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

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 SiO2 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 micromol/L to 1000 micromol/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 micromol/L in the fertile group (n=30), 270.00+/-83.92 micromol/L in asthenozoospermia group, 187.97+/-43.90 micromol/L in oligozoospermia group and 175.65+/-67.07 micromol/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.