Quantitative determination of salicylic acid and metabolites in animal tissues by liquid chromatography–tandem mass spectrometry Original Research Article

In order to study the excretion pattern of salicylic acid and its metabolites in animals, a liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed. The method was used to study the biotransformation of salicylic acid in poultry (chicken and pigeon) after therapeutic use, i.e. in matrices such as plasma, excreta (=combined urine and faeces), kidney and liver. Thereafter, the method was also adopted for the analysis of residues of salicylic acid in porcine tissues, such as muscle, kidney, liver and skin + fat. For the biotransformation study, salicylic acid, the oxidation and amino acid conjugation metabolites such as gentisic acid, salicyluric acid, and a double conjugated ornithine metabolite, together with the internal standard phenoxyacetic acid were extracted from 1.0 g of tissue or 100 μl of plasma into ethyl acetate, after acidifying using 1 M HCl. After centrifugation and for kidney and liver tissue, the analytes were back-extracted and subjected to a solid-phase clean-up on SAX columns. For the analysis of excreta, the sample preparation included only a dilution step, performed before LC–MS/MS analysis. For the residue study, salicylic acid and the internal standard phenoxyacetic acid were extracted from 1.0 g of porcine muscle, liver, kidney and skin + fat with 6 ml of diethyl ether, after acidifying using 3 ml of 1 M HCl. After extraction and centrifugation, the ether phase was evaporated under N2 at 40 °C. The residue was redissolved with 500 μl of 0.1% acetic acid in water. For skin + fat and when needed for the other tissues, a supplemental extraction of the redissolved residue with 500 μl of hexane was performed. The analysis of the extracts was done on a Nucleosil 100-5 C18 column, using a gradient elution with 0.1% acetic acid in water and methanol. A Quattro Ultima® triple quadrupole instrument was used, equipped with an ESI z-spray source, which was operated in the negative ion MS/MS mode. The methods were validated for the linearity (100–1000 ng ml−1 and 10–50 μg ml−1 for plasma; 5–250 μg g−1 for excreta; 100–1000 ng g−1 and 5–25 μg g−1 for the biotransformation study in poultry kidney and liver; 25–1000 ng g−1 for the residue study in porcine tissues); trueness and precision; specificity; limit of detection and limit of quantification. The limit of quantification for the residue analysis of salicylic acid in porcine tissues was set at 50 ng g−1.