LC-MS method development for analysis of lipid peroxidation products
Layer, Daniel Michael, III
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Background . Lipid peroxidation (LPO) is a prominent manifestation of oxidative stress in biological systems and the levels of LPO products in human blood are advocated to be useful markers of systemic oxidative stress. Linoleic acid peroxides are the predominant LPO products in human blood serum and the goal of this project is to transition an existing high performance liquid chromatography-photodiode array (HPLC-PDA) methodology to a liquid chromatography-mass spectroscopy (LC-MS) platform. It is anticipated that an LC-MS method will provide greater sensitivity and specificity in comparison to HPLC-PDA. Methods . Flow injection analysis using pure standard compounds was used to identify the optimum ionization interface (APCI or ESI), mode (positive or negative) and preferred mobile phase solvent for MS detection. Resolution of standard compound mixtures in reversed phase chromatography was systematically optimized by proportioning the mobile phase composition of water and acetonitrile. A sample preparation methodology was developed to include a deuterated internal standard and the method performance characteristics including linearity, recovery (by standard additions methodology) and imprecision were evaluated. The ability of the method to respond to known oxidative stress was examined in human blood experimentally oxidized by Fenton reaction using copper ions. Results . ESI interface in negative ion mode produced abundant, predictable, M-1 molecular ions and was selected for MS detection. Acetonitrile containing 40% water at a flow rate of 0.3 mL/min, without acid or based modifiers, achieved a compromise between optimal chromatographic resolution and optimal MS detection. The linear rage of the LC-MS method was 0.056 to 1600 nM which was 15X more sensitive than HPLC-PDA. Sample extraction recovery ranged from 75–103% for authentic lipid analytes and 72–81% for deuterated internal standard compound d4-13-hydroxy-octadecadienoic acid. The imprecision of the assay was estimated to be 15–18% coefficient of variation. Copper ion Fenton reaction oxidation of human serum resulted in a 10-fold increase in the level of the target compound 9-hydroxy-octadecadieneoic acid. Conclusion . LC-MS provides a more sensitive platform for measurement of total linoleic acid peroxidation products relative to HPLC-PDA. However, the mobile phase volatilization requirements of the MS detector limit the water content and flow rate of the LC system and thereby the chromatographic resolution. The limited chromatographic resolution results in equivocal MS peak integration and marginal analytical imprecision. Future method development should focus on increasing the chromatographic resolution of the reported methodology.