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)

A highly sensitive method for the determination of D-lactic acid and 3-hydr oxybutyric acid (3-HB) in rat plasma was developed using high-performance l iquid chromatography with octadecylsilica (ODS) connected to a chiral colum n. At first, (D + L)lactic acid and 3-HE in the plasma were derivatized wit h a fluorescent reagent, 4-nitro-7-piperazino-2, 1,3-benzoxadiazole (NBD-PZ ), separated on the ODS column and determined fluorimetrically at 547 nm wi th 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 beta -cyclodextrin chiral column by changing the flow of the eluent via a six-port valve. Then, D-lactate derivative was separat ed enantiomerically from the L-lactate derivative, and the enantiomeric rat io was determined from the chromatogram. Intra- and inter-day accuracy valu es for the determination of D-lactic acid in 10 muL of rat plasma were 97.8 -109.2 and 98.4-109.9%, and those for 3-HE were 99.8-108.4 and 99.8-103.8%. respectively. The intra- and inter-day precision values were within 4.6 an d 5.1% for D-lactic acid, and 2.7 and 2.4% for 3-HE, respectively. The dete ction limits for D-lactic acid and 3-HE were approximately 2.0 and 0.04 muM , respectively (signal-to-noise ratio 3). The proposed method was applied t o the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D-lactic acid and 3-H E concentrations were observed in the diabetic rats as compared to the norm al rats. Copyright (C). 2001 John Wiley & Sons, Ltd.