Evaluation of Micro Extraction by Packed Sorbent, Liquid-Liquid Extraction and Derivatization Pretreatment of Diet-Derived Phenolic Acids in Plasma by GC-MS/MS

Claudia Mardones, Instrumental Analysis Departament, Faculty of Pharmacy, University of Concepción, Concepcion, Chile
Luis Bustamante, Instrumental Analysis Departament, Faculty Of Pharmacy, University Of Concepción, Concepcion, Chile
Dietrich von Baer, Instrumental Analysis Departament, Faculty Of Pharmacy, University Of Concepción, Concepcion, Chile
Daniel Duran, Clinical Biochemistry And Immunology Departament, University Of Concepcion, Concepcion, Chile
Edgar Pastene, Departament Of Pharmacy, Faculty Of Pharmacy, University Of Concepción, Concepcion, Chile

Miniaturized sample pretreatments for the analysis of phenolic metabolites in plasma involving protein precipitation, enzymatic deconjuation, extraction procedures (microextraction by packed sorbent (MEPS)) and liquid - liquid extraction (LLE)) and different derivatization reactions were systematically evaluated. The analysis were conducted by gas chromatography coupled to a triple quadrupole mass detector operating in multiple reaction monitoring mode for the evaluation of 43 possible diet-derived phenolic acids. A previous enzyme purification step was necessary for the phenolic deconjugation before extraction. BSTFA/TMCS and two different tetrabutylammonium (TBA) salts for derivatization reactions were compared. The optimum reaction conditions were 50 µL of BSTFA/TMCS at 90 °C for 30 min, while TBA salts were associated with loss of sensitivity due to rapid activation of the inert GC liner. Phenolic acids extractions from plasma, using MEPS and LLE, were optimized in order to achieve reproducible quantification. Optimal MEPS performance was achieved using a C-18 packed bed, and better results for less polar compounds such as methoxylated derivates were observed. Despite the low recovery for some analytes, the repeatability obtained using the automated extraction procedure in the GC inlet was 2.5%. Instead, using LLE, better recoveries (80-110%) for all studied analytes were observed, however, at the expense of repeatability (3.8-18.4%). The analytical investigation of phenolic compounds in biological samples such as gerbil plasma collected before and after the administration of a calafate extract was tested. The miniaturized procedures allow to work with small sample volumes, which is imperative in metabolomic studies using plasma from small laboratory animals