γ-Hydroxybutyrate analysis in human serum with liquid chromatography-tandem mass spectrometry on the basis of MS3 mass transition

An LC-MS/MS/MS GHB quantification method in human serum supported by the adduct formation process with the components of the mobile phase was developed and validated. The continuous infusion of GHB made the identification of a GHB MS3 mass transition possible (185/103/85). A Luna 5 μm C18 (2) 100Å, 150 mm × 2 mm analytical column and the elution with a programmed flow of the mobile phase consisting of 10% A (H₂O/methanol=95/5, v/v) and 90% B (H₂O/methanol=3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH=3.2) were used. The analytical sample preparation consisted of protein precipitation with 1 mL of the mobile phase B. The calculated limit of detection/quantification was 0.28/0.96 μg/mL, respectively. The study presented demonstrated that the process of analyte adduct formation with the components of the mobile phase can be used successfully to generate MS3 transitions in the case of very small molecules which cannot be detected in this way by the application of conventional analytical strategies.