Mass spectrometry applications for characterization and quantitation in biological matrices

Author(s)

Andréia de Melo Porcari

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2012-12-03

Examining board members

Marcos Nogueira Eberlin; Isabel Cristina Sales Fontes Jardim; Ed Hoffman Madureira

• Advisor: Marcos Nogueira Eberlin

Abstract

This research uses mass spectrometry (MS) as a tool to characterize and quantify different analytes in biological matrices. At first, the development of a technique for direct analysis of triacylglycerols (TAG) in meats and animal tissues is shown. This technique allows sample characterization through its lipid profile. It starts with a photo-heating process which aims to extract, almost instantaneously, TAG from the matrix, using very little amounts of solvent. The extract is collected on a paper which is then analyzed by EASI-MS (easy ambient sonic-spray ionization), thus revealing the TAG profile in a few seconds, without the use of hydrolysis, derivatization or exhaustive extractions. Thermally-imprinted EASI-MS (T-EASI-MS) is able to differentiate kinds of meats and has been shown to be in agreement with previous reported data and results from traditional techniques used for lipid analysis of the same samples. In a second phase, this research uses MS as a tool for quantitative analyses, through the development of two analytical methods for cortisol analysis in bovine plasma and milk, using a LC-MS/MS (liquid chromatography - tandem mass spectrometry) system. These methods were fully validated using the matrix matched methodology for plasma analysis and a non-extracted calibration curve (prepared in solvent) for milk analysis. Both methodologies use small amounts of sample and achieved very low limits of quantification (0.1 ng mL and 0.15 ng mL of cortisol for plasma and milk, respectively). The methods were applied to the analysis of samples from two veterinary experiments. In the first one, the aim was to investigate the correlation between bovine plasma and milk cortisol concentrations, as well as to determine if the milking process can change basal cortisol level in these fluids. In the second experiment, the aim was to evaluate whether milk cortisol concentrations varied or not in cows with or without sub-clinical mastitis. The samples of the second experiment were also analyzed by ELISA (enzime-linked immunosorbent assay) in order to compare the results with those from LC-MS/MS (AU)

• FAPESP's process: 10/03938-1 - Development and validation of a LC-MS/MS quantification method for cortisol determination in order to study animal welfare in cattle
• Grantee: Andréia de Melo Porcari
• Support Opportunities: Scholarships in Brazil - Master