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 |
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Authors: | Pastorini Elisabetta Locatelli Marcello Simoni Patrizia Roda Giulia Roda Enrico Roda Aldo |
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Institution: | Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy. |
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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 17.5mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2mL/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 50ng/mL for both analytes (0.2ng injected) and matrix-matched standard curves showed linearity up to 4000ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively =6.3% and =11% for 5-ASA and =8.0% and =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 4000ng/mL) were respectively >90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.%=10%). The method was applied to evaluate the pharmacokinetic of 5-ASA after a single oral dose administration of this compound (1200mg) to 24 healthy volunteers. The mean maximum concentration levels were 680ng/mL for 5-ASA and 1240ng/mL for N-Ac-5-ASA and the kinetic profiles were in agreement with previous studies. |
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