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 (CH3ONH2) and 160 mg Na2SO4 to 0.5 mL plasma diluted to 1 mL with water in an extraction vial followed by HS-SPME using Polydimethylsiloxane/Divinylbenzene fiber at 750 rpm and 77 °C for 15 min.  The developed HS-SPME-GC-MS method was validated according to FDA guidelines in SIM mode and applied for targeted determination of lipid peroxidation aldehyde metabolites in plasma of 24 cardiovascular patients vs 20 healthy controls.  The run time of the GCMS method was less than 15 min and the LOQ of the 10 targeted aldehydes were 0.5 nM for decanal, 5 nM for hexanal, heptanal, octanal, citronellal and citral, 7 nM for malondialdehyde, 35 nM for 4- hydroxynonenal, 105 nM for 4- hydroxyhexenal and 500 nM for glyoxal.  Hexanal, Malondialdehyde and Hydroxynonenal concentrations were significantly higher in patients (p-value<0.05) in the targeted study, while citral was significantly lower as obtained from the untargeted study.  Reporting an aldehydic profile signature -whether predictive or diagnostic-for cardiovascular patients would support proper medical intervention at the initiation or progression phases of the disease when expanded on larger number of subjects.
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