Comparison of Six Methylation Methods for Fatty Acid Determination in Yak Bone Using Gas Chromatography

Yak bone is rich in nutrition but not used efficiently. Six methylation methods (M1: acetyl chloride-methanol method, M2: H₂SO₄-methanol method, M3: HCl-methanol method, M4: KOH-methanol method, M5: M2 in combination with M4, and M6: M3 in combination with M4) with (system 1 (S1)) or without (system 2 (S2)) prior acid hydrolysis for fatty acid (FA) determination using gas chromatography in yak bone were comprehensively compared. The contents of total FA determined by using M1 and M2 were significantly higher (p < 0.05) than those by using M3 for both S1 and S2 (119.83 versus 118.34 versus 38.31 mg/g in S1; 104.03 versus 93.73 versus 30.08 mg/g in S2 for M1 versus M2 versus M3, respectively). The result determined by M4, M5, and M6 was less than 5 mg/g. Validation results of M1 and M2 indicate that for most (>90%) of the FA, the intra-day and inter-day CVs were <9%. Recoveries for more than 90% of the FA were within a range of 85–117%. In addition, C18:1n9c, C16:0, C18:0, and conjugated linoleic acid were the major FAs in yak bone, approximately accounted for 50, 20, 10, and 10% of the total FA, respectively. And the ratio of saturated and unsaturated FA in yak bone was about 1:2. The acid hydrolysis/H₂SO₄-methanol esterification method would be more efficient for the accurate determination of FA in yak bone, whereas the direct acetyl chloride-methanol method would be used as an alternative for the fast analysis.