Fluorometric determination of 3,5-dimethy-4, 6-diphenyl-tetrahydro-2H-1,3, 5-thiadiazin-2-thion in rat serum by high performance liquid chromatography with post column derivatization

Abstract

A rapid and sensitive method for the determination of 3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazin-2-thion (NIP-200) in rat peripheral serum was developed by using high performance liquid chromatography, with post column derivatization with NaC1O and o-phthal aldehyde (OPA) followed by detection with fluorometer. The NIP-200 was separated by the reversed phase on Zorbax ODS (5 µm, 4.6 mmϕ×150 mm). In the first reaction coil, NIP-200 was decomposed to methylamine, then, in the second reaction coil, the methylamine was made to react with OPA. The OPA derivative was detected fluorometrically(Ex 335 nm/Em 450 nm). The optimum concentration of OPA was in the range from 0.01% to 0.02%. A sample of 0.5 ml of rat serum was transferred to a 10 ml conical centrifuge tube. Then 5 ml of chloroform was added and the two phases were mixed thoroughly. The mixed solution added 1 ml of water was mixed softly, and centrifuged at 3000 rpm for 10 min. After 4 ml of the organic phase transferred was evaporated at 40°C, 50 µl of methanol was added, and 10 µl of the final solution was injected into a Chromatograph. The determination was made by measurement of the peak height of the NIP-200 derivative. The practical lower limit of the determination was 2.5 ng/ml serum (S/N=3). The relative standard deviations for 2.5 ng/ml and 5.0 ng/ml NIP-200 were 6.97% and 5.02%, respectively. The calibration curve was linear in the range from 0.2 ng to 10 ng of NIP-200. © 1986, The Japan Society for Analytical Chemistry. All rights reserved.