Using a non-natural fatty acid methyl ester (FAME) as an internal standard we evaluated the ability of easy ambient sonic-spray ionization mass spectrometry (EASI-MS) to quantitate the unsaturated FAME levels in B100 biodiesel from soybean, canola and Jatropha curcas. The ability of EASI(+)-MS to discriminate between saturated versus unsaturated FAME was also evaluated. Calibration curves for the major FAME, those from linolenic, linoleic and oleic acids, showed good linearity with coefficient correlation > 0.99 with the EASI(+)-MS method. The spike recoveries ranged from 76% to 127% with good repeatability and reproducibility as indicated by the relative standard deviation (RSD), which ranged from 5.1% to 17.7%. The accuracy of the method was established using a standard reference material (SRM). Real biodiesel samples were also quantitated via EASI(+)-MS, and the unsaturated FAME content was found to correlate adequately with the data obtained from the iodine number. Therefore, EASI(+)-MS was shown to provide fast, direct, linear and accurate quantitation of the unsaturated FAME in biodiesel samples. (C) 2014 Elsevier Ltd. All rights reserved.