Quality-by-design approach for development of aqueous headspace microextraction GC-MS method for targeted metabolomics of small aldehydes in plasma of cardiovascular patients

Lipid peroxidation products, such as short chain aldehydes, are powerful biomarkers of oxidative stress, due to the advantage of long lifetime compared to other metabolites of the lipidome. This work proposes an advanced combined derivatization/solvent-less extraction procedure from plasma followed by rapid Gas Chromatography with Mass Spectrometric detection (GC-MS). A new sample pretreatment protocol is presented which is based on a combination of aldehyde derivatization with methoxyamine under fully aqueous-based conditions of diluted plasma samples followed by headspace solid-phase microextraction (HS-SPME) which is faster compared to methods in the literature serving the same purpose. Being the smallest oximation reagent, methoxyamine derivatization does not require a silylation step of hydroxyl groups as customary and made it possible to have the shortest run times for this series of aldehydes by GC-MS. A Response Surface Methodology (RSM) is employed to optimize the HS-SPME of the aldehyde methoximes to provide insights into the Design Space (DS) of HS-SPME of aldehydes of variable chain lengths and unsaturation. The workflow includes a Quality by Design (QbD) approach for optimization of sample microextraction and derivatization methodology under fully aqueous conditions, in contrast to all reported non-aqueous tedious and long extraction methods in the literature followed by development of a rapid GC-MS assay. The optimal sample preparation obtained from the RSM, and multiple linear regression procedure involved addition of 15 mg methoxyamine (CH