The mystery of post-mortem Gamma-hydroxybutyrate formation - method development and validation for the detection of endogenous GHB and its related compounds.

Interpreting γ-hydroxybutyrate (GHB) concentrations in post-mortem samples remains challenging, due to the potential for formation both in corpore after death and in vitro after sampling. The possible influence of metabolically-related endogenous substances on post-mortem GHB levels has not yet been clarified. To address this, an analytical method was developed and validated for the simultaneous detection of GHB, γ-butyrolactone (GBL), and eight related endogenous compounds: succinic semialdehyde, γ-aminobutyric acid, putrescine, α-hydroxybutyrate (AHB), β-hydroxybutyrate (BHB), L-glutamic acid, succinic acid, and GHB-glucuronide (GHB-Gluc). Sample preparation involved protein precipitation using acetonitrile/methanol (85:15), followed by solid-phase extraction. Chromatographic separation was achieved using a reversed-phase C-18 analytical column with a 33-minute gradient run employing water and acetonitrile, both containing 0.1 % formic acid, as mobile phases. Human whole blood served as the matrix for calibration and quality controls, with endogenous levels corrected using matrix blanks. The method achieved limits of detection and limits of quantification of 0.5 µg/mL for all analytes. Calibration ranges extended up to 75 µg/mL, depending on the substance. Linear regression was applicable for most analytes, except BHB, GHB, putrescine, and succinic acid. Accuracy and precision were satisfactory (< +/- 10 %) across all concentration levels. The LC-MS/MS method allows for comprehensive quantification of GHB and related endogenous substances potentially involved in its post-mortem increase. Application to real post-mortem samples will help clarifying the role of these compounds in GHB formation after death and support more accurate interpretation of forensic toxicological findings.