Identification of the Positional Isomers of 2-Fluorobenzoic acid 1-O-Acyl Glucuronide by Directly Coupled HPLC-NMR

Abstract

Directly coupled HPLC-lH NMR was used in the "stopfiaw"mode to separate and rapidlyidenwan equilibrated mixture of ester glucuronide isomers formed spontaneously by intramolecular rearrangement reactions ( i t e d acyl migration and mutarotation) of 2-fluorobenzoic acid B-1-glucuronide (1-0-(2-fluorobenzoyl)-~-glucopyranuronic acid). The equilibrated mixture of isomers was obtained by incubation of the synthetic 2-fluorobenzoic acid glucuronide in buffer solution (PH 7.4) at 25 "C for 24 h. The B-anomer of the 1-0-acyl glucuronide, and the 2-, 3-, and 4-positional glucuronide isomers (all three as both a-and B-anomers) present in the equilibrium mixture, were all characterized after separation in an isocratic chromatographic system containing phosphate buffer at pH 7.4 and 1% acetonitrile in the mobile phase. The HPLC-NMR investigations also elucidated the mutarotation of the positional glucuronide isomers as well as showing the bene& of the HPLC-NMR technique as a primary analytical tool. This HPLC-NMR method will be of particular value in studies on the acyl migration reactions of nonsteroidal ant3da"atory drug glucuronides which may be related to their toxicological properties. Abstract published in Advance ACS Abstracts, August 15, 1995.

Separation of isomers of acyl glucuronides using micellar electrokinetic capillary chromatography in dynamically coated capillaries

“…However, as the mass over charge for a pair of anomers is very similar a migration order in the MEKC system corresponding to the order of elution obtained in reversed-phase HPLC can be expected. The elevated baseline between two anomers is due to the anomerization process taking place during electrophoresis [17].…”

“…Until now only high-performance liquid chromatography (HPLC) combined either with ultraviolet (UV) light absorbance [7,[10][11][12][13], fluorescence [14], mass spectrometry [15] or with nuclear magnetic resonance (NMR) [16][17][18][19][20][21][22] detection have been used for this purpose. The detection by NMR has the major advantage that the full structure of each peak may be assigned including the anomerization status and the isomer position.…”

Methods of Glycoconjugate Analysis

“…For example, to follow reactions in vitro such as acyl migration, mutarotation etc. Sidelmann et al (1995) have reported that low rates of 1 ml/min is optimal with respect to filling the NMR flow cell (a lH flow probe of 3mm i.d. with a volume of lOOpl).…”

Systematic Investigation into the Differences in the (+) APPI Efficiencies of Positional (Ortho, Meta, and Para) Isomers

“…For this reason, many studies aimed at understanding the APPI ionization mechanism have been conducted in order to better use this technique. [39][40][41][42][43] However, the separation and detection of isomers present great challenges in terms of analytical resolution and quantification using MS-based techniques. Isomers can behave differently in chemical reactions, which is why, for example, one isomer can be a pharmaceutical and another is harmful to living organisms.…”

“…Isomers can behave differently in chemical reactions, which is why, for example, one isomer can be a pharmaceutical and another is harmful to living organisms . Consequently, the ability to separate and identify positional isomers is important in many fields . However, the separation and detection of isomers present great challenges in terms of analytical resolution and quantification using MS‐based techniques …”