doi:10.1016/j.chemphyslip.2006.01.007 
Copyright © 2006 Elsevier Ireland Ltd All rights reserved.
NMR and HPLC-MS/MS analysis of synthetically prepared linoleic acid diol glucuronides
Department of Pharmaceutical Sciences (JY, DH, DFG) and Chemistry (MDM), University of Connecticut, Storrs, CT 06269, United States
Received 30 September 2005;
revised 18 January 2006;
accepted 19 January 2006.
Available online 9 February 2006.
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Abstract
Hydroxylated fatty acids are important mediators of many physiological and pathophysiological processes in a variety of human tissues. Recent evidence shows that in humans many of these are ultimately excreted in the urine as the glucuronide conjugates. In this paper we describe a general approach for the chemical synthesis of glucuronide conjugate derivatives of fatty acids. The synthesis strategy employs three steps (epoxidation, hydrolysis and glucuronidation) using methyl linoleate as a model non-hydroxylated starting compound. Hydroxylated starting compounds would require only the glucuronidation step. NMR and HPLC-MS/MS experiments were used to help determine the structure of the synthesized glucuronide conjugates and to identify fragmentation product ions useful for discriminating positional isomers in biological samples. This synthetic strategy should prove useful for generating analytical standards in order to identify and quantify glucuronide metabolites of hydroxylated fatty acids in humans.
Keywords: Linoleic acid diols; Hydroxylated fatty acids; Glucuronides; NMR; HPLC-MS/MS
Article Outline
- 1. Introduction
- 2. Experimental procedures
- 2.1. Synthesis procedures
- 2.1.1. Methyl-d-glucuronate (2)
- 2.1.2. 1,2,3,4-Tetra-O-(3-fluorobenzoyl)-d-methyl glucuronate (3)
- 2.1.3. 2,3,4-Tri-O-(3-fluorobenzoyl)-α-d-methylglucuronide bromide (4)
- 2.1.4. 9,10- and 12,13-monoepoxide of methyl linoleate (6)
- 2.1.5. 9,10- and 12,13-dihydroxy methyl linoleate (7a and 7b)
- 2.1.6. 9,10-Dihydroxy methyl linoleate (7a)
- 2.1.7. 12,13-Dihydroxy methyl linoleate (7b)
- 2.1.8. 2,3,4-Tri-O-(3-fluorobenzoyl)-β-d-glucuronides of 9,10- and 12,13-dihydroxy methyl linoleate (8)
- 2.1.9. β-Glucuronides of linoleic acid diol (9)
- 3. Results and discussion
- 3.1. Theoretical consideration of structural isomers and stereoisomers
- 3.2. Characterization of glucuronide derivatives of dihydroxy methyl linoleate (8) and glucuronide derivatives of dihydroxy linoleic acid (9)
- 3.2.1. α-Glucuronide or β-glucuronide
- 3.3. Positional isomer assignment and stereochemistry for β-glucuronides of methyl linoleate (8)
- 3.4. HPLC-MS/MS investigation of synthesized compounds
- Acknowledgements
- References
Fig. 1. 1H NMR spectra of deprotected final products. 9b is 13-glucuronide substituted dihydroxy linoleic acid. 9d is 12-glucuronide substituted dihydroxy linoleic acid. 9h is 10-glucuronide substituted dihydroxy linoleic acid. 9i is 9-glucuronide substituted dihydroxy linoleic acid.
Fig. 2. NOESY data from 9i, 9h, 9d and 9b. The crosspeaks from the olefinic region to those peaks around 2 ppm and from 3.5 to 4.4 ppm and 2 ppm are used to determine stereochemistry.
Fig. 3. A typical mass spectrum of glucuronide positional isomers. (A) All isomers, the cone voltage is 70 V and the collision energy is 25 V, (B) the 9-glucuronide, (C) the 10-glucuronide, (D) the 12-glucuronide and (E) the 13-glucuronide. The cone voltage and collision energy for B–E are 70 and 35 V, respectively.
Fig. 4. Proposed fragmentation mechanism for the formation of the m/z 313 ion in the CID spectrum of 9-glucuronide-10-hydroxy-12-linoleic acid and 12-glucuronide-13-hydroxy-9-linoleic acid.
Fig. 5. Proposed fragmentation mechanism for the formation of the m/z 201 and m/z 171 ion from the m/z 313 ion fragment in the CID spectrum of 9-glucuronide-10-hydroxy-12-linoleic acid and 10-glucuronide-9-hydroxy-12-linoleic acid. (Charge-remote allylic fragmentation as described Wheelan et al., 1996.)
Fig. 6. Proposed fragmentation mechanism for the formation of the m/z 201 and m/z 171 ion from the m/z 313 ion fragment in the CID spectrum of 12-glucuronide-13-hydroxy-9-linoleic acid and 13-glucuronide-12-hydroxy-9-linoleic acid. (Charge-remote allylic fragmentation as described Wheelan et al., 1996.)
Table 1.
13C NMR chemical shift assignment of the 2,3,4-tri-O-(3-fluorobenzoyl)-d-glucuronides of 9,10- and 12,13-dihydroxy methyl linoleates (in CDCl3)a
a All FBz
13C resonate between 130.3 and 116.6 ppm. All methyl protecting groups at 6′ resonate at 53.0 ± 0.2 ppm. All methyl protecting groups at 1 resonate at 51.4 ± 0.1 ppm.
Table 2.
1H NMR chemical shift assignment and coupling-constants of the 2,3,4-tri-O-(3-fluorobenzoyl)-d-glucuronides of 9,10- and 12,13-dihydroxy methyl linoleates (in CDCl3)a
a All protons of FBz protect group resonate between 7.78 and 7.14 ppm. All protons on carbon 3–7 and 14–17 resonate from 1.68 to 1.14 ppm. All protons in methyl protecting groups at 1 resonate at 3.68 ± 0.01 ppm. All protons in methyl protecting groups at 6′ resonate at 3.73 ± 0.02 ppm.
Table 3.
13C MR chemical shift assignment of the β-glucuronides of 9,10- and 12,13-dihydroxy linoleic acid (in d-methanol and CDCl3)
Table 4.
1H NMR chemical shift assignment and coupling-constants of the β-glucuronides of 9,10- and 12,13-dihydroxy linoleic acid (in d-methanol and CDCl3)
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