Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography–high‐resolution mass spectrometry

Rationale High‐resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1 mL of human serum using gas chromatography (GC) coupled to HRMS. Methods Analytes were extracted from serum after deproteinization and liquid–liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full‐scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2 ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated. Results T esters showed a linear response between 0.25 and 10 ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10 ng/mL and isocaproate between 2 and 10 ng/mL); detection limits remained between 0.1 and 0.5 ng/mL and accuracy between 81% and 119%. The MA (MEW = 10 ppm) was maintained between −2.4 and 4.8 ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17‐OH‐progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5 ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3 ppm) was maintained between −0.47 and 0.12 ppm. Conclusions The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10 ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T‐isocaproate based on the MEW of 10 ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary.