title: Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high-pressure liquid chromatography with post-column hydrolysis and fluorescence detection.
abstract: A selective, sensitive and rapid high-performance liquid chromatography method with post-column hydrolysis and fluorescence detection was developed for the simultaneous quantification of acetylsalicylic acid and its metabolite salicylic acid in human plasma. Following the addition of 2-hydroxy-3-methoxybenzoic acid as internal standard and simple protein precipitation with acetonitrile, the analytes were separated on a ProntoSIL 120 C18 ace-EPS column (150 × 2 mm, 3 µm) protected by a C8 guard column (5 µm). The mobile phase, 10 mm formic acid in water (pH 2.9) and acetonitrile (70:30, v/v), was used at a flow rate of 0.35 mL/min. After on-line post-column hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) by addition of alkaline solution, the analytes were measured at 290 nm (λex ) and 400 nm (λem ). The method was linear in the concentration ranges between 0.05 and 20 ng/μL for both ASA and SA with a lower limit of quantification of 25 pg/μL for SA and 50 pg/μL for ASA. The limit of detection was 15 pg/μL for SA and 32.5 pg/μL for ASA. The analysis of ASA and SA can be carried out within 8 min; therefore this method is suitable for measuring plasma concentrations of salicylates in clinical routine.
authors.affiliation: Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Eva-Luise
authors.initials: EL
authors.lastname: Hobl
authors.forename: Bernd
authors.initials: B
authors.lastname: Jilma
authors.forename: Josef
authors.initials: J
authors.lastname: Ebner
authors.forename: Rainer W
authors.initials: RW
authors.lastname: Schmid
mesh_terms: D001241:Aspirin / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D015203:Reproducibility of Results; D020156:Salicylic Acid / Q000097:blood*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D020156:Salicylic Acid; D001241:Aspirin