Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high‐pressure liquid chromatography with post‐column hydrolysis and fluorescence detection

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 (λ_ex) and 400 nm (λ_em). 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. Copyright © 2012 John Wiley & Sons, Ltd.     

Enzyme-assisted extraction of arsenic species from plant material

Investigations regarding the transfer and metabolism of arsenic species in plants require mild extraction conditions to conserve the original composition of arsenic species. Beside the use of water or water/methanol for extraction of arsenic species from plant samples, enzymes can assist this procedure by digestion of cellulose and other constituents of cell walls, resulting in a faster, more efficient extraction technique which preserves the arsenic species. The investigations presented here were focused on the stability of certain arsenic species in enzymatic solutions, optimal conditions for their chromatographic separation and detection namely by means of ion chromatography–inductively coupled plasma mass spectrometry and improvements with respect to extraction efficiency. With commercially available enzymes and enzyme mixtures, the digestion rate of soluble starch as model cellulose was determined using high-performance anion exchange chromatography–pulsed amperometric detection analysis of glucose as the major digestion product. The most effective digestion rate (80% within 4?h) was obtained with Viscozyme®. This enzyme mixture was applied to extracted arsenic species from algae and terrestrial plant materials. Qualitative and quantitative differences in the results between enzyme-assisted and water extractions were obtained and discussed. The results show that the application of enzymes in mild extraction protocols should be evaluated as an additional step for the identification of As-metabolics in organisms. Careful selection of suitable enzyme mixtures can overcome the disadvantage that extraction efficiency is very organism-specific.