Abstract: Odd and branched-chain fatty acids (OBCFAs) possess functions such as maintaining intestinal health and exhibiting anti-cancer and anti-inflammatory effects. In recent years, they have become a research hotspot. However, the quantitative analysis methods for OBCFAs in bovine milk and dairy products still require further optimization. In this study, a gas chromatography-flame ionization detector (GC-FID) method was established for the analysis of 39 fatty acids including seven major OBCFAs (iso-C15:0, anteiso-C15:0, C15:0, iso-C16:0, iso-C17:0, anteiso-C17:0, and C17:0) commonly found in milk fat. Samples were directly methylated and detected by GC-FID and quantified by an internal standard method. Liquid milk and milk-based formula were selected as representative matrices to comprehensively validate the method’s specificity, precision, accuracy, and quantitation limits. The precision and accuracy were further confirmed using the standard reference material NIST SRM 1869. This method was also applied to compare the fatty acid compositions of 12 milk samples (organic and non-organic) from six brands across three sources: China, Europe, and Australia/New Zealand. The results showed good resolution of all 39 fatty acids. The seven OBCFAs exhibited no significant impurity interference at their peak positions, meeting the specificity requirements. The recoveries of the seven OBCFAs in milk and milk powder were 98%–102% and 99%–104% at spiked levels of 50% and 100%, respectively, with relative standard deviation (RSD) ranging from 1% to 4%. The limits of quantitation (LOQ) ranged from 0.340 to 0.837 mg/100 g for milk and from 3.063 to 7.530 mg/100 g for milk powder, indicating high precision, accuracy, and sensitivity. The measured values for 21 fatty acids with certified values in NIST SRM 1869 were within the certified ranges, with RSD of 1%–5%, demonstrating that this method also has excellent precision and accuracy for other fatty acids. With respect to fatty acid composition, milk from Australia/New Zealand contained higher relative contents of OBCFAs and ω-3 polyunsaturated fatty acids (PUFAs), as well as a lower ω-6/ω-3 PUFA ratio, compared with milk from China and Europe. Furthermore, in Australia/New Zealand, the total OBCFA relative content in organic milk was higher than that in non-organic milk from the same brand. Additionally, for the same brands in all three regions, organic milk contained higher levels of α-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, and ω-3 PUFA, and a lower ω-6/ω-3 PUFA ratio, compared to non-organic milk. This method is characterized by simple pretreatment, good specificity, high sensitivity, and excellent accuracy and precision, and can be widely applied to nutritional function research and quality evaluation of milk and dairy products.

Key words: bovine milk and dairy products; organic and non-organic milk; odd and branched-chain fatty acids; quantitative analysis; direct methylation; gas chromatography-flame ionization detector