LC- 1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 24; no. 3; pp. 477 - 485
• Main Authors: Mortensen, Rasmus W., Corcoran, Olivia, Cornett, Claus, Sidelmann, Ulla G., Troke, Jeff, Lindon, John C., Nicholson, Jeremy K., Hansen, Steen Honoré
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 2001; Elsevier Science
• Subjects: Acyl-migration; Analysis; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; General pharmacology; Glucuronide; Glucuronides - chemistry; Glucuronides - isolation & purification; HPLC elution order; HPLC-NMR; Magnetic Resonance Spectroscopy - methods; Medical sciences; Naproxen - chemistry; Naproxen - isolation & purification; Pharmacology. Drug treatments; Protons; Reactive metabolites; S-naproxen; Spectrophotometry, Ultraviolet; Stereoisomerism; HPLC elution order; HPLC-NMR; Reactive metabolites; Acyl-migration; Glucuronide; S-naproxen; Solid phase extraction; Metabolite; HPLC chromatography; Acyl; NMR spectrometry; Metabolism; Isocratic condition; Glucuroconjugate; Isomer; Non steroidal antiinflammatory agent; Reversed phase chromatography; Ultraviolet detector; Naproxen; Arylpropionic acid derivatives; Quantitative analysis
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(00)00453-2

The reactive metabolite S-naproxen-β-1- O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC- 1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-β-1- O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase I (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4- O-acyl isomers, β-1- O-acyl glucuronide, 3- O-acyl isomers, 2- O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: α/β-4- O-acyl isomers, S-naproxen aglycone, β-1- O-acyl glucuronide, 3- O-acyl isomers, and α/β-2- O-acyl isomers. In both systems the elution order for the 2-, 3- and 4- O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC- 1H NMR [U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113–122]. The α-1- O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-β-1- O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37°C. In both HPLC systems it eluted just before the β-1- O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a α-1- O-acyl isomer when studying glucuronide reactivity and degradation.