Determination of free and total L-carnitine in human serum by HPLC method and Its clinical application

Abstract

Objective To develop and validate a simple and reliable HPLC method for the analysis of flee and total carnitine in human serum and to investigate its clinical significance. Methods After proteins in serum were precipitated with a precipitating reagent, carnitine in serum was derivatized to form its ester. HPLC separation of the sample solution was performed on a Lichrospher SiO2 column and detected by ultraviolet absorbance at 260 nm. A mobile phase composed of acetonitrile-citric acid-triethanolamine was found to be the most suitable for this separation at a flow rate of 1.2 ml/min and enabled the baseline separation of the carnitine from interferences with isocratic elution. The free and total carnitine levels in serum were studied in 347 subjects. Results Under the chromatographic conditions described, the carnitine derivative had a retention time of approximately 10 min. Good separation and detectability of carnitine in human serum sample were obtained. The method proved to be linear in the range of carnitine from 0 μmol/L to 400 μmol/L The relative standard deviations of within-assay ( n = 5 ) for free and total carnitine analysis were 3.36% and 1.97% , respectively, and between-assay (n =7) for free and total carnitine analysis were 3.04% and 1.77%, respectively. The average recovery was 98. 2% for free camitine and 96.3% for total carnitine, respectively. The average L-carnitine concentrations in the 347 subjects were as follows: total carnitine (52. 2 ± 8. 6 ) μmol/L, free carnitine ( 42. 3 ± 8. 3 ) μmol/L and acylcarnitine ( 9. 9 ± 2. 9 )μmol/L in the male group ( n = 182), and total carnitine (48.2 ± 9. 9 ) μmol/L, free camitine ( 37.9 ±8. 7) μmol/L and acylcarnitine ( 10. 3 ± 3.5 ) μmol/L in the female group ( n = 165 ). Statistical analysis showed that total and free carnitine levels of male were higher than that of female ( P 0. 05). Conclusion The method has been successfully applied to the simultaneous determination of free and total carnitine in serum with good sensitivity, specificity and repeatability, and this is a useful guidance for the clinic therapy and the mechanism study on the diseases associated with carnitine. Key words: Carnitine; Chromatography,high pressure liquid ; Reference values