Simultaneous determination of D-lactic acid and 3-hydroxybutyric acid in rat plasma using a column-switching HPLC with fluorescent derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ)

• Published in: Biomedical chromatography 2001-05, Vol.15 (3), p.189-195
• Main Authors: Fukushima, Takeshi, Lee, Jen-Ai, Korenaga, Takanori, Ichihara, Hideaki, Kato, Masaru, Imai, Kazuhiro
• Format: Article
• Language: English
• Subjects: 3-Hydroxybutyric Acid - blood; Animals; Chromatography, High Pressure Liquid - methods; Diabetes Mellitus, Experimental - blood; Fluorescent Dyes; Lactic Acid - blood; Male; Oxadiazoles; Piperazines; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.60

A highly sensitive method for the determination of D‐lactic acid and 3‐hydroxybutyric acid (3‐HB) in rat plasma was developed using high‐performance liquid chromatography with octadecylsilica (ODS) connected to a chiral column. At first, (D + L)‐lactic acid and 3‐HB in the plasma were derivatized with a fluorescent reagent, 4‐nitro‐7‐piperazino‐2,1,3‐benzoxadiazole (NBD‐PZ), separated on the 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 derivative was introduced directly to a phenylcarbamoylated β‐cyclodextrin chiral column by changing the flow of the eluent via a six‐port valve. Then, D‐lactate derivative was separated enantiomerically from the L‐lactate derivative, and the enantiomeric ratio was determined from the chromatogram. Intra‐ and inter‐day accuracy values for the determination of D‐lactic acid in 10 µL of rat plasma were 97.8–109.2 and 98.4–109.9%, and those for 3‐HB were 99.8–108.4 and 99.8–103.8%, respectively. The intra‐ and inter‐day precision values were within 4.6 and 5.1% for D‐lactic acid, and 2.7 and 2.4% for 3‐HB, respectively. The detection limits for D‐lactic acid and 3‐HB were approximately 2.0 and 0.04 µM, respectively (signal‐to‐noise ratio 3). The proposed method was applied to the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D‐lactic acid and 3‐HB concentrations were observed in the diabetic rats as compared to the normal rats. Copyright © 2001 John Wiley & Sons, Ltd.