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Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 6924263, 7 pages https://doi.org/10.1155/2023/6924263 Research Article A New Method for Ivermectin Detection and Quantification through HPLC in Organic Matter (Feed, Soil, and Water) 1 2 2 Alicia Maria Carrillo Heredero , Giulia Segato , Simonetta Menotta , 2 1 1 1 Elena Faggionato , Alice Vismarra , Marco Genchi , and Simone Bertini Department of Veterinary Sciences, University of Parma, Strada del Taglio 10, Parma 43126, Italy Food and Feed Chemical Department, Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna, Via Antonio Bianchi, 7/9, Brescia 25124, BS, Italy Correspondence should be addressed to Alicia Maria Carrillo Heredero; firstname.lastname@example.org Received 13 December 2022; Revised 6 February 2023; Accepted 15 February 2023; Published 1 March 2023 Academic Editor: Serban C. Moldoveanu Copyright © 2023 Alicia Maria Carrillo Heredero et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ivermectin is a macrocyclic lactone widely used in veterinary medicine for its broad-spectrum antiparasitic properties. It has been proven to be efective and safe. Te purpose of this study was to develop a high-performance liquid chromatography method with a diode array detector for ivermectin screening in feed and water for animal consumption. Furthermore, the objective was to quantify ivermectin levels that were higher than 0.5 mg/kg in solid matrixes and 0.1 mg/kg in water. Doramectin was used as process standard. Samples were extracted using solid phase extraction with silica and C-18 columns. Te method involved the use of high-performance liquid chromatography (HPLC) with a diode array detector (DAD). Te results were interpreted using a calibration curve built with ivermectin standards at multiple concentrations (0.5, 1, 2, 5, and 12.5 mg/kg). Statistical evaluation of data was done using ANOVA. Te data analysis showed that the linear regression was highly signifcant (P < 0.001), the intercept values were not signifcantly diferent from zero, and the correlation coefcient values (>0.999) indicated excellent linearity. Further tests demonstrated that this method is also useful when studying soil matrixes. Te soil was dried and analyzed in the same way as feed; the same recoveries were realized on the spiked samples. Te method is easy, inexpensive, precise, and repeatable; it requires very small amounts of sample. Ivermectin is authorized for oral, topical, and parenteral 1. Introduction (subcutaneous) administration [1, 2, 5]. Te European Ivermectin is a macrocyclic lactone widely used in veterinary Medicine Authority has authorized ivermectin for multiple medicine for its broad-spectrum antiparasitic properties. species: cattle, pigs, sheep, goats, horses, reindeers, mice, Ivermectin is naturally produced by the yeast Streptomyces rats, dogs, cats, primates, and humans. Ivermectin is mar- avermitilis, and it consists of a mixture of two compounds: keted either alone or in combination with other antiparasitic 22,23-dihydroavermectin β1a (C H O , B ) and 22,23- compounds [6, 7]. In farm animals, doses can vary according 48 74 14 1a dihydroavermectin β1b (C H O , B ). Ivermectin B to the route of administration and range from 200 µg per kg 47 72 14 1b 1a makes at least 80% of the mixture, while no more than 20% is of body weight in cattle, sheep, and goats to 300 µg per kg of made of ivermectin B . It is active against both arthropods body weight in pigs [6, 8]. 1b and nematodes [1, 2], causing their paralysis and subsequent It is estimated that between 80 and 98% of the drug is death by blocking chloride channels . Like all avermec- excreted in feces, without being metabolized, thus reaching tins, ivermectin is highly liposoluble . the environment intact. 2 Journal of Analytical Methods in Chemistry Maximum residual limits (MRLs) have been established (1757). It was equipped with a chromatographic precolumn for all mammalian food-producing species, except animals (Supelguard for inverse phase, C-18, 2 cm long, internal producing milk for human consumption. Te marker res- diameter: 4.6 mm, and particle mean diameter: 5 µm, idue is 22,23-dihydroavermectin B1a. Te MRLs are re- Supelco, Bellefonte, PA, USA) and a chromatographic stricted in the targeted tissues in all species as follows: 30 µg/ column (Supelcosil for inverse phase, C-18, 25 cm long, kg in the kidney and 100 µg/kg in the fat and liver [8, 9]. Te internal diameter: 4.6 mm, and particle mean diameter: Italian National Reference Center for the surveillance and 5 µm, Supelco, Bellefonte, PA, USA). control of feed for animals (CReAA—Centro di Referenza Smaller equipment employed consisted of plastic Nazionale per la Sorveglianza e il Controllo degli Alimenti adapters for the reservoirs, 50 mL polypropylene tubes with per gli Animali) has established a detection limit of less than pressure caps (EuroClone S.p.A. Milan, Italy), 50 mL plastic 1 mg/kg in feed. reservoirs, a manifold system suited for solid phase ex- Although ivermectin has a wide safety margin in traction (SPE) (Supelco, Bellefonte, PA, USA), pipettes mammals [1, 2], it has been shown to be toxic in numerous (10–1000 µL) (Gilson Middleton, WI, USA), 500 mg/6 mL SPE C-18 ENVI columns (Supelco code: 57064, Supelco, invertebrate species. Mesa et al. reported possible ecological consequences in aquatic ecosystems . Johnson-Arbor Bellefonte, PA, USA), and 500 mg/6 mL SPE silica LC-Si has raised environmental concerns about ivermectin resi- columns (Supelco code: 505374, Supelco, Bellefonte, PA, dues . Terefore, an accurate system to monitor iver- USA). Disposable materials used were 0.45 μm flters mectin levels in organic substrates would therefore be of (Incofar, Modena, Italy), 6 mL reservoirs with 13 mm in- crucial importance. ternal diameter (Strata, Phenomenex, Torrance, CA, USA), Diferent methods for ivermectin detection have been frits (Phenomenex, Torrance, CA, USA), 10 mL plastic sy- reported by several authors. Asbakk et al. used high- ringes (Rays SpA, Osimo, AN, Italy), and 250 mL plastic performance liquid chromatography (HPLC) with fuores- centrifuge bottles with screw caps (Nalgene, Rochester, cence detection to detect ivermectin in feces from reindeers NY, USA). . Iglesias et al. used the same method for soil and cattle Glassware used consisted of 10 mL v-bottom graded test tubes, 10 mL u-bottom test tubes, 10 mL–20 mL amber test feces . Liquid chromatography-tandem mass spec- trometry (LC-MS-MS) has also been shown in several tubes with screw caps, class A or B glass pipettes, Pasteur glass studies to be useful for multiple matrixes such as plasma, pipettes, graded cylinders, class A fasks, and amber vials. milk, liver, and whole blood [14–20]. Liquid chromatography-electrospray ionization-mass spectrome- 2.2. Chemicals and Reagents. All reagents had known ana- try/mass spectrometry (LC-ESI-MS/MS) has also been used lytical purity. Te water used was deionized and ultra- to detect macrocyclic lactones in fsh muscle . fltrated water. Tis study aims to develop a HPLC method with a diode Methanol for analysis from Carlo Erba Reagents (DASIT array detector (DAD) for screening for ivermectin in feed Group S.p.A., Milan, Italy) was used for the extraction, while and water for animal consumption. Furthermore, we wanted Aluminium oxide 90 (neutral alumina) from Merck (Merck to quantify ivermectin levels higher than 0.5 mg/kg in solid KGaA, Darmstadt, Germany) was used for the preliminary matrixes and 0.1 mg/kg in water. We used doramectin as chromatography phase. Te following solvents were used for process standard . SPE column activation and elution as well as components of the HPLC mobile phase: methanol for HPLC from Carlo 2. Materials and Methods Erba Reagents (DASIT Group S.p.A., Milan, Italy), aceto- nitrile for HPLC from Carlo Erba Reagents (DASIT Group 2.1. Instruments. Instruments employed for the experiment S.p.A., Milan, Italy), and dichloromethane for analysis from consisted of ultrasound water bath (SONICA Sweep System Carlo Erba Reagents (DASIT Group S.p.A., Milan, Italy). Mod. 5300EP, SOLTEC, Milan, Italy), homogenizer type Ivermectin with declared purity from Dr Ehrenstorfer (LGC Grindomix (RETSH Grindomix Mod.GM200, Verder sci- Labor GmbH Augsburg, Germany) and doramectin with entifc, Haan, Germany), fask shaker (Janke and Kunkel declared purity from Sigma-Aldrich (Merck KGaA, Mod. HS501 digital, IKA Labortechnik, Staufen, Germany), Darmstadt, Germany) were used, respectively, for the for- centrifuge (Eppendorf Mod, 5804 R, Merck KGaA, Darm- tifed specimen and as an internal standard. stadt, Germany), vacuum pump (KNF Neuberger, Mod. For each new experiment, a mixture of solvents for ° ° Laboport N820 FT.18, Milan, Italy), water bath at 50 C± 5 C dilution made of acetonitrile (56%), methanol (37%), and with nitrogen current device (Buchi Mod. B-461 water bath, water (7%) (v/v) and the activated neutral alumina (100 g of GEMINI LAB sustainable equipment, Apeldoorn, the neutral alumina moisturized with 6 mL of deionized water) Netherlands), vortex (Ika MS2 Munishaker, IKA Labor- were prepared before use. technik, Staufen, Germany), and scales (Mettler Toledo Mod. All solvents and solutions used for the HPLC system XPE12025, Columbus, OH, USA; Sartorius Mod.ME235P, were fltered on a 0.2 μm flter. Sartorius, Go¨ttingen, Germany). Of the two scales used, one had 10 mg sensitivity and one had 0.01 mg sensitivity. Te system for high-performance liquid chromatogra- 2.3. Preparation of Standard and Quality Control Samples. All standards and controls were prepared just before use. A phy included a degasser, pump, autosampler, diode array detector, and software Agilent ChemStation Rev. A.10.02 stock solution of ivermectin and one of doramectin was Journal of Analytical Methods in Chemistry 3 prepared in a 10 mL fask, considering the declared purity, at For activation, the SPE silica columns were inserted into a concentration of 1000 µg/mL. Te solution was made with the manifold, one for each sample plus the blank and for- tifed controls. We percolated 5 mL of acetonitrile for HPLC methanol for HPLC and stored in the freezer in an amber tube for no more than 12 months . by gravity in columns and then dried them for 1 minute An intermediate solution was made for each compound using vacuum. 5 mL of dichloromethane for HPLC was at 10 µg/mL, starting from the stock solution. In a 10 mL leached and dried for 1 minute after applying vacuum. fask, the stock solution was added to the solvent mixture For conditioning, the SPE C-18 columns were inserted (acetonitrile (56%), methanol (37%), and water (7%)) at into a second manifold, one for each sample plus the blank a concentration of 1 :100. and fortifed controls. 5 mL of dichloromethane was per- From the intermediate solutions, ivermectin was diluted colated by applying vacuum and dried for 1 minute; then with the solvent mixture to obtain fve working solutions at 5 mL of a water/acetonitrile mixture (50 : 50 v/v) was per- 0.1, 0.2, 0.4, 1, and 2.5 µg/mL for calibration points. Another colated, and the column was kept wet. working solution (0.05 µg/mL) was made by adding 50 µL of A 10 mL syringe was applied to the SPE C-18 columns using the appropriate adapter. Simultaneously, 5 mL of the water/ the intermediate solution of ivermectin and doramectin to 9.95 mL of the solvent mixture. acetonitrile mixture (50 : 50 v/v) and 2 mL of each sample were For each analytical session, a blank control and a fortifed put in each syringe using a calibrated class A pipette. Te fuid sample at 0.1 mg/L were set up for water samples. In a 10 mL was allowed to percolate, keeping the column wet. Columns fask partially flled with deionized water, 100 µL of in- were then washed with 1 mL of the water/acetonitrile mixture termediate solution at 10 µg/mL was added, made up to (50 : 50 v/v) that was left to percolate by gravity and then dried volume, and diluted in a ratio of 1 : 2 with a dilution mix. with vacuum for 15 seconds. Te walls of the columns were For each analysis session, a blank control and a fortifed washed by adding 2 mL of the water/acetonitrile mixture (50 : 50 control were set up for solid samples (feed or soil). Negative v/v) and by letting it fow with the use of the vacuum pump. control (blank) consisted of 10 g of an ivermectin-free Finally, the column was dried for 1 min, maintaining the sample (feed or soil). Te fortifed sample (0.5 mg/kg) was vacuum. Te SPE C-18 columns were inserted into the conditioned made of 10 g of an ivermectin-free sample (feed or soil) to which 500 µL of the intermediate solution at 10 µg/mL was SPE silica columns with the appropriate adapter. 5 mL of the added and left for at least 10 minutes. dichloromethane/acetonitrile mixture (9 :1 v/v) was added to the All samples (controls and tests) were subjected to the SPE C-18 columns and left to percolate, maintaining the vacuum same extraction, purifcation, and analysis procedure. for 1 minute; another 5 mL of the mixture was subjected to the same procedure. Te SPE C-18 columns were discarded, and ivermectin was eluted from the silica SPE column with 5 mL of 2.4. Experimental Design. Te solid samples were extracted acetonitrile for HPLC by gravity, without letting it go dry. A with methanol and purifed using a solid phase extraction further 2 mL of acetonitrile for HPLC was added and eluted by (SPE) technique. Te purifed extracts were analyzed using applying vacuum. Te eluate was collected in a conical tube and HPLC after dilution. ° ° dried in a nitrogen stream at 50 C± 5 C. Te eluate was then diluted with 2 mL of the solvent mixture and vortexed for 2.4.1. Extraction and Purifcation of the Sample. Te water 1 minute, and the samples were fltered on 0.45 pm flters. samples were diluted in a ratio of 1 : 2 with the diluting Approximately 1.5 mL was placed in amber vials. solution, and 500 µL of the intermediate solution of the internal standard (doramectin) was added at 10 µg/mL; 1.5 mL was transferred in an amber vial. 2.4.2. Analysis by High-Performance Liquid Soil samples were dried in a heater at 45 C prior to Chromatography. Reverse-phase liquid chromatography was processing. At least 2/3 of the total solid samples (feed or carried out. Te mobile phase consisted of a solution con- soil) were shredded to obtain a homogeneous powder. Using taining acetonitrile (56%), methanol (37%), and water (7%) in a scale with a sensitivity of 0.01 g, 10 g of ground samples an isocratic process. Te stationary phase consisted of a C-18 were transferred to a conical fask. 50 mL of methanol and precolumn and a C-18 column, respectively, a Supelguard 500 µL of the intermediate solution of the internal standard reverse-phase chromatographic column C-18 (2 cm long, (doramectin) at 10 µg/mL were added to the sample, and the 4.6 mm internal diameter, and 5 µm average particle di- fask was capped. Te fasks were placed in a water bath with ameter) and a C-18 Supelcosil reverse-phase chromato- ultrasound for 20 minutes and then shaken on the shaker for graphic column (25 cm long, 4.6 mm internal diameter, and 5 µm average particle diameter). one hour at 700 oscillations/minute. 40 mL of supernatant was placed in plastic test tubes with a pressure cap and All HPLC analyses were conducted at room temperature centrifuged at 3000 rpm for 5 minutes. A frit was put inside (20 C). Before injecting the samples, the system was equili- each 6 mL reservoir, then 5 g of activated neutral alumina brated for 30 minutes under the conditions described above. were added, and another frit was put on top. Te 50 mL Te fow was set at 1.2 mL/min, and the wavelength at reservoirs were then fxed on top with the appropriate 245 nm. A volume of 100 µL was injected for each sample run. adapters. 15 mL of centrifugate supernatant was added to the Te ivermectin standard (0.05 µg/mL), the internal 50 mL reservoir. Te frst 5 mL was discarded, and the standard (0.05 µg/mL), the blank (Figure 1), and the fortifed remaining volume was collected in glass tubes. controls (Figure 2) were analyzed frst. Subsequently, all 4 Journal of Analytical Methods in Chemistry DAD1 A, Sig=245, 10 Ref=of mAU -1 0 2 4 6 8 10 12 14 min Figure 1: Chromatogram of a blank sample. At ivermectin retention times, no peak was registered, while doramectin’s peak is indicated with number 1 (RT 6.929). samples were run and the standard injections were repeated of a known sample extract together with a 50 µL volume of every three samples. one of the working solutions of ivermectin. Te working Te analysis was continuously monitored by Accredia, solution is chosen so that it corresponds to the same amount the Italian accreditation body, which certifes the quality of in ng of analyte comparable to that present in the sample. laboratory practices and conformity of the results obtained. Te perfect overlap of the standard and sample confrms the association when the peak has a good shape, and it is symmetrical without splitting or any deformations that may 3. Results and Discussion indicate the presence of an interferent and not of an analyte. Te area under the peak will be equal to the sum of half of the As can be seen in Figure 3, the calibration line had the standard and half of the sample because 50 µL of each was nanograms injected on the x-axis and the area under the injected instead of a full injection of 100 µL. Te spectrum of peak on the y-axis. Te calibration curve and instrument the sample peak was also compared with the spectrum at sensitivity are acceptable when intermediate concentration, evaluating the overlap factor (1) Te correlation of determination (r ) is greater than (match factor), which must not be less than 900/1000. or equal to 0.99. st (2) Te peak relative to the 1 point of the calibration curve is quantifable. 3.1. Precision and Accuracy. Te quantitative confrmatory method validation was performed according to the Annex Te concentration of ivermectin present in the sample III of the EU Regulation 2017/625. Te measurement un- was calculated based on the photometric response obtained certainty was expressed, at the concentration subject to from the chromatogram report, as can be seen in Figure 4. In validation (detection limit of 0.5 mg/kg), using the β error the case of positive samples above the maximum concen- (false negative) result less than or equal to 5% (statistical tration, the sample was diluted to remain in the scope. certainty 1 − β � 95%). Te concentration of ivermectin in the sample was calcu- lated as shown in equation (1), where A is the injected amount of ivermectin (ng), P is the weight of the sample (g), and 2 is the 3.1.1. Specifcity, Detection, and Quantifcation. Specifcity result obtained from the simplifcation of the calculation: was assessed by applying the entire test method to 20 blank ng injected x 1000 µI x 50 mL x 2 mL/100 µL x P x 2 mL samples for both solid and liquid samples. None of the x 1000. samples showed chromatographic peaks at the analyte retention time. mg A Concentration � . (1) To verify detection capability, extracts of 20 blank sample kg (P∗ 2) and the relative 20 strengthened at a value of 0.5 mg/kg were Te concentration of ivermectin added to the fortifed treated and injected in 2 analytical sessions. In all the for- sample was calculated by interpolation on the tifed samples, the chromatographic peak at the ivermectin calibration line. retention time was highlighted, while the blank samples did To qualitatively confrm the presence of ivermectin, co- not show any chromatographic peak at the ivermectin re- chromatography was carried out to make sure that the tention time. Terefore, the maximum tolerable error of 5% analyte in the suspect sample is indeed the one that is being was respected. Te limit of detection (LOD) was set at investigated. Te chromatography is run by injecting 50 µL 0.25 mg/kg and tested experimentally. Journal of Analytical Methods in Chemistry 5 DAD1 A, Sig=245, 10 Ref=of mAU -1 0 2.5 5 7.5 10 12.5 15 17.5 min Figure 2: Chromatogram of the spiked sample. Te numbers indicate the peaks of interest at respective retention time (RT). Number 1 indicates the peak of doramectin (RT 5.990), while number 2 is ivermectin (RT 8.100). 3.1.3. Uncertainty. Uncertainty was expressed as the max- Area imum extended relative uncertainty. In expressing the un- certainty, the actual degrees of freedom (v), calculated using the Welch–Satterthwaite formula, the calculated coverage factor (k), and a 95% confdence level (p) were considered. Tese parameters are summarized in Table 1. From the above uncertainty, we calculate the uncertainty of the result (C) using the following equation: U(y) r) � C∗ . (2) U(y 50 2 + 3.2. Method Validation. Te method was validated for both -- screening and confrmatory purposes. 0 100 200 For the screening validation, the selectivity, specifcity, Amount (ng/ul) and quantifcation limit were verifed by the 20 independent analyses of blank feed samples and 20 independent analyses Figure 3: Calibration curve. of blank feed samples spiked at the quantifcation limit concentration settled at 0.5 mg/kg. Te same was done for drinking water: 20 independent analyses of blank water Te limit of quantifcation (LOQ) was established at samples and 20 independent analyses of blank water samples 0.5 mg/kg. It was experimentally verifed through tests on spiked at the quantifcation limit concentration settled at fortifed feed at the limit of quantifcation in six independent 0.5 mg/kg. Te method was found to be applicable also to replicas. this matrix. Te applicability of the method was studied for feed 3.1.2. Accuracy. Accuracy was assessed through the 3-level matrixes, and the concentration range was settled from the recovery rate within the scope of the method. Precision was limit of quantifcation, 0.5 mg/kg, up to 10 mg/kg, consid- assessed by calculating the standard deviation and the co- ering the content of ivermectin in medicated feed. Further tests demonstrated that this method is also useful when efcient of variation (%) by conducting 6 replicate tests for each of the concentration levels. studying soil matrixes. Indeed, when the soil was dried and Linearity was evaluated to understand the feld of ap- analyzed in the same way as feed, sufcient recovery rates plication of the method (0.5–10 mg/kg). During calibration, were met in spiked sample. r obtained was 0.9999 for ivermectin. Specifcity was Linearity was evaluated to cover the entire range of assessed by applying the entire test method to 6 blank application of the method. For each calibration curve samples. Te tests conducted made it possible to verify the (Figure 3), fve concentration levels were chosen (0.5, 1, 2, 5, absence of an instrumental response around the and 12.5 mg/kg) and every level was replicated three times. retention time. Te calibration curves were obtained using least squares 6 Journal of Analytical Methods in Chemistry DAD1 A, Sig=245, 10 Ref=of mAU -1 0 2 4 6 8 10 12 14 16 18 min Figure 4: Chromatogram of a positive sample. Number 1 indicates doramectin peak (RT 6.372) and number 2 indicates ivermectin peak (RT 8.782). Table 1: Summary of uncertainty parameters. involving LC in diferent matrixes have reached lower Extended Degrees sensitivity by using diferent detectors, for example, MS was Validation relative of Coverage used by Morbidelli et al. in plasma to reach a 0.5–20.0 ng/mL Analyte range (mg/ uncertainty freedom factor (k) kg) range , by Durden et al. who saw in dried blood spots U (ӯ) (v) concentration of 1 ng/mL , and by Duthal et al. who Ivermectin 0.5–10 12.56 24 2.06 could detect a range of e 0.5–60 ppb in milk . Asbakk et al. and Iglesias et al. used a fuorescence detector reaching sensitivity at 5−2000 ng/g and 1–2000 ng/g, respectively Table 2: Data elaboration for each concentration level. [12, 13]. Number Relative Coefcient To determine trueness, precision, recovery, and re- Concentration Recovery of standard of variation peatability, spiked blank feed samples were used because no level (mg/kg) rate (%) replicas deviation (%) certifed reference material was available for ivermectin. In 0.5 6 91.8 2.99 3.26 detail: blank feed samples were spiked, before the beginning 2 6 98.3 1.67 1.70 of the extraction procedure, with ivermectin at three dif- 10 6 96.1 3.10 3.20 ferent concentration levels, and 6 independent replicas were carried out for each spiking level. Te results obtained are shown in Tables 2 and 3. Table 3: Total elaboration of 18 spiked feed samples. Ruggedness and reproducibility were evaluated using Total Total relative standard Total coefcient of ongoing data. Ruggedness was verifed by comparing recovery rate (%) deviation variation (%) processed quality control samples made by diferent op- 95.4 3.77 3.95 erators under diferent environmental conditions and at diferent times with diferent batches of standards and laboratory materials. Te analytical method was found to regression analysis. Te dataset obtained was investigated be rough, as the quality controls results are stable under the using the ANOVA approach. Te elaboration showed that planned operating conditions. Te reproducibility CV%, the linear regression was highly signifcant (P < 0.001), the calculated from 40 ongoing quality control data, is 8.40%. intercept values were not signifcantly diferent from zero, and the correlation coefcient values (>0.999) indicated 4. Conclusions excellent linearity. Selectivity and specifcity were evaluated by analyzing six Te HPLC method presented here allows the detection and samples from diferent kinds of feed and by confrming the quantifcation of ivermectin levels in diferent matrixes by absence of analytical substances with retention times similar creating a calibration curve and using doramectin as to ivermectin. a process standard. Tis validated method is easy, cheap to Te detection limit was fxed and verifed at half of the use, precise, and repeatable, and it needs very small amounts LOQ value by analyzing six spiked feed samples at 0.25 mg/kg. of samples. Te quantifcation limit was fxed and verifed at 0.5 mg/ It could be easily used to detect ivermectin in solid and kg at half of the minimum quantifcation level concentration liquid matrixes for monitoring and environmental pur- recommended by CReAA. Other validated methods poses. In fact, this method could be a reference method for Journal of Analytical Methods in Chemistry 7 Products, Animal Species, Route of Administration, Recom- ecotoxicity studies on the control of environmental pol- mended Dose and Marketing Authorization Holders in the lutants based on ivermectin. Further studies could use the Member States, European Medicine Agency (EMA), same analysis to extend the applicability to other mac- Amsterdam, Netherlands, 2009. rocyclic lactones or similar molecules in the same  Committee for medicinal products for veterinary use matrixes. (CVMP), “Ivermectin: modifcation of maximum residue limits,” summary report (5), European Medicines Agency, Abbreviations Amsterdam, Netherlands, 2004.  European Medicines Agency (Ema), Committee for Medicinal CV: Coefcient of variation Products for Veterinary Use European Public MRL Assessment DAD: Diode array detector Report (EPMAR), European Medicines Agency, Amsterdam, ESI: Electrospray ionization Netherlands, 2014. 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Oksanen, “Ivermectin in reindeer feces: determination by HPLC,” Journal of Agricul- Te data used to support the fndings of this study are tural and Food Chemistry, vol. 47, no. 3, pp. 999–1003, 1999. available from the corresponding author upon request.  L. E. Iglesias, C. Saumell, F. Sagu¨es, J. M. Sallovitz, and A. L. Lifschitz, “Ivermectin dissipation and movement from Conflicts of Interest feces to soil under feld conditions,” Journal of Environmental Science and Health, Part B, vol. 53, no. 1, pp. 42–48, 2017. Te authors declare that they have no conficts of interest.  I. C. Moschou, M. E. Dasenaki, and N. S. Tomaidis, “Ion- ization study and simultaneous determination of avermectins and milbemycines in fsh tissue by LC-ESI-MS/MS,” Journal Acknowledgments of Chromatography B, vol. 1104, pp. 134–140, 2019.  P. Tipthara, K. C. Kobylinski, M. 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Journal of Analytical Methods in Chemistry – Hindawi Publishing Corporation
Published: Mar 1, 2023
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