Desenvolvimento de métodos de extração e derivatização para análises cromatográficas dos ácidos biliares e aminoácidos em estudos metabolômicos

Abstract

This work presents the development and validation of chromatographicmethods for quantification of bile acid in blood plasma and amino acids in cerebrospinal fluid. Sample preparation methods based on microwave assisted derivatization were developed for analyzing bile acids and amino acids. The derivatization of bile acids was performed using the two-step process of methoximation and silylation. The reaction time, power and quantity of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) + 1 % trimethylchlorosilane (TCMS) were optimized to improve the derivatization. The optimized derivatization conditions required 210 W for 3 min. The MAD method exhibited linearity with respect to cholic acid between 0.78 and 20.0 g mL-1 with an LOQ of 0.23 g mL-1 and a precision ranging from 1.08 to 9.32% CV. The amino acids samples were analyzed by gas chromatography with a mass spectrometric detector. The validated methodology for determination of amino acids was applied to real samples of cerebrospinal fluid from hospitalized patients with suspicion of fungical infection. The experimental design used to optimize the conditions showed that the optimal derivatization time was 3 min with a microwave power of 210 W. The sensitivity was very good, with limits of detection (LDs) ranging from 0.01 mol L1 to 4.24 mol L1 and limits of quantification (LQs) ranging from 0.02 to 7.07 mol L1. The precision, measured using the relative standard deviation (RSD), ranged from 4.12 to 15.59% for intra-day analyses and from 6.36 to 18.71% for inter-day analyses. The coefficients of determination (R2) were above 0.990 for all amino acids. Next, a hollow fiber-liquid phase microextraction method was developed for sampling and preconcentration of bile acids present in plasma samples. The analyses were performed by high performance liquid chromatography coupled to mass spectrometry. The optimized two-phase LPME procedure for the extraction of bile acids showed limits of detection between 2.07 and 2.66 nmol L-1 and limits of quantification between 10.35 and 13.20 nmol L-1. The intra-assay precision ranged from 3.3 to 11.9% (25.0 mg L-1) and 2.1 to 8.3% at the concentration of 100.0 mg L-1. The developed method was linear over the range of 10.35 to 531.0 nmol L-1 for all analytes. The methodology was validated and applied to the analysis of bile acids in plasma of workers exposed to solvents, and volunteers not exposed. The developed methods shown to be appropriate for both determinations inplasma and cerebrospinal fluid. The amino acid method using microwave assisted derivatization shows to be suitable for bio monitoring analysis, easy, quick, sensitive, and selective. The method for bile acids analysis by hollow fiber liquid phase microextraction showed high sensitivity, low solvent consumption, and good recovery.