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.