title: Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma.
abstract: A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5 mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2 mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50 ng/mL for both analytes (0.2 ng injected) and matrix-matched standard curves showed linearity up to 4000 ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively < or = 6.3% and < or = 11% for 5-ASA and < or = 8.0% and < or = 10% for N-Ac-5-ASA. For both analytes the within- and between-batch accuracy (bias%) values ranged respectively from -8.4% to 7.9% and from -7.9% to 8.0%. The overall recoveries (n=6) at three tested concentration levels (i.e. 100, 1000 and 4000 ng/mL) were respectively >90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.% < or = 10%). The method was applied to evaluate the pharmacokinetic of 5-ASA after a single oral dose administration of this compound (1200 mg) to 24 healthy volunteers. The mean maximum concentration levels were 680 ng/mL for 5-ASA and 1240 ng/mL for N-Ac-5-ASA and the kinetic profiles were in agreement with previous studies.
authors.affiliation: Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pastorini
authors.forename: Marcello
authors.initials: M
authors.lastname: Locatelli
authors.forename: Patrizia
authors.initials: P
authors.lastname: Simoni
authors.forename: Giulia
authors.initials: G
authors.lastname: Roda
authors.forename: Enrico
authors.initials: E
authors.lastname: Roda
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
mesh_terms: D000636:Aminosalicylic Acids / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D019804:Mesalamine / Q000097:blood* / Q000493:pharmacokinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000636:Aminosalicylic Acids; D019804:Mesalamine; C028836:N-acetyl-5-aminosalicylic acid