A method for the fractionation and determination of carnitines in milk and milk products

Some analytical conditions were investigated in order to develop a more appropriate analytical method for the fractionation and determination of carnitines in milk and milk products. On fractionation into acid-soluble and acid-insoluble carnitines, a 6% concentration of perchloric acid was selected to obtain a clear milk supernatant, since concentrations of 2-6% perchloric acid made no significant difference to the recovery of acid-soluble and acid-insoluble carnitines. The acid-soluble fraction yielded, most of the octanoyl-, about half of the decanoyl-, a small part of the lauroyl- and none of the myristoyl carnitine. The effect of potassium hydroxide concentration on the hydrolysis of carnitine in milk was investigated, and 2.3N potassium hydroxide was found to be most suitable for the acid-soluble fraction of milk, while 3.5N was the most suitable concentration for insoluble milk fractions. Acetyl CoA with a high specific radioactivity of at least 0.05 pCi/5 nmol in the reaction mixture was required for determination of small amounts of carnitine in milk. It was found that phosphate buffer was superior to Tris buffer, since the radioactive noise resulting from the use of Tris in the reaction mixture caused a higher blank value. In this analytical method, thelower detection limit of carnitine in milk was 0.05 nmol, and the coefficients of variation were estimated to be 1.0% for total carnitine, 1.6% for free carnitine, 1.0% for acid-soluble carnitine and 6.2% for acid-insoluble carnitine.