Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.
Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.
Shi
G
G
Bristol-Myers Squibb Company, Drug Metabolism and Pharmacokinetics, Stine-Haskell Research Center, 1090 Elkton Road, Newark, DE 19711, USA. gshi@palatin.com
Lloyd
T L
TL
Sy
S K B
SK
Jiao
Q
Q
Wernicki
A
A
Mutlib
A
A
Emm
T A
TA
Unger
S E
SE
Pieniaszek
H J
HJ
D000578:Amidines / Q000097:blood* / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007555:Isoxazoles / Q000097:blood* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008698:Mesylates / Q000097:blood*; D010456:Peptides, Cyclic / Q000097:blood*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results
D000578:Amidines; D007555:Isoxazoles; D008698:Mesylates; D010456:Peptides, Cyclic; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C085286:DMP 728; C118235:roxifiban