Untargeted Profiling of Biodiesel Oxidation Products by UHPLC-HRMS and Multivariate Analysis

Biodiesel undergoes oxidative degradation, losing stability
upon
exposure to heat, oxygen, and metallic traces. To investigate these
alterations, six commercial B100 samples and one laboratory-synthesized
sample (hereafter referred to as “originals”) were subjected
to two standardized oxidation assays: RapidOxy (ASTM D7545) and Rancimat
(EN 14112); and subsequently analyzed using ultrahigh-performance
liquid chromatography coupled to high-resolution mass spectrometry
(UHPLC-HRMS). An untargeted analytical approach initially detected
7,722
molecular features. A Python-based script was employed to remove redundancies,
resulting in 3,131 unique variables. Following data standardization,
supervised modeling was conducted using Partial Least Squares Discriminant
Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminant
Analysis (OPLS-DA). These models, validated through 200 permutation
tests, exhibited determination coefficients (R2) close to 1.0 and group-based cross-validated accuracy (Q2) exceeding 0.8, effectively distinguishing
nonoxidized from oxidized fuels. Features with Variable Importance
in Projection (VIP) scores greater than 2.0 in PLS-DA and above 1.5
in OPLS-DA were subjected to the Wilcoxon–Mann–Whitney
test. Resulting p-values were adjusted using the Benjamini–Hochberg
procedure and combined with a |log2 fold-change| threshold
of ≥2.0, yielding 12 statistically robust candidate molecular
features. Individual Receiver Operating Characteristic (ROC) curves
showed area under the curve (AUC) values ranging from 0.80 to 1.00,
confirming their strong discriminatory power. Their chemical compositions
clearly distinguish degradation pathways: the RapidOxy assay produced
hydroperoxy-epoxydienoates and furan-fatty acids, whereas the Rancimat
test yielded hydroxy-stearates, dihydroxypalmitates, and the lactone
curvulalide. These findings support the differentiation between oxidation
profiles and may offer insights into the predominant pathways involved
in biodiesel oxidation.