Optimization of a miniaturized microwave-assisted saponification and extraction method to evaluate mosh and MOAH in biological samples

• The miniaturized saponification enables MOH analysis in ≤400 mg of adipose tissue. • A re-extraction step was critical for accurate MOAH quantification. • The method led to an LOQ decrease of about 4 times compared to traditional methods. • Validated method meets JRC criteria for recovery and repeatability. • The method reduces sample size, solvent use, and processing time significantly. Mineral oil hydrocarbons (MOH) are a group of lipophilic contaminants composed of thousands of different compounds. The human toxicokinetics (absorption, metabolization, distribution and elimination) of these contaminants depends on their molecular structure but information are still lacking. The primary gap to be filled is the optimization and validation of a method to treat a very small amount of samples, as the ones produced during in-vitro studies. The aim of this study is to develop and validate a miniaturized microwave-assisted saponification and extraction (MASE) method for the evaluation of MOH subclasses in low amounts of biological samples (<400 mg). The method was validated using an LC/GC×GC-FID/MS system in mono- and two-dimensional modes. The results demonstrated excellent recovery (≥94%), repeatability, and compliance with JRC (Joint Research Center) guidance criteria for internal standard ratios. Furthermore, the method achieved a limit of quantification as low as 0.4 µg of MOH in the sample, outperforming conventional extraction methods.