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Development and validation of a LC-MS/MS method for determination of eight PEth homologues in whole blood
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Resumo(s)
Phosphatidylethanol (PEth) is a group of direct ethanol biomarkers used to distinguish between different
drinking patterns, such as heavy and social drinkers. So far, the PEth homologue most used for this
purpose is PEth 16:0/18:1. This homologue is also the one determined in most of the LC-MS/MS
methods developed for PEth analysis. Often one or a few PEth homologues are quantified in LC-MS/MS
methods published, but recently a LC-MS/MS method for the determination of six PEth homologues
was developed. The main aim of the dissertation was to develop a LC-MS/MS method for the
quantitative determination of eight of the most common PEth homologues in whole blood. This can be
helpful, especially for the determination of PEth in blood from people with high alcohol consumption.
Chromatographic separation of the PEth homologues was tested on five UHPLC columns. Different
mobile phases were investigated to optimize separation. Best chromatographic separation of the PEth
homologues was achieved using an Acquity BEH C18 column (100 2.1 mm I.D., 1.7 µm particles)
and a mobile phase consisting of 5 mM ammonium acetate pH 5 (Solvent A) and methanol (Solvent B).
The total run time was 5.7 minutes. MS/MS parameters were optimized for each of the eight PEth
homologues and internal standards. Two MRM transitions ions, those with the highest responses, were
chosen for each compound. Isotope labeled internal standards were used for five of the eight PEth
homologues. Two sample preparation techniques, liquid-liquid extraction (LLE) and 96-well supported
liquid extraction (SLE), were investigated to extract the PEth homologues from blood. The extraction
recoveries obtained were within 45-82% when LLE was used and within 68-80% when 96-well SLE
was used. Matrix effects were studied and showed ion suppression for the majority of the PEth
homologues, and ion enhancement for PEth 17:0/18:1. For compounds with their own internal standards,
the matrix effects were corrected for their internal standard. An LC-MS/MS method for the
determination of eight PEth homologues was developed and validated. The validation showed that
UHPLC-MS/MS method is precise, accurate, and sensitive for its purposes.
Descrição
Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2022
Palavras-chave
PEth homólogos LC-MS/MS Extração líquido-líquido (LLE) Extração sólido-líquido em 96-well (96-well SLE) Validação do método Teses de mestrado - 2022