Determination of serum D-lactic and L-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

D-Lactic and L-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography ( HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3- benzoxadiazole ( NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica ( Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor ( a) of 1.32 and a resolution (R-s) of 1.98. Moreover, the features of the first elution of D-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum D-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5 - 101.2 and 89.0 - 100.7%, and the intra-day and inter-day precisions were 0.3 - 1.2 and 0.4-4.8%, respectively. The proposed method was applied to determine D-lactic and L-lactic acids in human serum of normal subjects and diabetic patients, showing that both D-lactic and L-lactic acid concentrations were significantly increased in the serum of diabetic patients (n= 31) as compared with normal subjects ( n= 21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine D-lactic and L-lactic acid simultaneously. Finally, serum D-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.