Integrated HPLC-MS and ¹H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides

Abstract

1. Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics.

2. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-β-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an α,α-dimethylated ibuprofen analogue.

3. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37°C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and ¹H-NMR spectroscopy and the results compared.

4. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, ¹H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of ¹H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than ¹H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-β-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers.

5. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

Keywords::

• High-performance liquid chromatography-mass spectrometry (HPLC-MS)
• nuclear magnetic resonance (NMR) spectroscopy
• acyl glucuronide
• hydrolysis
• transacylation
• plasma
• ibuprofen
• ibufenac

Acknowledgements

This work was supported by the BBSRC, EPSRC and AstraZeneca PLC through the award of CASE studentships to Caroline H. Johnson and Lisa Iddon.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.