Determination of free l-carnitine in human seminal plasma by high performance liquid chromatography with pre-column ultraviolet derivatization and its clinical application in male infertility

Abstract

Background To develop and validate a simple and reliable high performance liquid chromatographic (HPLC) method for the analysis of free l-carnitine in human seminal plasma and to investigate its clinical significance as a potentially additional means of evaluating the infertile male. Methods After proteins in seminal plasma are precipitated with a mixture of acetonitrile and methanol (9:1; v/v), free l-carnitine in seminal plasma was derivatized to form its UV-absorbing ester. HPLC separation of the sample solution was performed on a Lichrospher SiO 2 column and detected by ultraviolet absorbance at 260 nm. A mobile phase composed of acetonitrile-citric acid buffer (containing 12 mmol/L triethanolamine, pH 5.0) 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 free l-carnitine from interferences with isocratic elution. The free l-carnitine levels in seminal plasma were studied in both 30 control subjects and 87 patients with infertility. Ejaculates were classified into studied subgroups and defined as: asthenozoospermia ( n = 29), oligozoospermia ( n = 19) and oligoasthenoteratozoospermia ( n = 39). Results Under the chromatographic conditions described, the free l-carnitine derivative had a retention time of approximately 13 min. Good separation and detectability of free l-carnitine in human seminal plasma sample were obtained. The method proved to be linear in the range of l-carnitine from 0 μmol/L to 1000 μmol/L. The relative standard deviations of within- and between-assay for free l-carnitine analysis were 1.23 and 1.36 %, respectively. The recoveries were 91.6–96.5% for the human seminal plasma samples. Free l-carnitine concentrations in the populations were 392.66 ± 107.18 μmol/L in the fertile group ( n = 30), 270.00 ± 83.92 μmol/L in asthenozoospermia group, 187.97 ± 43.90 μmol/L in oligozoospermia group and 175.65 ± 67.07 μmol/L in oligoasthenozoospermia group. The large difference ( P < 0.01) between the fertile and infertile populations is evident and the difference between the subdivided groups in the infertile group is not significant ( P > 0.05). Conclusion The determination of free l-carnitine level in seminal plasma may prove useful as a potentially biochemical marker of fertility and this is a useful guidance for the clinic therapy and the mechanismic study on the male reproduction.