Current separations 20-2.cdr

Color profile: Generic offset separations profileBlack 150 lpi at 45 degrees S.Bolze ,
1 O.Lacombe ,
1 G.Durand ,
2 P.Chaimbault ,
2 F.Massière ,
3 C.Gay-Feutry ,
3 N.Bromet *
T.Hulot1
1. LIPHA, 115, avenue Lacassagne, 69003 Lyon, France2. BIOTEC CENTRE, 10 avenue Claude Guillemin, 45071 ORLEANS CX 2, France3. BIOPREDIC, 14-18 rue du Professeur Jean Pecker, 35000 RENNES, France Standardization of a LC/MS/MS Method for the
Determination of Acyl Glucuronides and Their Isomers
Acyl glucuronides of carboxylic acids are unstable and reactive metabolites that canisomerize (by acyl migration) and hydrolyze at physiological pH. They also can bindto proteins under these conditions. The reactivity of acyl glucuronides wasdetermined in vitro by monitoring the formation of isomers and hydrolysis productsof 1-O-b-acyl glucuronide produced by human microsomes. This required thedevelopment of a standard analytical method for acyl glucuronides. Although ionspray-tandem mass spectrometry could be used for specific detection of acylglucuronides, the isomers showed similar fragmentation pathways. Consequently,chromatographic separation was also required. The chromatographic method wasbased on a single stationary phase with variation of the percentage of the organicmodifier in mobile phase (ammonium acetate 10mM - acetonitrile) in gradientelution mode. This method was used to resolve the acyl glucuronide isomers of eightdrugs and to quantify the unmetabolized aglycones. The relative amounts of thedifferent isomers were used to elucidate the mechanism of the isomerization reaction.
This method can readily be extended to study the reactivity of the acyl glucuronidemetabolites of new chemical entities. Introduction
M a ny a c i d i c d r u g s c o n t a i n i n g Materials and Method
e i g h t a c i d i c d r u g s : To l m e t i n , In vitro biosynthesis of
acyl glucuronides
F1 . These drugs have
to bind covalently to proteins in vitro and in vivo causing potential toxicity 17-22.prnC:\work\current separation\cs20-2\current separations 20-2.cdrWednesday, August 28, 2002 9:34:42 AMPlate: 1 of 12 Color profile: Generic offset separations profileBlack 150 lpi at 45 degrees Molecular structures of the eight drugs studied.
LC/MS/MS analytical method
acetate ammonium buffer (70:30, v:v) +0.5% acid acetic for solvent A and adjusted for each compound with a flowrate of 1 mL/min and run time was Applied Biosystems, Toronto, Canada).
Mass spectrometry conditions for aglycone detection. Ion Spray voltage (V); DeclusteringPotential (V) or Orifice Voltage; 3Focusing Potential (V) or Focusing Ring Potential; 4Entrance remaining after the four hour incubationperiod. The 1-O-b -acyl glucuronidepeak was identified by monitoring itsdisappearance after two hours ofi n c u b a t i o n glucuronidase at 37° C. Finally, thepercentage of the different acylglucuronide isomers was assessed usingpeak area ratios.
Discussion
Proposed major fragmentation pathway for acyl glucuronides (e.g., Zomepirac) using TIS/MS/MS inpositive ionization mode. Major fragments are the protonated aglycone ion (m/z = 292) and a fragment of the aglycone (m/z = 139).
T1 .
for the acyl glucuronide for the different ionization modes are outlined in F2
(positive ionization mode) and F3
17-22.prnC:\work\current separation\cs20-2\current separations 20-2.cdrWednesday, August 28, 2002 9:34:46 AMPlate: 3 of 12 Color profile: Generic offset separations profileBlack 150 lpi at 45 degrees Conclusion
37 °C allowed identification of the 1-O- b acyl glucuronide (F4 and
F5
d e t e r m i n e t h e h y d r o l y s i s a n d T3
separation of acyl glucuronide isomers.
T4
T2 . Some samples
coefficient of 0.9976 or better for all the References
1. H. Spahn-Langguth and L.Z. Benet, 2. H. Spahn-Langguth, M. Dahms and A. Proposed major fragmentation pathway for acyl glucuronides using TIS/MS/MS in negative ionization mode. Major fragments include the deprotonated aglycone ion and the aglycone fragment 4. M.L. Hyneck, P.C. Smith, A. Mufano, generated by the loss of CO2. The charge can also be located on the glucuronic acid moiety (m/z = F.A. McDonagh and L.Z. Benet, Clin. Pharmacol. Ther. 44 (1998) 107-114. [Aglycone-H ]
5. J. Hasegawa, P.C. Smith and L.Z. Benet, Drug. Metab. Disp., 10 (1982) 469-473. 6. A. Kretz-Rommel and A. Boelsterli, Drug Metab. Disp. 22 (1994) 956-961. 7. C. Volland, H. Sun, J. Dammeyer and L.Z. Benet, Drug Metab. Disp. 19 (1991) 8. N. Dubois, F. Lapicque, M.H. Maurice, [Aglycone-CO - ]
M. Pritchard, S. Fournel-Gigleux, J. Magdalou, M. Abiteboul and G. Siest,Drug Metab. Disp. 21 (1993) 617-623 9. M. Castillo and P.C. Smith, Drug Metab. LC gradient elution profile for tested drug.
10. P.C. Smith and J.H. Liu Xenobiotica 23 Eluent A: ACN-acetate ammonium buffer 10 mM (70:30, v:v) +0,5% acetic acid Eluent B: ACN-acetate ammonium buffer 10 mM (4:96, v:v) 11. T. Mizuna, L.Z. Benet, and E.T. Lin, Biopharm. Drug Disp. 20 (1999) 131-136. 17-22.prnC:\work\current separation\cs20-2\current separations 20-2.cdrWednesday, August 28, 2002 9:34:50 AMPlate: 5 of 12 Color profile: Generic offset separations profileBlack 150 lpi at 45 degrees LC/MS/MS chromatogram for the achiral compound Zomepirac (top) and identification of the 1-O-b acyl glucuronide peak following hydrolysis byb -glucuronidase (bottom).
LC/MS/MS chromatogram for the chiral compound Ketoprofen (top) and identification of the 1-O-bacyl glucuronide peak following hydrolysis by b -glucuronidase (bottom).
17-22.prnC:\work\current separation\cs20-2\current separations 20-2.cdrWednesday, August 28, 2002 9:34:51 AMPlate: 7 of 12 T3.
Determination of unmetabolized aglycone and percentage of biosynthesized acyl glucuronides.
T4.
Relative proportions of acyl glucuronides after four hours (other isomers are acyl migrated isomers
of 1-O-b acyl glucuronide but their exact chemical structure have not been determined).

Source: http://www.currentseparations.com/issues/20-2/20-2e.pdf