An improved LC–MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids
Section snippets
Reagents
PGE2, PGD2, d4-PGD2, d4-PGE2, and antibodies against COX-1, COX-2, lipocalin prostaglandin D synthase (L-PGDS), hematopoietic prostaglandin synthase (H-PGDS), cytosolic prostaglandin E synthase (cPGES), microsomal prostaglandin E synthase (mPGES)-1 and -2 for Western blots were purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and citric acid and butylated hydroxytoluene (BHT) were obtained from
Method validation
The deprotonated molecules of PGE2 and PGD2 were detected at m/z 351 during negative ion electrospray mass spectrometry. Collision-induced dissociation of the [M-H]− ions produced abundant fragment ions of m/z 333, 315, 271, and 233 for both species, corresponding to [M-H-H2O]−, [M-H-2H2O]−, [M-H-2H2O-CO2]−, and [M-H-hexanal-H2O]−, respectively [28], [29]. The product ion tandem mass spectra of PGE2 and PGD2 are shown in Fig. 2. Because the most abundant product ion of both analytes was m/z
Acknowledgments
This research was supported by the Department of Veterans Affairs and grants HL-075557 and HL-66196 from the National Heart, Lung, and Blood Institute (to J.W.C.), American Heart Association National Scientist Development grant 0230279N and University of Illinois at Chicago Campus Research Board grant S06-118 (to L.X.), and National Institutes of Health grant P50 AT00155 jointly funded by the Office of Dietary Supplements, the National Center for Complementary and Alternative Medicine, and the
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