A novel method for the determination of isoxazoline derivatives in plasma by ultra-high performance liquid chromatography–tandem mass spectrometry: validation and applicability to screening tests

The diseases, some of which are parasitic, transmitted by arthropods such as ticks or fleas pose a serious health threat to pets and farm animals. Antiparasitic drugs are one of the largest groups of veterinary medications (9). Antiparasitic compounds include the isoxazoline derivatives that have been developed in the last 25 years. These include fluralaner, sarolaner, lotilaner, afoxolaner and esafoxolaner. A single oral dose of isoxazoline derivatives provides effective protection against arthropods for up to 12 weeks. These drugs have a rapid onset of action, which reduces the risk of disease transmission by ectoparasites (18, 19). In recent years, single and combined isoxazoline products have been approved by the American Food and Drug Administration and the European Medicines Agency, and these drugs are widely used to treat ectoparasite infections. Fluralaner, sarolaner and lotilaner have been approved for use in dogs and cats, afoxolaner has been approved for dogs (19), esafoxolaner for cats (15), fluralaner for hens (7, 11, 14) and lotilaner for humans (13). Despite the growing popularity of isoxazoline derivatives, very few studies have investigated their presence in biological matrices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (1, 2, 5, 10, 16, 17). Most of the described analytical methods support the determination of only one isoxazoline derivative in a given biological matrix (2, 5, 10, 16). These methods are also characterised by low repeatability due to incomplete information about equipment settings and extraction techniques. Therefore, the first aim of this study was to develop a sensitive, simple, inexpensive and rapid method for analysing isoxazoline compounds. The second aim was to revalidate the proposed method for use in human plasma and the plasma of three animal species, as well as in the first screening test of plasma obtained from a small animal clinic. Ultra-high performance (UP) LC-MS/MS was the technique selected to achieve the aims.

The mass spectrometer was operated in electrospray positive ionization (ESI+) mode. The spectra were traced in multiple-reaction-monitoring mode at 556.06 m/z to 400.0 and 457.0 for fluralaner, 581.03 m/z to 457.0 and 415.9 for sarolaner, 595.98 m/z to 439.75 and 166.03 for lotilaner and 625.73 to 469.89 and 195.97 for afoxolaner (Table 1).

The method developed in this study was fully validated in laying hen plasma. Additionally a validation outcome emerged from the results for total recovery and the matrix effect from samples of canine, feline and human plasma; these results are shown in Table 3. The results of the validation protocol met all acceptance criteria (6) (Table 2). The values of r and r² in the linear regression analysis of the calibration curve were above 0.99 (Tables 4–7), and the entire calibration range was 0.001–1.5 μg/mL. Accuracy and precision were estimated for all QCs and the LLOQ, both for individual samples and for the six samples prepared over a period of three days. Intra-day precision was 1.79% to 14.97%, and accuracy was 1.33% to 11.67% for all QC and LLOQ (Tables 4–7).

Inter-day precision was 1.75% to 11.70%, and accuracy was 1.67% to 7.78% for all QC and LLOQ (Tables 4–7). The LOD was set at 0.5 ng/mL ± 0.38 (with a minimum signal-to-noise ratio of 10 : 1) and the LLOQ was 1.0 ng/mL ± 0.04–0.14 (Tables 4–7). The analysis of the selectivity/specificity of the method revealed no significant peaks during the retention of isoxazoline derivatives in drug-free plasma separated from the blood of clinically healthy animals. No significant carry-over of fluralaner, sarolaner, lotilaner or afoxolaner was observed in analyses involving high concentrations of these analytes. All analytes appeared to be stable after 3 h of storage at sample processing temperature; after 24 h of storage in an autosampler at 4°C; after 24, 48, 72 and 480 h of thawing and freezing cycles; and after 168 h of refrigerated storage at 2°C (Tables 4–7). The mean matrix effect for all species was 11.99%.

The proposed method was successfully applied to analyse the plasma of randomly selected patients of a small animal clinic for the presence of isoxazoline derivatives. The concentrations of isoxazoline compounds were within the analytical range of the method in 15 out of 20 dogs (1.0–434.6 ng/mL) and in 2 out of 10 cats (23.8–233.3 ng/mL) (Table 8).

The present study was undertaken to develop the first ever method for determining four isoxazoline compounds in plasma samples. The proposed method was developed in several steps. The optimal detector parameters were selected in the first step; the optimal chromatographic conditions were selected in the second step; and a quick, effective, easy and inexpensive analyte extraction method was developed in the third step. In the fourth step, the proposed method was validated in laying hen plasma and revalidated in canine, feline and human plasma. The validated method was successfully used in a screening study to determine the concentrations of isoxazoline derivatives in dogs and cats that were randomly selected in a private veterinary clinic.

The molecular weight of isoxazoline derivatives was 556.29 g/mol for fluralaner, 581.36 g/mol for sarolaner, 596.76 g/mol for lotilaner and 625.87 g/mol for afoxolaner. These values were used to determine the parent ions of the analysed molecules on the assumption that each substance was ionised only once (m/z = 1/1). The optimal chromatographic conditions were selected in the following stage of the study. First, the column was selected based on the properties of isoxazoline derivatives which are lipophilic compounds (XLogP3 of 5.6 for fluralaner, 3.4 for sarolaner, 6.6 for lotilaner and 6.7 for afoxolaner). Shoop et al. (15) used a Zorbax SB C18 column (2.1 mm × 50 mm, 5 μm) to determine the plasma concentrations of afoxolaner, whereas Toutain et al. (16) determined the plasma levels of lotilaner using a Daicel Chiralpak IA-3 column (150 × 4.6 mm) with a much larger particle size than that applied in the current study. A Zorbax Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm) was used only by Wu et al. (17) to measure the concentrations of all isoxazoline compounds, but in their study, tissues rather than plasma were used as the matrix. In contrast, the column used in the present study had a smaller particle size to achieve higher analytical sensitivity. The Acquity HSS T3 column was also chosen because it is compatible with 100% aqueous solvents. A similar mobile phase to that used in the present experiment was applied by Sari et al. (8), but they used methanol instead of acetonitrile. Methanol was not used in this study because it leads to a maximal reduction of baseline resolution. Wu et al. (17) used a mobile phase composed of 5 mmol/L of aqueous ammonium formate solution and MeOH. In the present study, however, a buffer was not used to avoid crystallisation, which may occur under mass spectrometry conditions due to solvent evaporation and elevated temperatures in the ion source.

Sample preparation is a critical step in analyses of drug residues. An adequate sample preparation protocol removes matrix interference and improves the sensitivity and robustness of the method. The main goal of this study was to develop a quick, easy, short, sensitive, and low-cost method of plasma purification. Protein precipitation is a short procedure that was well suited to the needs of this study. To date, a short and easy method for extracting lotilaner from plasma has only been proposed by Toutain et al. (16). In turn, Kilp et al. (2) and Sari et al. (8) used pure ACN for protein precipitation and determined the plasma levels of fluralaner by solid-phase extraction. Wu et al. (17) also used ACN for extraction, followed by a cleanup step involving a PRiME (protein removal in mass spectrometry experiments) HLB (hydrophilic–lipophilic balance) cartridge, and they analysed four isoxazoline compounds (similarly to the present study), but in tissues, not in plasma, which explains the additional cleanup step. For this reason, the method developed by the cited authors is longer, consists of more steps and is more expensive to validate when adopted for tissue analysis because of the high cost of tissue samples.

The method was successfully validated according to Kruve et al. (3, 4). The calibration curves showed linearity in a sixfold wider range than other methods (1, 8, 17). The validation confirmed sensitivity several times higher than that described by other authors (1, 17). The validated method demonstrated acceptable inter-day precision and accuracy across all QC levels and LLOQ. In contrast, Elliot et al. (1) reported lower precision, with inter-day values exceeding 19.5% and falling out of the ranges of all parameters except LLOQ in our protocol. The developed method demonstrated high analyte stability under all tested conditions, whereas Elliot et al. (1) and Wu et al. (17) did not include stability testing, for example during freeze–thaw cycles, which may limit the long-term reliability of their quantification results. In contrast to Wu et al. (17), Sari et al. (8) and Elliot et al. (1), we evaluated the selectivity of our method. This avoided any potential interferences and carry-over effects during analysis, especially because our calibration curve included very high and very low concentration points. The mean matrix effect for all species was not quite 12%, confirming that the developed method was influenced by matrix components to an acceptable extent by the criteria established.

The developed analytical method met all validation criteria, demonstrating superior sensitivity, precision and stability compared with data available in the literature. Minimal matrix effect, low LOD/LLOQ and comprehensive stability tests indicated greater reliability of this method for accurately measuring isoxazoline derivatives. The proposed method was successfully used to analyse the plasma of randomly selected cats and dogs from a small animal clinic for the presence of isoxazoline derivatives. This screening confirmed that the method worked well under real clinical conditions, and could be taken up for the detection and measurement of isoxazoline residues in plasma collected during routine veterinary visits. Such a screening test would reveal whether antiparasitic drugs had been used and give a veterinarian reliable information when there was no record of treatment. Therefore, the presented method can be useful not only in pharmacokinetic studies, but also for control purposes, especially in cases of suspected drug side effects or misuse, or of their undocumented administration. Given the growing use of isoxazoline-based treatments, developing a fast, cost-effective and reproducible screening method is essential for accurate monitoring and regulatory compliance.

Although the developed method met the criteria selected by the authors, the main limitation of the study was that the MS/MS protocol was designed to examine each isoxazoline compound separately. Another is that no isotopic internal standard was used in the study although such a standard would have been a better choice in tandem mass spectrometry to obtain more consistent results. In addition, the sample preparation method used in the study is a “dirty” technique that can shorten the life of the column. Despite these limitations, the proposed method is a reliable solution for determining isoxazoline derivatives in plasma. Further research is needed to address the identified shortcomings and achieve more satisfactory results.

This is the first study to propose and validate a UPLC-MS/MS method for quantifying all isoxazoline derivatives in the blood plasma of laying hens, dogs, cats and humans. The results indicate that the developed method is replicable, precise, accurate, selective and sensitive. The main advantages of the method are its simplicity and effectiveness, as well as quick sample preparation, which decreases costs and facilitates rapid and precise determination of the analytes in plasma. Despite the existence of cleaner techniques of plasma purification than protein precipitation, the proposed method had a high recovery rate, and the matrix effect was small enough for the method to be validated with sufficient accuracy and precision. The method seems to be suitable for practical use, and was successfully applied to test for the isoxazoline derivatives fluralaner,sarolaner, lotilaner and afoxolaner in animals that had been orally administered isoxazolines.