A liquid chromatography and tandem mass spectrometry method for the determination of potential biomarkers of cardiovascular disease

A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitation of α-ketoglutaric acid (α-KG), l-carnitine (l-CAR) and acetyl-l-carnitine (acetyl-l-CAR) in human urine as potential biomarkers of cardiovascular disease. The separation was performed using an isocratic elution of 0.1% formic acid in water and acetonitrile (97:3, v/v) on an Acclaim 120 C8 column (150 mm × 4.6 mm, 3.0 μm). The flow rate of the mobile phase was 1.2 mL/min and the total assay run time was 3 min. Detection was performed on a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode via an electrospray ionization (ESI) source in positive and negative ion modes. This method covered a linearity range of 0.1–500 ng/mL for l-CAR and acetyl-l-CAR and 1–1000 ng/mL for α-KG with lower limits of quantification (LLOQ) of 0.08 ng/mL for l-CAR, 0.04 ng/mL for acetyl-l-CAR and 0.8 ng/mL for α-KG. The intra-day and inter-day precision and accuracy of the quality control samples exhibited relative standard deviations of less than 5.54% and relative error values from −5.95% to 3.11%. Analyte stability was evaluated under various sample preparation, analysis and storage conditions and varied from −9.89% to −0.47%. A two-step solid-phase extraction (SPE) procedure using silica gel and quaternary amine cartridges was used for urine sample cleanup. The average recoveries for all analyzed compounds were better than 86.64% at three concentrations. The method was successfully applied for the quantitation of α-KG, l-CAR and acetyl-l-CAR in human urine samples.