Direct Analysis of Synthetic Phenolic Antioxidants, and Fatty Acid Methyl Ester Stability in Biodiesel by Liquid Chromatography and High-Resolution Mass Spectrometry

Methods to identify and quantify synthetic phenolic antioxidants, 3-tert-butyl-4-hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl-hydroquinone (TBHQ) and propyl gallate (PG), in biodiesel samples by using reversed-phase liquid chromatography (LC) were developed. Using a C18 phase with LC and UV detection showed co-elution between BHT and fatty acid methyl esters (FAME) in the biodiesel sample, whereas an alkyl phenyl modified stationary phase resulted in good separation of all antioxidants from the fatty acid matrix, and allowed more accurate quantification of antioxidants in biodiesel samples. The latter column was applied for further study. Calibration curves for the four antioxidants were constructed, and the limit of detection estimated. Good calibration linearity was observed over the investigated concentration range of 10–80 ppm, with correlation coefficients (R2) ranging from 0.9986 to 0.9995 for all antioxidants. LOD values of 0.010, 0.015, 0.0125 and 0.030 ppm, and recoveries of 70 ± 2, 85 ± 2, 103 ± 2 and 92 ± 4% for PG, TBHQ, BHA and BHT at injected concentrations of 35 ppm were established, respectively. The method was applied for quantification of antioxidants in biodiesel without addition of spiked antioxidants, then for biodiesel spiked with the four antioxidants, and a commercial source of biodiesel with BHT addition. Identification of FAME in the biodiesel samples was performed by using an instrument capable of ultra-high performance LC hyphenated with an electrospray Orbitrap mass spectrometer (UHPLC–ESI-OrbitrapMS). The stability of antioxidants and FAME in different samples was then investigated. Total FAME C18 content decreased to 52 ± 4% w/w after 1 week, and 29 ± 6% w/w after 8 weeks in the test sample without antioxidants; FAME content and antioxidant composition were stable in the samples with antioxidants added, even after 8 weeks exposure to sunlight.