Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids

Abstract

Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (18∶2) purported to be anticarcinogenic, low levels of essential fatty acids, and trans fatty acids that increase when essential fatty acids are increased in dairy rations. Milk and rumen fatty acid methyl esters (FAME) were prepared using several acid‐(HCl, BF 3, acetyl chloride, H 2 SO 4) or base‐catalysts (NaOCH 3, tetramethylguanidine, diazomethane), or combinations thereof. All acid‐catalyzed procedures resulted in decreased cis/trans (Δ9 c, 11 t‐18∶2) and increased trans/trans (Δ9 t, 11 t‐18∶2) conjugated dienes and the production of allylic methoxy artifacts. The methoxy artifacts were identified by gas‐liquid chromatography (GLC)‐mass spectroscopy. The base‐catalyzed procedures gave no isomerization of conjugated dienes and no methoxy artifacts, but they did not transesterify N‐acyl lipids such as sphingomyelin, and NaOCH 3 did not methylate free fatty acids. In addition, reaction with tetramethylguanidine coextracted material with hexane that interfered with the determination of the short‐chain FAME by GLC. Acid‐catalyzed methylation resulted in the loss of about 12% total conjugated dienes, 42% recovery of the Δ9 c,11 t‐18∶2 isomer, a fourfold increase in Δ9 t,11 t‐18∶2, and the formation of methoxy artifacts, compared with the base‐catalyzed reactions. Total milk FAME showed significant infrared (IR) absorption due to conjugated dienes at 985 and 948 cm−1. The IR determination of total trans content of milk FAME was not fully satisfactory because the 966 cm−1trans band overlapped with the conjugated diene bands. IR accuracy was limited by the fact that the absorptivity of methyl elaidate, used as calibration standard, was different from those of the other minor trans fatty acids (e.g., dienes) found in milk. In addition, acid‐catalyzed reactions produced interfering material that absorbed extensively in the trans IR region. No single method or combination of methods could adequately prepare FAME from all lipid classes in milk or rumen lipids, and not affect the conjugated dienes. The best compromise for milk fatty acids was obtained with NaOCH 3 followed by HCl or BF 3, or diazomethane followed by NaOCH 3, being aware that sphingomyelins are ignored. For rumen samples, the best method was diazomethane followed by NaOCH 3.