Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography-tandem mass spectrometry.
A column-switching liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 microl) were injected onto a C18-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0-acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C18 column, with a mixture of 20 mM ammonium acetate-acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M-H]- were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M-H] in MS-MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M-H-Glu]-, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025-100 microg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78-103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.
Keski-Hynnilä
H
H
Department of Pharmacy, University of Helsinki, Finland.
Raanaa
K
K
Forsberg
M
M
Männistö
P
P
Taskinen
J
J
Kostiainen
R
R
D000818:Animals; D002396:Catechols / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000097:blood*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D009570:Nitriles; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002396:Catechols; D004791:Enzyme Inhibitors; D009570:Nitriles; C071192:entacapone