9 nM) of the existing ALAS activity assays. Thus, the proposed method successfully quantifies the 5-ALA concentrations in cell lysates after the ALAS reaction.
