Analysis of γ-(Cholesteryloxy)butyric Acid in Biologic Samples by Derivatization with 5-(Bromomethyl)fluorescein Followed by High-Performance Liquid Chromatography with Laser-Induced Fluorescence Detection

Abstract

This report describes the first application of 5-(bromomethyl)fluorescein (5-BMF) for the quantitation of a pharmaceutically relevant carboxyl-containing analyte in a biological matrix. An analytical method for quantitation of γ-(cholesteryloxy)butyric acid (CBA), a relatively new antitumor agent, in different tissues of Sprague-Dawley rats was developed. 5-BMF was employed to form a stable and spectrally well-characterized conjugate of CBA. The derivatization yield was maximized by optimizing several reaction variables. The conjugate was separated by high-performance liquid chromatography and quantitated by a laboratory-constructed argon ion laser fluorometer. The detection limits for CBA were 4.6 × 10 -9 and 6.34 × 10 -11 M by conventional and laser-induced fluorescence (LIF), respectively. A derivatization limit of detection of 1.85 × 10 -9 M was achieved by LIF for the conjugate. The analytical method was useful for quantitation of CBA in various tissues in the picogram per milliliter range.

Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as labeling reagent

“…Therefore, the feasibility of TMPAB‐I as a derivatizing reagent in CE‐LIF has been evaluated by the simultaneous detection of six LMM thiols. Under optimized derivatization and separation conditions, a baseline separation of all the six derivatives was achieved within 18 min and the limits of detection (signal‐to‐noise ratio = 3) are from 0.04 to 0.07 nM, which are lower than reported methods . The practical applications of the proposed method have been validated by the determination of thiols in human plasma samples and plum flower with recoveries of 92.0–109.4%.…”

Capillary electrophoresis in the evaluation of aminothiols in body fluids

“…The dye labels both thiols and carboxylic groups; besides the free carboxyl group of aminothiols can react with the CH 2 Br moiety of 5-BMF to form a fluorescent conjugate which can be monitored by an argon ion laser fluorometer [73].…”

6‐Oxy‐(acetyl ethylenediamine) fluorescein, a novel fluorescent derivatization reagent for carboxylic acids and its application in HPLC

“…So far, 5-BMF has been studied in detail to detect carboxylic acids in HPLC [28 -30] and CE [31,32] acid derivatives are observed [30] and it is not used in the separation of multicarboxylic acids and in real sample. With CE method, 5-BMF is used to detect the fatty acids in serum [31].…”