| Summary: | 碩士 === 台北醫學院 === 藥學研究所 === 90 === D-Lactate level is an useful indicator for the degree of the microbial contamination, and it has some relations to the pathophysiology of human diseases such as encephalopathy, diabetes and acidosis, in which plasma D-lactate levels were increased.
For more effective quantitative determination of D-lactate, an HPLC method with pre-column fluorescent derivatization was developed. D,L-Lactate were fluorimetrically derivatized by 4-nitro-7-piperazino- 2,1,3-benzoxadiazole (NBD-PZ) and 4-(N,N- dimethylaminosulfonyl)-7- piperazino-2,1,3-benzoxadiazole (DBD-PZ). The following chiral columns were selected: Chiralcel OD-RH, Chiralcel OJ-R and Chiralpak AD-RH. The capacity factor, the separation factor and the resolution were investigated by modifying the compositions of the mobile phases. According to the results, a highly sensitive method which includes fluorescent derivatization of D,L-lactate with NBD-PZ was established. The elution order of D,L-lactate derivatives was L and D by recent literatures, but the elution order in our method was inverse. The inversion of the elution order can solve the problem of large peak of L-lactate covers over the peak of D-lactate when a small amount of D-lactate with a large excess of L-lactate in the biological samples.
The NBD-D,L-lactate is separated by HPLC on an octadecylsilica (ODS) column and determined fluorimetrically at 547 nm with 491 nm of excitation wavelength. During the separation step on the ODS, the peak fraction of (D + L)-lactate derivatives was introduced directly to an amylose chiral column by switching the flow of the elute via a six-port valve. Then, D-lactate derivative was separated enantiomerically from that of L-lactate, and the enantiomeric ratio was determined from the chromatogram. The inter-day and intra-day levels of precision were acceptable, with coefficients of variation from 0.80% to 14.44%.
The proposed method was applied to the urine of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of D-lactate related to creatinine were observed in the diabetic rats as compared to the normal rats. It should be necessary to pursue the changes of D-lactate concentrations under the pathological conditions.
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