Adduct supported analysis of γ-hydroxybutyrate in human serum with LC-MS/MS

Abstract

To avoid the detection of small fragmentation products of γ-hydroxybutyrate (GHB), a liquid chromatography-tandem mass spectrometry GHB quantification method in human serum supported by adduct formation was developed and validated. The continuous infusion of GHB/GHB-D6 made the identification of two adducts possible and GHB/GHB-D6 sodium acetate adduct fragmentation was used as target mass transition. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and 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. Protein precipitation with 1 mL of the mobile phase B was used as the sample preparation. The calculated limit of detection/quantification was 1 μg/mL. The presented study shows that the fragmentation of GHB sodium acetate adducts is an effective way of quantification of this small molecule and is an interesting alternative to other methods based on the detection of ions smaller than 85 Da. This fact together with the short analysis time of 3 min and the fast sample preparation make this method very attractive for forensic/clinical application.

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

“…In comparison to the LC/MS/MS method for GHB analysis in human serum on the basis of analyte adduct formation with the components of the mobile phase [6], a better LOD/LOQ could be achieved with the MS3 method presented here. The results of the validation experiments demonstrated, that the presented analytical strategy based on LC-MS/MS/MS and the GHB adduct formation/fragmentation process can be applied in the field of forensic toxicology and clinical chemistry as an alternative to common GHB analytical methods in human serum.…”

“…To ensure the best comparison between GHB detection with MS2 and MS3 the chromatographic conditions applied were the same as presented in the MS2 analytical method published earlier [6]. Therefore, the separation was performed with a Luna 5 m C18 (2) 100Å, 150 mm × 2 mm column (Phenomenex, Aschaffenburg, Germany) and the elution with a mobile phase consisting of 10% A (H 2 O/methanol = 95/5, v/v) and 90% B (H 2 O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH = 3.2).…”

“…This fact together with the similarity to other endogenous substances makes the ␥-hydroxybutyrate determination with LC-MS/MS difficult and is the reason why other MRM strategies like the application of the analyte adduct formation/fragmentation process were developed [6,7]. The strategy of analyte adduct application showed, that to avoid the detection of small GHB fragmentation products, fragmentation products with higher masses of appropriate GHB adducts can be used in the LC-MS/MS analytical method.…”

LC–MS n Small-Molecule Drug Analysis in Human Serum: Could Adducts have Good Prospects for Therapeutic Applications?

“…So far, it has been demonstrated that controlled analyte adduct formation is an interesting alternative (and sometimes even the only effective possibility) for the LC-MS n quantification of relevant small-molecule drugs [7,9,[11][12]15]. Although it was already demonstrated that analyte adducts can be observed for numerous drugs with appropriate molecular structure and therefore the possibilities for its application can be defined as wide, analyte adduct formation seems to be applicable only if the conventional strategy of LC-MS n analysis does not bring the expected results [17].…”

“…2 applications To avoid the detection of small ion fragments, an analyte adduct formation/fragmentation strategy was developed for the generation of bigger ion fragments that could be used for LC-MS 2 analysis of γ-hydroxybutyrate (GHB), which was used as a model drug [7]. A GHB sodium acetate adduct ion was described as a target, of which the fragmentation was the basis for LC-MS 2 analysis to avoid the detection of ions smaller than 85 Da as described previous [8].…”