LC-MS/MS profiling of urinary metabolites of nitroaspirin (NCX 4016) in healthy volunteers following oral administration

The urinary metabolic profile of nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs), has been studied for the first time in a Phase I, open, single oral dose (1600 mg, sachet) metabolism and excretion study involving 8 healthy male Caucasian subjects, mean age 27 (range 22-39), mean BMI 23 (range 19-27). Urine samples were collected before treatment (pre-dose) and at the following time intervals: 0-6, 6-12, 12-24, 24-48 and 48-72 h. Characterization of metabolites was achieved by reverse-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection. All analyses were performed on urine samples extracted (2 vol) with CH3CN: 2M H3PO4 (99:1; v/v) using a ThermoFinnigan Surveyor system equipped with a quaternary pump, a Surveyor UV/VIS diode array programmable detector (6000 LP), a Surveyor autosampler, a vacuum degasser and connected to a Thermo Finnigan LCQ Advantage ion trap mass spectrometer. Separations for MS analysis were done in gradient elution from 95% A (H2O:CH3CN: CH3COOH; 95:5:0.1 v/v/v) to 62 % B (CH3CN) in 30 min (flow rate 0.2 ml min-1). For quantitation of metabolites (UV-DAD detection), CH3COOH in phase A was replaced by H3PO4 (0.1%, v/v). LC-MS/MS analysis indicates that no unchanged drug and free metabolites are excreted in urines at any observation time; 4 main conjugated metabolites have been characterized, arising from different metabolic steps involving a) cleavage of the ester bond with formation of salicylic acid (SA) and benzenemethanol,3-hydroxy--nitrate (NCX4015); b) alcohol oxidation; c) conjugation with glucuronic acid or aminoacids. Metabolites 1 and 2 have been identified as glycine conjugate derivatives of SA (salicyluric acid) and of 3-hydroxybenzoic acid, metabolite 3 as glucuronic acid derivative of NCX 4015, and metabolite 4 as a mercapturic acid derivative, arising from NCX4015 through the nucleophilic displacement by GSH of the leaving ONO2- group. Quantitative determination indicates that the urinary excretion of the metabolites in the overall 72 h-interval accounts for approximately 51.2% of the administered dose (range 30-75%), the bulk of excretion being within 12 h.