engleski

Development of analytical method based on ultra performance liquid chromatography (UPLC) tandem QTRAP mass spectrometry for determination of antihelmintic drugs in surface waters

Introduction Pharmaceuticals are natural or synthetic chemicals designed to treat disease and fight infections both in humans and in animals. In case of veterinary pharmaceuticals they are not only used to treat disease in animals but they are also added to the feed to improve growth rate and feed efficiency. For veterinary pharmaceuticals well-established pathways to enter the aquatic environment are: (i) drugs used in fish farms reach surface waters after direct application in aquaculture or through local water treatment plant at fish farm ; (ii) drugs used for therapeutical treatment or as growth promoters reach surface (ground) waters directly via urine and/or feces of animals kept outdoors or via manure applied as fertilizer dispersed on the fields [1, 2, 3]. Depending on their physicochemical properties they may end up in ground water or, after heavy rain, may enter surface waters as run-off from the soil. Therefore aquatic organisms are directly exposed to influence of pharmaceuticals and the problem lies in the fact that little is known about ecotoxicological effects of pharmaceuticals on wildlife. [4] Not only the animal life is affected by the negative influence of pharmaceuticals in the environment but they also pose risk for human health. The presence of pharmaceuticals has been reported in treated sewage water, rivers and streams, seawater, ground water and drinking water. Anthelmintics are a class of antiparasitic pharmaceuticals and their task is to control endo- and ecto-parasitic organisms of wide range veterinary important animals in agriculture and aquaculture. They are categorized into eight groups by their molecular structure: benzimidazoles, diphenylsulfides, imidazothiazoles, hexahydropyrazines, macrocyclic lactones, salicylanilides, tetrahydropyrimidines and others. The use of antihelimintic is extensive in all branches of animal farming and therefore the awareness of their appearance in animal tissue has been noted. As the result the maximum residue limits of anthelmintic in animal tissue such as skin, fat, liver, kidney, muscle, eggs and also in milk have been regulated [5]. On the other hand the information about the presence of anthelmintics in the environment is very scarce. Their presence in the environment can have unwanted effect on the parasites in the way that the strains of parasites appear that are more resistant. Method development The aim of this work was to develop a sensitive and selective method for determining anthelmintic pharmaceuticals in surface waters. Samples were prepared using solid phase extraction (SPE) as a technique which is simple to perform, the costs of apparatus and the solvents used are quite low and the technique is efficient for clean-up of water samples. The chromatographic separation of analytes was achieved on ultrapreformance liquid chromatography (UPLC). With this technique it is possible to achieve good chromatographic separation in short period of time. As the detector the QTRAP mass spectrometer was used in order to obtain good selectivity and sensitivity. Analyzed anthelmintics were chosen from different groups: macrocyclic lactone group (moxidectin), benzimidazole gropup (flubendazole, fenbendazole, mebendazole, oxibendazole, albendazole, thiabendazole, triclabendazole), diphenylsulfid group (febantel), hexahydropyrazine group (praziquantel) and imidazothiazole group (levamisole). Stock solutions of each compound were prepared in methanol in concentration of 100 mg/L. The first step was to optimize the conditions on the mass spectrometer QTRAP. Standard solution of each compound was introduced into the instrument with continuous flow of 0.4 mL/min while the parameters for the source were optimized. The parameters optimized were ion spray, nebulization gas flow, turbo gas flow and temperature. The next step was to optimize collision energy for each compound. After the fragmentation process two transitions with the highest abundance were chosen in order to use them for identification purpose. After optimization the parameters on the mass spectrometer the chromatographic parameters were investigated. As the stationary phase in chromatographic system C18 column was used to separate analytes. This column was chosen due to its characteristics which allow analyzing simultaneously a big number compounds that have various physicochemical properties For the mobile phase different combinations of solvents were used. For the water phase 0.1 % formic acid or 5 and 10 mmol/L of ammonia acetate buffer were used. For the organic phase 0.1 % formic acid in methanol and acetonitrile were used. All above mentioned mobile phases were used in gradient elution which was also adjusted to give the optimum peak height and shape. The final step of optimization was optimization of sample preparation procedure. The solid phases were Oasis HLB and MCX. Oasis HLB covers wider range of compounds because it is used for acids, bases and neutrals whereas MCX is mixed mode cation exchange sorbent used for bases. Experiments were conducted under different pH of water to see if the pH affects the efficiency of sample preparation procedure. The last step of sample preparation is elution of the components of interest. Methanol and acetonitril were used as eluents. Combinations of parameters mentioned above were investigated in order to find the suitable combination that gave the optimal recovery for all the anthelmintics that were chosen for this analysis. Recovery was calculated for each analyte in order to show that the extraction process gave satisfying results. In each step of the analysis the parameters were chosen to give the best peak abundance and peak shape for every anthelmintic investigated. Some compromise had to be made because anthelmintic analyzed were from different groups and therefore they have different physicochemical properties. The sample of total ion chromatogram (TIC) of analyzed anthelmintics is shown on Figure 1. SPE – UPLC – QTRAP analytical method was validated before it was used for analyzing selected analyites in real water samples. The parameters of validation were linearity, limit of detection, limit of quantitation, repeatability and reproducibility. Calibration was done for all the analytes and the calibration curves fitted the experimental data well (R2 > 0.99). Limits of detection of analytes were in the range from 0.05 to 1.7 ng/L except for the moxidectin that has limit of detection of 0.6 μg/L. Recoveries calculated after the sample preparation procedure are for most of the analytes more than 70%. The validated method was then used to analyze real samples that were collected from Llobregat river which is a recipient of waste water of some animal farms.

anthelmintic drugs; UPLC; QTrap mass spectrometry

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