Combining quantitative and qualitative approaches using Sequential Window Acquisition of All Theoretical Fragment-Ion methodology for the detection of pharmaceuticals and related compounds in river fish extracted using a sample miniaturized method

Abstract

A fast method for analysis of 47 pharmaceuticals active compounds (PhACs) in fish muscle has been developed and validated addressing the parameters accuracy, precision (intraday and interday), matrix effect at three spiking levels: 5, 25 and 50 ng PhAC g−1 in fish as well as linearity, limit of detection (LOD), limit of quantification (LOQ). Sixteen protocols were performed varying extraction techniques, solvents, sample filtration and clean-up step. The selected method was based on an ultrasound extraction with acidified mixture of acetonitrile and isopropyl alcohol followed by a clean-up step using Z-Sep/C18 sorbents. Quantitative analysis of the PhACs in the extracts was accomplished by UPLC- QTOF-MS using Sequential Window Acquisition of All Theoretical Fragment-Ion, SWATHTM acquisition technology. 90% Of the compounds presented extraction recoveries between 60 and 130% with LOQ between 0.2 and 11 ng g−1. The validated method was applied to the analysis of 32 muscle samples from thirteen different species of fish collected in four European river basins (Adige, Evrotas, Llobregat and Sava). A total of ten compounds were found in fish samples with diltiazem as the most frequently detected one followed by carbamazepine and caffeine. Additionally, by taking advantage of the information-rich mass spectral data from the SWATH mode acquisition, the raw data were reprocessed for the presence of the most prescribed 250 pharmaceuticals, metabolites, and drugs of abuse previously reported to occur in the aquatic environment. By considering the mass errors of the molecular ion (˂± 3 ppm) and one characteristic fragment ion (˂±10 ppm) as well as the Library score and the Formula Finder score of the data processing software six compounds were retrieved, and eventually four of them confirmed with authentic standards: cocaine and its metabolite benzoylecgonine, the stimulant nicotine, and the antibiotic ofloxacin. Two lipid regulators, lovastatin and simvastatin, were determined as a false positive.