A method is developed for the simultaneous extraction of gentisic (GA), salicyluric (SUA) and salicylic acid (SA) in human plasma from Willow Bark extract, by solid phase extraction (SPE) using Waters Oasis HLB (divinylbenzene-n-vinylpyrrolidone copolymer) cartridges. Also, a method is optimized comprising of reversed-phase (RP) high-performance liquid chromatography (HPLC) in connection with electrospray ionization mass spectrometry (ESI-MS), fluorescence detection (FLD) and photo diode array detection (DAD) to identify and quantify GA, SUA and SA in the SPE effluents. An improved sensitivity regarding the lower detection limit (LOD) of < 7 ng/ml, the limit of quantitation (LOQ) of 20 ng/ml and short analysis times of < 15 min is required. The validated SPE method shows linearity in the range of 9.0-58.2 ng/ml for GA, 9.4-191.5 ng/ml for SUA and 12.8-1101.6 ng/ml for SA. The correlation coefficient values are > 0.9994 and 0.99 for fluorescence detection (FLD) and electrospray ionization mass spectrometry (ESI-MS), respectively. The recoveries are from 91.3-102.1% for gentisic acid (GA), 86.8-100.5% for salicyluric acid (SUA) and 75.8-81.4% for salicylic acid (SA) depending on the starting concentrations. RP-LC-ESI-MS/MS studies using collision induced dissociation (CID) confirm that the investigated analytes are not artifacts and facilitate further specific identification in addition to the determination of the parent ion mass even in the presence of co-eluting peaks. The established method is also used to analyze gentisic (GA), salicyluric (SUA) and salicylic acid (SA), not only after intake of Willow Bark capsules (Assalix, BNO 1455) but also as naturally occurring constituents in human plasma after the intake of salicylic acid containing foods.
Copyright 2004 Elsevier B.V.