title: Simultaneous quantification of PGI2 and TXA2 metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method.
abstract: The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland; Chair and Department of Toxicology, Faculty of Pharmacy, Jagiellonian University, Medical College, Medyczna 9, 30-688 Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kij
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Mateuszuk
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Sitek
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
authors.forename: Kamil
authors.initials: K
authors.lastname: Przyborowski
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zakrzewska
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Wandzel
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland; Chair and Department of Toxicology, Faculty of Pharmacy, Jagiellonian University, Medical College, Medyczna 9, 30-688 Krakow, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Walczak
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland; Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, 31-531 Krakow, Poland. Electronic address: stefan.chlopicki@jcet.eu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Chlopicki
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D011464:Epoprostenol / Q000097:blood* / Q000652:urine*; D005260:Female; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000172:deficiency*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013928:Thromboxane A2 / Q000097:blood* / Q000652:urine*
chemical_list: D009569:Nitric Oxide; D013928:Thromboxane A2; D011464:Epoprostenol
keywords: Eicosanoids; NO-deficient mice; Prostacyclin; Thromboxane A(2); UHPLC/MS/MS method