Sensitive, selective detection and differentiation of salicylates and metabolites in urine by a simple HPTLC method

We present a method for salicylates which is slightly more labor intensive than the usual manual Trinder's test, but is much more sensitive and able to identify individual drugs or metabolites. A 2-mL acidified urine aliquot is briefly extracted with 5 mL ether, and the residue from evaporating the ether under nitrogen is chromatographed on a 250-microns silica gel HPTLC plate using benzene-acetic acid-diethylether-methanol (60:9:30:5) as mobile phase and 5% aqueous ferric chloride as chromogen. The hardiness of the method is evidenced by the Rf values, which vary by no more than 3% over a four-month period. The Rf values are 0.70 for salicylic acid and diflunisal, 0.67 for aspirin and methyl salicylate, 0.60 for gentisic acid, 0.57 for p-aminosalicyclic acid, and 0.40 for salicyluric acid. Detection limits of 1 ppm or less for all the analytes compared favorably to limits of more than 20 ppm for Trinder's test. Separations and spot shapes are sufficiently good to make instrumental quantitation potentially applicable. Sensitivity is sufficient to give clearcut, positive test results 48 h after a single 80-mg dose of ASA by mouth or a 100-mg dose of methyl salicylate by skin injection with a muscle rub, and more than 96 h after a 660-mg oral aspirin dose. Thus, the test is useful for detection and a good degree of differentiation, even in patients using subtherapeutic doses of these salicylates or in those with trace residues from significantly remote full therapeutic or larger doses prior to specimen collections.