Simultaneous Determination of Free and Total Carnitine in Human Serum by HPLC with UV Detection

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method for simultaneous determination of free and total carnitine in human serum has been developed, and its clinical significance has been investigated. After proteins in serum were precipitated, carnitine in serum was derivatized to form its ester. HPLC separation of the sample solution was performed on a SiO(2) column and detected by UV absorbance at 260 nm. A mobile phase composed of acetonitrile-citric acid-triethanolamine was found to be the most suitable for this separation. The free and total carnitine levels in serum were studied in 347 subjects. The method proved to be linear in the range of carnitine from 5 micromol/L to 400 micromol/L. The relative standard deviations of within-assay for free and total carnitine analyses were 3.36% and 1.97%, respectively; and between-assay for free and total carnitine analyses were 3.34% and 1.77%, respectively. The average recovery was 98.2% for free carnitine and 96.3% for total carnitine, respectively. The method has been applied to the simultaneous determination of free and total carnitine in serum. Statistics analysis showed that total and free carnitine levels of males were higher than that of females (p < 0.01) while acylcarnitine level had no statistically significant difference (p > 0.05).