Selection of the derivatization reagent—The case of human blood cholesterol, its precursors and phytosterols GC–MS analyses

Phytosterols (PS; β-sitosterol and campesterol) and cholesterol precursors (CP; desmosterol and lathosterol) have been suggested as important biochemical markers of cholesterol intestinal absorption and liver biosynthesis, respectively. Given that these compounds appear in human blood in low amounts, sensitive and accurate methodology is required, such as gas chromatography–mass spectrometry (GC–MS) the most frequently used. One of the most critical factors of the GC analytical determination is the step of derivatization. Thus, the main objective of the present study was compare and select the better (one out of three) silylation mixtures as follows: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide/ammonium iodide (MTBSTFA:NH4I), N-O-bis-(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane (BSTFA:TMCS), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide/1,4-dithioerythritol/trimethyliodosilane (MSTFA:DTE:TMIS). The results of this study are discussed and accompanied by a brief review on the importance and principles of derivatization process, specifically in silylation reactions in GC–MS sterols analyses. Furthermore, a scrutiny of some published results is presented, along with additional information about mass spectral data of these potentially useful compounds. Interestingly, the results of the study showed that from the three validated methodologies, the selected one, based on the best relation specificity/sensibility, is MSTFA:DTE:TMIS. With this silylation procedure for simultaneous determination of PS and CP in human serum by GC–MS in selected ion monitoring (SIM) mode, good linearity (r2 ≥ 0.931), precision (repeatability ranging from 0.92 to 3.91 CV and intermediate precision ranging from 5.12 to 6.33) and recoveries (≥93.6%) were obtained. Thus, it proved to be a helpful methodology in the quantification of predominant serum cholesterol origin in each patient.

► Simultaneous derivatization of cholesterol precursors and phytosterols in serum.
► MSTFA/DTE/TMIS was selected as the best silylation reagent.
► Assessment of biomarkers for cholesterol absorption/synthesis ratio determination.
► Better control of hypercholesterolemia.
► Contribution for a better drug prescription and for the elaboration of diets.