Depletion of [³H] Amitraz in Sheep

Depletion of [³H] Amitraz in Sheep | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Depletion of [³H] Amitraz in Sheep Zehra Hajrulai-Musliu, Stefan Jovanovski, Aleksandar Janevski, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7792400/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Dec, 2025 Read the published version in Journal of Radioanalytical and Nuclear Chemistry → Version 1 posted You are reading this latest preprint version Abstract The depletion of amitraz in sheep is investigated following intramuscular administration of the tritium-labelled [³H] drug. A validated liquid scintillation counting (LSC) method was employed to quantify residues in multiple biological matrices from sheep, including blood, urine, milk, feces, and tissues such as kidney, liver, muscle, and fat. Excellent linearity (R² ≥ 0.991), low detection limits (2–4 × 10⁻⁵ µCi/mL) and very good recovery rates ranging from 72% to 104% were determined. The radioactive drug was distributed rapidly in the sheep, peaking in blood and excreta in a few hours. The amitraz residue levels in edible tissues dropped to trace or undetectable levels by day 28 after treatment. Fat tissue retained the residues the longest, while levels in milk and muscle remained low throughout the experiment. The study findings are relevant for regulatory decisions and responsible use of pesticides such as amitraz for food production. Amitraz [³H] amitraz sheep residue depletion liquid scintillation counting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Amitrazalso known as (methylimino) dimethylidyne) di-2,4-xylidine is a formamidine acaricide and insecticide widely used in agriculture. Farmers and veterinarians rely on it to control pests such as ticks, mites, and lice in animals, particularly sheep and cattle. Amitraz has become increasingly important for use on farms worldwide because of effectives against a range of parasites (Pass and Mogg, 1995 ). The presence of amitraz residues in foods such as meat has been reported. These can be a consumer and trade concern should levels exceed the legal limitssuch as the 50 µg/kg set by the EU or 100 µg/kg permitted by Codex Alimentarius for certain tissues. Some studies have found violations where amitraz residues exceeded these standards (European Food Safety Authority [EFSA], 2021; Pohorecka et al., 2018 ).Appropriates testing and monitoring of residues and promoting of withdrawal period based on findings of depletion studies is necessary (CIR (EU) 2021/808; VICH GL49). Depletion studies entail measurement of radioactivity in blood, tissues, and other biological samples using tools such as the liquid scintillation counter (LSC), which is commonly employed in pharmacokinetic and biodistribution research (Füll, Wallasch, Hilton, & Planz, 2022 ). Liquid scintillation counting (LSC) enables detection of low levels of radioactivity in biological samples and is widely used in depletion studies and quantification of radiolabeled drugs and their metabolites (Temple, 2015 ; FDA, 2022), including complex biological samples (Wang et al., 2022 ). The LSC method validated following established guidelines (ICH, 2005). Similar validation strategies have been employed in other studies using LSC, such as Jalal and Bondarenko ( 2025 ), who quantified radiolabeled agrochemicals with high sensitivity; Benito et al. ( 2012 ), who demonstrated LSC’s versatility in detecting β-emitting isotopes across biological matrices; and the National Diagnostics Laboratory ( 2004 ), which outlines foundational principles for reliable LSC operation. Our study had two primary goals. First, to ensure that our LSC method could accurately measure tritiated ([³H]) amitraz in sheep tissues and fluids. Second, to track the decrease in [³H] amitraz and its residues over time after injection in sheep. The study findings would support regulation and good production practices. Experimental Study Design Twenty-four adult sheep ( Ovis aries ), each weighing approximately 65 ± 3 kg were included in the study. They were divided into seven groups (n = 3 each) with the last two groups (Groups 6 and 7) comprising lactating ewes for milk radioactive testing. Each sheep received a single intramuscular dose of amitraz at 2 mg/kg, (~ 130 mg per animal). This was prepared by diluting a commercial 12.5% amitraz solution (125 mg/mL) with sterile saline to a final concentration of 10 mg/mL. For analysis, 1.61 mL of [³H]-amitraz (1000 µCi/mL) was added to the commercial solution, resulting in a total volume of 300 mL and a nominal radioactive concentration of 5.38 µCi/mL. Each sheep received a final dose of 70 µCi by intramuscular injection of 13 mL, divided equally between the gluteal and cervical muscles to minimize local irritation. The activity of the [³H]-amitraz standard was verified by liquid scintillation counting in March 2025, using a tritium calibration standard and applying instrument efficiency (63.53%) and background corrections. Throughout the study, animals were monitored for clinical signs and injection-site reactions. In lactating ewes, milk samples were periodically collected to detect amitraz and radiolabel residues. All procedures complied with institutional ethical standards and national regulations governing the use of radiolabeled substances in animal testing. The study adhered to established veterinary protocols and followed VICH guidelines, specifically VICH GL48, which outlines methods for assessing drug metabolism and residue depletion in food-producing animals. This involved examining marker residue depletion to establish withdrawal periods and ensure appropriate systemic absorption of amitraz. The study protocol received ethical approval from the Macedonian Agency for Food and Veterinary Affairs under Decision No. 11-1340/1 dated 03 July 2025. This approval complies with the national regulations on animal protection and welfare (Official Gazette Nos. 149/14, 149/15, 53/16, and 65/23) and the Law on Veterinary Health Protection (Official Gazette No. 124/15). The project was carried out at the Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, involving 24 sheep, registered under institutional protocol No. 11–378/1, dated 27 January 2025. Sheep were euthanized according to animal care protocols at intervals of 1, 3, 5, 7, 14, 21, and 28 days post-treatment. Sample Preparation for Total Radioactivity Assay Whole blood (2 mL), urine (5 mL), milk (5 mL) and feces (1 g) were digested in open vessels with 2 mL 65% HNO₃ (ROTIPURAN® ≥ 65%, p.a., ISO, Carl Roth) and 3 mL 30% H₂O₂ (ROTIPURAN® 30%, p.a., ISO, stabilized, Carl Roth) at 100°C for 30–60 min, until no dense NO₂ fumes were observed. Liver, muscle, kidney, and fat were processed in the same way in Kjeldahl flasks. After cooling, each digest was brought to volume with deionized water, centrifuged at 3,000 rpm (ambient), and then passed through 0.45 µm syringe filters. An aliquot of the clear filtrate was mixed with 10 mL Ultima Gold™ scintillation cocktail. Samples were measured on a Packard Tri-Carb 4810 TR liquid-scintillation counter (PerkinElmer, Waltham, MA, USA) in ³H mode with tSIE/AEC quench monitoring. Photomultiplier gains were stabilized daily to place the external ³H peak at channel 40. Spectra were acquired for 1 min in Region A (0–18.6 keV). Region B (2.0–18.6 keV) was recorded concurrently to check spillover, using an 18 ns coincidence window and the factory static-gain controller. Counting efficiency was corrected automatically with the instrument’s external standard channels-ratio quench curve, and activity was calculated as the blank-subtracted mean of duplicate determinations. Immediately before analysis, a four-point external calibration (0.01–1 µCi/mL) was prepared from freshly made [³H]-amitraz standards, yielding excellent linearity (R² >0.990) and agreeing with the sealed ³H/¹⁴C reference within ± 1%. Detector efficiency for ³H exceeded 60% throughout, while background never surpassed 10 cpm. Results and discussion Method Validation The LSC method developed for quantifying [³H] amitraz in sheep biological matrices demonstrated strong analytical performance across all tested sample types. To prepare the matrix matched calibration curve, blood, urine, milk, feces, and tissues (kidney, liver, muscle and fat tissues) were spiked with varying volumes of a 5 µCi/mL working solution, with concentrations ranging from 0.001 to 1.0 µCi/mL. Calibration curves for blood, urine, milk, feces, kidney, liver, muscle, and fat showed excellent linearity, with coefficients of determination (R²) ranging from 0.991 to 0.997 (Fig. 2 ). The results indicated that the analytical limits of detection (LODs) and limits of quantification (LOQs) varied significantly when comparing pure solutions to different matrices, as summarized in Table 1 . In solution, the LODs ranged from 2.0 × 10⁻⁵ to 4.0 × 10⁻⁵ µCi/mL and LOQs from 7.0 × 10⁻⁵ to 1.2 × 10⁻⁴ µCi/mL. While in matrix-matched samples, the LOD 0.001 µCi/mL (all matrices tested, except for blood(0.013 µCi/mL) and milk (0.014 µCi/mL). Both blood and milk are probably slightly more complex matrices for the LSC. The LOQ values in matrix-matched conditions, were consistently at 0.001 µCi/mL across most matrices although the values for blood, milk and urine were 0.309 µCi/mL, 0.319 µCi/mL and 0.327 µCi/mL, respectively. These results demonstrate the need to pay attention to the matrix even with sensitive analytical instrumentation. Table 1 Calibration curves and corresponding R 2 ;LOD and LOQ values for different matrices Concentration, µCi/mL Blood Urine Milk Faeces Kidney Liver Muscle Fat Intensity (cpm) 0 400 350 350 500 450 350 400 550 0.01 20420 19463 17552 16276 15455 15246 16732 14205 0.1 239182 217641 203513 183116 171522 174925 182942 161287 0.5 1283782 1128662 1073652 969242 904997 963805 964801 861681 1 2913239 2704850 2635069 2284026 2152232 2241009 2371000 2006749 Slope 2890857 2670004 2598045 2258661 2126602 2220983 2337006 1986760 Intercept -39451 -45548 -50543 -36657 -35835 -36089 -45341 -30842 Correlation coefficient 0.998 0.997 0.996 0.997 0.997 0.998 0.996 0.998 Correlation coefficient, R 2 0.997 0.993 0.992 0.994 0.994 0.995 0.991 0.995 LOD, analytical, µCi/mL 0.00002 0.00002 0.00002 0.00003 0.00003 0.00003 0.00003 0.00004 LOQ, analytical, µCi/mL 0.00007 0.00007 0.00007 0.00010 0.00010 0.00008 0.00009 0.00012 LOD, sample, µCi 0.013 0.013 0.014 0.001 0.001 0.001 0.001 0.001 LOQ, sample, µCi 0.309 0.327 0.319 0.001 0.001 0.001 0.001 0.001 For the recovery blood, urine, milk, feces, kidney, muscle, liver, and fat (5 each, 1 g or equivalent) were individually spiked with varying volumes of a 5 µCi/mL working solution of [³H] amitraz. The samples were processed and analyzed by LSC. Recovery rates were calculated by comparing measured values to expected concentrations derived from the calibration curves. The values ranged from 71.8% to 103.6%, as detailed in Tables 2 , 3 , 4 and 5 . These values fall within the acceptable range defined by international guidelines [ (EU) 2021/808; VICH GL49] which recommend recoveries between 70% and 120%. The highest recovery (up to 103.6%) was for kidneys and slightly lower (72–85%) for muscles, which demonstrates matrix-dependent extraction and binding affinity. These outcomes are consistent with previous studies that have demonstrated the robustness and reliability of the liquid scintillation counting (LSC) method for analyzing radiolabeled compounds in complex biological matrices (Chughtai et al., 2025 ; Zirena Vilca et al., 2020 ; International Atomic Energy Agency, 2016 ; Rogliardo et al., 2025 ). Harper et al. ( 2019 ) demonstrated that liquid scintillation counters (LSCs) reliably measure both parent compounds and metabolites across various animal tissues, making them well-suited for comprehensive residue depletion and pharmacokinetic studies. The technique is suitable for monitoring tritiated and carbon-14 labeled substances (Eckert & Ziegler, 2020). Table 2 Recovery rates for blood, milk and urine samples, standard deviation and relative standard deviation. Nominal Concentration, µCi/mL Blood Milk Urine Blood Milk Urine Between matrixes Estimated concentration, µCi/mL Recovery (%) Standard deviation, µCi/mL Relative standard deviation, % 0.02 0.019 0.021 0.017 92.7 102.7 85.4 0.0017 9.25 0.04 0.032 0.035 0.033 80.0 87.8 81.7 0.0016 4.91 0.06 0.045 0.050 0.047 75.2 82.8 77.7 0.0023 4.94 0.08 0.063 0.067 0.059 78.7 83.4 74.0 0.0037 5.94 0.1 0.071 0.080 0.097 70.7 80.5 97.0 0.0133 16.05 Table 3 Recovery rates for faeces in different concentration levels Nominal Concentration, µCi/kg Feces Estimated concentration, µCi/kg Recovery (%) 6250 5532.6 88.5 12500 10983.5 87.9 25000 17685.5 70.7 62500 54479.0 87.2 Table 4 Recovery rates for muscle, kidney and liver samples, standard deviation and relative standard deviation. Nominal Concentration, µCi/kg Muscle Kidney Liver Muscle Kidney Liver Between matrices Estimated concentration, µCi/kg Recovery (%) Standard deviation, µCi/kg Relative standard deviation, % 125 105.8 100.3 103.8 84.6 80.2 83.0 2.8 2.7 1250 1063.7 987.9 1063.4 85.1 79.0 85.1 43.7 4.2 12500 8974.0 11359.5 12433.7 71.8 90.9 99.5 1770.8 16.2 25000 20421.2 24086.8 25888.8 81.7 96.3 103.6 2786.2 11.9 Table 5 Recovery rates for fat tissue samples in different concentration levels Nominal Concentration, µCi/kg Fat Estimated concentration, µCi/kg Recovery (%) 1250 1174.9 94.0 12500 11309.1 90.5 25000 23963.6 95.9 62500 54104.9 86.6 Harper et al. ( 2019 ) reported similar recovery performance using LSC although for [¹⁴C]-labeled antiparasitic agents in bovine tissues. Depletion Kinetics in Biological Matrices Radioactivity levels in faeces peaked rapidly (mean 0.407 µCi at 3 h) and then declined steadily to low levels by day 28 (mean 0.018 µCi). This, consistent with amitraz’s partial biliary excretion (Chou et al., 2001 ).Radioactivity in blood reached a maximum at 5 h post-dose (mean 28.46 µCi) and declined sharply (mean 3.70 µCi at 24 h), followed by a later increase (mean 68.37 µCi at 672 h), indicating redistribution rather than continuous elimination. Radioactivity in tissues was highest early, especially in fat (mean 1.36 µCi), reflecting amitraz’s high lipophilicity (Pass and Mogg, 1995 ). Muscle and organs showed much lower levels, and all tissues demonstrated substantial depletion by day 28. Radioactivity in milk remained low throughout the study, with a maximum mean of 0.249 µCi at 3 h followed by a rapid decline, suggesting minimal risk of exposure to high levels of amitraz in milk ( do Monte Barretto et al., 2017 ). Radioactivity in urine peaked at 3 h (mean 4.66 µCi) and then declined rapidly, confirming renal excretion as a primary elimination route (de Roode, Joosten, & Behe, 2024 ). Our studies demonstrates the importance of using radiolabelled compounds to understand their absorption, distribution, metabolism, and excretion (ADME). Penner et al. ( 2012 ) reporting radiolabeling in mapping ADME pathways, particularly in preclinical drug development. Fazaeli et al. ( 2024 ) also reported the use [⁶²Zn]-labeled amoxicillin in fish with rapid clearance of the compound Rapid distribution and excretion of radioactivity were evident in blood, urine, feces, and edible tissues, with concentrations declining to trace or undetectable levels by day 28. These findings are consistent with previous pharmacokinetic profiles reported by Hugnet et al. (1996), which also documented swift clearance of amitraz and its metabolites in food-producing animals. Notably, fat tissue retained higher residue levels than other matrices perhaps due to amitraz’s lipophilic nature (Pass and Mogg, 1995 ). However, even in fat, residue concentrations dropped below quantifiable limits. The low and transient presence of amitraz in milk and muscle tissues suggests minimal risk of consumer exposure through these products. Nanjundappa et al. ( 2021 ) reported low transfer rates of amitraz into milk. Conclusion The depletion of [³H] amitraz in sheep has been investigated by analyzing tissues and matrices such as liver, kidney, muscle, fat, blood, urine and faeces, using a validated liquid scintillation counting methods. Rapid distribution and excretion of the formamidine acaricide was observed in blood, urine, feces, and edible tissues so that levels were trace or undetectable levels by day 28. Fat retained the drug the longest. Tritium is often noted for its limitation in depletion studies compared to C-14, due to its apparent short half-life and tendency to be unstable. This was not a problem in this study, showing the possible use of tritium labelled drugs in other relevant studies. An innovative sample preparation technique was used in the absence of a sample oxidizer. Similar studies using other drugs of interested will be implemented following the procedures used in this research, with modifications. Declarations Author Contribution - Zehra H. Musliu (ZHM) – Conceptualization, Methodology, Supervision, Writing – Original Draft, Corresponding Author.- Stefan Jovanovski (SJ)– Investigation, Data Curation, Formal Analysis, Writing – Review & Editing.- Aleksandar Janevski (AJ)– Validation, Resources, Laboratory Support.- Elizabeta Dimitrieska Stojkovikj (EDS)– Project Administration, Sample Collection, Writing – Review & Editing.- Dea Musliu( DM)– Visualization, Data Interpretation, Quality Control.- James Jacob Sasanya (JJS)– Regulatory Insight, Writing – Review & Editing, Funding Acquisition. 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Supplementary Files CopyofRecoveryblood002.xlsx CopyofRecoveryfat.xlsx CopyofRecoveryfeces.xlsx CopyofRecoverykidny.xlsx CopyofRecoveryliver.xlsx CopyofRecoverymilk.xlsx CopyofRecoverymuscle.xlsx CopyofResultsUrine.xlsx CopyofResultsBlood.xlsx CopyofResultsfeaces.xlsx CopyofResultsMilk.xlsx CopyofResultsTissue.xlsx Cite Share Download PDF Status: Published Journal Publication published 24 Dec, 2025 Read the published version in Journal of Radioanalytical and Nuclear Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7792400","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":540967804,"identity":"ec07c534-5231-4ba9-90a3-ad64aae17b08","order_by":0,"name":"Zehra Hajrulai-Musliu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie2RsUoDQRBAZ1nYNJeUMucW9wu7HIjgiR9ik7DFNXcYEELSXQisvxC5aP7COrJgZ2rLg7QK6W3czSk27mlpsa8YZhjezgwLEAj8RxBAAJwC680bVwvmYtR2uhQEFhnxd6WNqFoFflOSetFczzQmg3jHjl/ApIOjDWneNFzG1c8KWT0J+axRaq4YL8CcMD6k8k5DWXumUBwKOddINL8yqVUyqzDet8q9R2GY751yoWNDv5Tee5cSYXGYMtJIye5zMUb7HYshFmNZbVHpSFlF5Cnjo0W82mJ56zk/WeYPsppk5+sbA6aYnsl1rR73r5OsXG48Y+w54js/pMQ97/1IB226uoFAIBCAD+1AS34QaCCVAAAAAElFTkSuQmCC","orcid":"","institution":"Faculty of Veterinary Medicine‑Skopje, University “Ss. 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Biological samples (blood, urine, milk, feces, kidney, liver, muscle, fat) were homogenized and measured. Solid matrices were solubilized with Soluene-350 at 55–60°C for 1–2 h, supplemented with hydrogen peroxide to minimize color quenching. After cooling, Hionic-Fluor scintillation cocktail was added, vortexed, and samples transferred to vials for radioactivity measurement on a Tri-Carb PerkinElmer LSC. Data were processed to obtain activity (cpm) and concentration values.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/b8e28db64a70ab2f36ac7067.jpg"},{"id":95400681,"identity":"d604ebb0-b912-4c21-aa9b-99cf4e817e8a","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39621,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves for all matrices, illustrating the direct linear relationship between [³H] amitraz concentration (µCi/mL) and cpm. Each matrix displayed a distinct but consistently linear response, with blood showing the highest slope and fat the lowest, reflecting matrix-specific sensitivity.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/fc3cdd72783fc35d3b20c685.jpg"},{"id":95400685,"identity":"032f0788-676f-451a-bb06-f8df1ad80852","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":191020,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery (%) of [³H] amitraz from urine, milk, and blood samples at various nominal activity levels (0.5–2.5 µCi). All matrices showed recovery rates above 70%.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/d87fce99f1ee5ba9eceb6012.jpg"},{"id":95526144,"identity":"872ccaa7-5449-4a01-949a-25d3c83e893c","added_by":"auto","created_at":"2025-11-10 10:06:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167285,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery (%) of [³H]Amitraz from faeces samples at different nominal activity levels. Recovery ranged from 70.7% to 88.5%.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/c7f80d3a5a36bcad914571d9.jpg"},{"id":95526762,"identity":"37be4c27-303b-4600-864a-6a1716843748","added_by":"auto","created_at":"2025-11-10 10:07:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247762,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery (%) of [³H]Amitraz from muscle, liver, and kidney samples at varying nominal activity levels. All tissues showed satisfactory recovery.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/fa2bf6ad3f1b578376c7bf15.jpg"},{"id":95400688,"identity":"d624c01f-0f79-460d-a03c-8fe666cafa9f","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":189677,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery (%) of [³H] amitraz from fat samples at different nominal activity levels. Recovery values ranged from 86.3% to 95.8%.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/86e9171c864e3f1c8416235e.jpg"},{"id":95400705,"identity":"51c3c183-4b39-49da-b0fd-9a66517eaf58","added_by":"auto","created_at":"2025-11-07 16:00:36","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":459219,"visible":true,"origin":"","legend":"\u003cp\u003eBlood concentration-time profile for [³H] amitraz shows a maximum at 5 hours post-exposure, followed by a decline after 7 hours and a sustained low level from 24 hours onward.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/55bf7eb0d3c6b49abf79d2f0.jpg"},{"id":95527125,"identity":"39b5821c-dd35-4250-b9d2-224a6262fa7f","added_by":"auto","created_at":"2025-11-10 10:11:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":330536,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual [³H] amitraz activity profiles in milk over time for each sheep. The highest activity is detected at 3–5 hours post-exposure, with a rapid decrease and persistent low activity after 24 hours. The X-axis break differentiates the early absorption and elimination phases from the prolonged low-level excretion\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/bafbbd4729ecb3ce3fb16855.jpg"},{"id":95526380,"identity":"e02efd64-9c7d-46f5-8c53-6e76d3deb018","added_by":"auto","created_at":"2025-11-10 10:06:54","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":386828,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual excretion profiles of [³H] amitraz in urine over time for each sheep. Peak urinary activity is observed at 3–5 hours post-exposure, followed by a rapid decline and low, steady excretion after 24 hours. The X-axis break distinguishes early timepoints from long-term elimination.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/c350c0b700cf31b180ef6919.jpg"},{"id":95400700,"identity":"ce4f1a85-53ef-4107-ab46-afb516aa3940","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":388683,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual [³H] amitraz activity profiles in feces over time for each sheep. Peak excretion in feces is observed at 3–5 hours post-exposure, followed by a pronounced decline and low, persistent excretion levels after 24 hours. The axis break emphasizes the difference between early and late timepoints\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/2493e12987f004365cfcc2e6.jpg"},{"id":95400703,"identity":"18895b4c-f93c-40af-9dc3-4fb27e711f1d","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":54427,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of [³H] amitraz in kidney, liver, muscle, and fat over 29 days post-exposure. Separate Y axes are used to display the distinct magnitudes and clearance kinetics among tissues. Kidney, liver, and muscle show rapid elimination and low residual radioactivity, while fat exhibits higher levels and more prolonged retention\u003c/p\u003e","description":"","filename":"Picture11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/a95fbc8ff9cc353a02897c11.jpg"},{"id":99172359,"identity":"42ef4732-ca72-4229-b069-d5e15db3cc96","added_by":"auto","created_at":"2025-12-29 16:08:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3387143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/a01138f6-8124-4861-be33-f0faf9323833.pdf"},{"id":95526423,"identity":"0386db4f-1fcb-4962-9381-6192ffed4834","added_by":"auto","created_at":"2025-11-10 10:06:57","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":159619,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoveryblood002.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/4e22a068c9409ab7b6730199.xlsx"},{"id":95527491,"identity":"38c01148-da42-4375-8c65-56ea186d65ba","added_by":"auto","created_at":"2025-11-10 10:13:50","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":131990,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoveryfat.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/a6d497f34b0536223a4f4fc1.xlsx"},{"id":95400690,"identity":"dbcafd35-b5a3-4f16-afce-c25814962424","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":152105,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoveryfeces.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/1083680b519633174928b6be.xlsx"},{"id":95400694,"identity":"8770eff1-06a3-49ba-b672-cf8e92eb7428","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":154924,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoverykidny.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/78b4b1b151258b657d208fc1.xlsx"},{"id":95527480,"identity":"fb588acd-36a7-4638-b6d5-a39ec0bfcc2e","added_by":"auto","created_at":"2025-11-10 10:13:49","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":154897,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoveryliver.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/1676c41b3ed26ec22f90cffe.xlsx"},{"id":95400702,"identity":"b478fc2f-6904-422c-af23-89caf56dba5c","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":159512,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoverymilk.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/10d87bebcc43c2ceea8b7039.xlsx"},{"id":95400704,"identity":"eff9d87e-f57b-4c59-8ce9-ba56b9abf16c","added_by":"auto","created_at":"2025-11-07 16:00:35","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":154923,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofRecoverymuscle.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/a6bcf363c8ddec1c304c0da7.xlsx"},{"id":95527181,"identity":"d0ef236a-4be9-421b-ac4c-134631466da0","added_by":"auto","created_at":"2025-11-10 10:11:18","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":309451,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofResultsUrine.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/69bb7d62e5e50c37fa59edba.xlsx"},{"id":95526499,"identity":"7e7989d8-a067-4200-9dbc-11851899ac2d","added_by":"auto","created_at":"2025-11-10 10:07:08","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":660951,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofResultsBlood.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/e703f707a286f8ea48febf6e.xlsx"},{"id":95526974,"identity":"5a4314a7-a19b-4f07-8158-89b0bcf98e58","added_by":"auto","created_at":"2025-11-10 10:08:57","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":300547,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofResultsfeaces.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/c06ad31b88e7af9c72c14327.xlsx"},{"id":95400716,"identity":"a9ab04ce-2fc0-42d2-a700-5c762aa9325a","added_by":"auto","created_at":"2025-11-07 16:00:36","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":253114,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofResultsMilk.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/d3f820681e74edb2164ead1b.xlsx"},{"id":95526542,"identity":"956b4cef-9353-4b35-ba3a-468356ed5678","added_by":"auto","created_at":"2025-11-10 10:07:13","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":355706,"visible":true,"origin":"","legend":"","description":"","filename":"CopyofResultsTissue.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7792400/v1/9401be2c704f0752030d0bd0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Depletion of [³H] Amitraz in Sheep","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmitrazalso known as (methylimino) dimethylidyne) di-2,4-xylidine is a formamidine acaricide and insecticide widely used in agriculture. Farmers and veterinarians rely on it to control pests such as ticks, mites, and lice in animals, particularly sheep and cattle. Amitraz has become increasingly important for use on farms worldwide because of effectives against a range of parasites (Pass and Mogg, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The presence of amitraz residues in foods such as meat has been reported. These can be a consumer and trade concern should levels exceed the legal limitssuch as the 50 \u0026micro;g/kg set by the EU or 100 \u0026micro;g/kg permitted by Codex Alimentarius for certain tissues. Some studies have found violations where amitraz residues exceeded these standards (European Food Safety Authority [EFSA], 2021; Pohorecka et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Appropriates testing and monitoring of residues and promoting of withdrawal period based on findings of depletion studies is necessary (CIR (EU) 2021/808; VICH GL49). Depletion studies entail measurement of radioactivity in blood, tissues, and other biological samples using tools such as the liquid scintillation counter (LSC), which is commonly employed in pharmacokinetic and biodistribution research (F\u0026uuml;ll, Wallasch, Hilton, \u0026amp; Planz, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLiquid scintillation counting (LSC) enables detection of low levels of radioactivity in biological samples and is widely used in depletion studies and quantification of radiolabeled drugs and their metabolites (Temple, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; FDA, 2022), including complex biological samples (Wang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The LSC method validated following established guidelines (ICH, 2005). Similar validation strategies have been employed in other studies using LSC, such as Jalal and Bondarenko (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who quantified radiolabeled agrochemicals with high sensitivity; Benito et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), who demonstrated LSC\u0026rsquo;s versatility in detecting β-emitting isotopes across biological matrices; and the National Diagnostics Laboratory (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), which outlines foundational principles for reliable LSC operation.\u003c/p\u003e\u003cp\u003eOur study had two primary goals. First, to ensure that our LSC method could accurately measure tritiated ([\u0026sup3;H]) amitraz in sheep tissues and fluids. Second, to track the decrease in [\u0026sup3;H] amitraz and its residues over time after injection in sheep. The study findings would support regulation and good production practices.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003eTwenty-four adult sheep (\u003cem\u003eOvis aries\u003c/em\u003e), each weighing approximately 65\u0026thinsp;\u0026plusmn;\u0026thinsp;3 kg were included in the study. They were divided into seven groups (n\u0026thinsp;=\u0026thinsp;3 each) with the last two groups (Groups 6 and 7) comprising lactating ewes for milk radioactive testing. Each sheep received a single intramuscular dose of amitraz at 2 mg/kg, (~\u0026thinsp;130 mg per animal). This was prepared by diluting a commercial 12.5% amitraz solution (125 mg/mL) with sterile saline to a final concentration of 10 mg/mL. For analysis, 1.61 mL of [\u0026sup3;H]-amitraz (1000 \u0026micro;Ci/mL) was added to the commercial solution, resulting in a total volume of 300 mL and a nominal radioactive concentration of 5.38 \u0026micro;Ci/mL. Each sheep received a final dose of 70 \u0026micro;Ci by intramuscular injection of 13 mL, divided equally between the gluteal and cervical muscles to minimize local irritation. The activity of the [\u0026sup3;H]-amitraz standard was verified by liquid scintillation counting in March 2025, using a tritium calibration standard and applying instrument efficiency (63.53%) and background corrections.\u003c/p\u003e\u003cp\u003eThroughout the study, animals were monitored for clinical signs and injection-site reactions. In lactating ewes, milk samples were periodically collected to detect amitraz and radiolabel residues. All procedures complied with institutional ethical standards and national regulations governing the use of radiolabeled substances in animal testing. The study adhered to established veterinary protocols and followed VICH guidelines, specifically VICH GL48, which outlines methods for assessing drug metabolism and residue depletion in food-producing animals. This involved examining marker residue depletion to establish withdrawal periods and ensure appropriate systemic absorption of amitraz. The study protocol received ethical approval from the Macedonian Agency for Food and Veterinary Affairs under Decision No. 11-1340/1 dated 03 July 2025. This approval complies with the national regulations on animal protection and welfare (Official Gazette Nos. 149/14, 149/15, 53/16, and 65/23) and the Law on Veterinary Health Protection (Official Gazette No. 124/15). The project was carried out at the Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, involving 24 sheep, registered under institutional protocol No. 11\u0026ndash;378/1, dated 27 January 2025. Sheep were euthanized according to animal care protocols at intervals of 1, 3, 5, 7, 14, 21, and 28 days post-treatment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample Preparation for Total Radioactivity Assay\u003c/h3\u003e\n\u003cp\u003eWhole blood (2 mL), urine (5 mL), milk (5 mL) and feces (1 g) were digested in open vessels with 2 mL 65% HNO₃ (ROTIPURAN\u0026reg; \u0026ge; 65%, p.a., ISO, Carl Roth) and 3 mL 30% H₂O₂ (ROTIPURAN\u0026reg; 30%, p.a., ISO, stabilized, Carl Roth) at 100\u0026deg;C for 30\u0026ndash;60 min, until no dense NO₂ fumes were observed. Liver, muscle, kidney, and fat were processed in the same way in Kjeldahl flasks. After cooling, each digest was brought to volume with deionized water, centrifuged at 3,000 rpm (ambient), and then passed through 0.45 \u0026micro;m syringe filters. An aliquot of the clear filtrate was mixed with 10 mL Ultima Gold\u0026trade; scintillation cocktail.\u003c/p\u003e\u003cp\u003eSamples were measured on a Packard Tri-Carb 4810 TR liquid-scintillation counter (PerkinElmer, Waltham, MA, USA) in \u0026sup3;H mode with tSIE/AEC quench monitoring. Photomultiplier gains were stabilized daily to place the external \u0026sup3;H peak at channel 40. Spectra were acquired for 1 min in Region A (0\u0026ndash;18.6 keV). Region B (2.0\u0026ndash;18.6 keV) was recorded concurrently to check spillover, using an 18 ns coincidence window and the factory static-gain controller. Counting efficiency was corrected automatically with the instrument\u0026rsquo;s external standard channels-ratio quench curve, and activity was calculated as the blank-subtracted mean of duplicate determinations. Immediately before analysis, a four-point external calibration (0.01\u0026ndash;1 \u0026micro;Ci/mL) was prepared from freshly made [\u0026sup3;H]-amitraz standards, yielding excellent linearity (R\u0026sup2; \u0026gt;0.990) and agreeing with the sealed \u0026sup3;H/\u0026sup1;⁴C reference within \u0026plusmn;\u0026thinsp;1%. Detector efficiency for \u0026sup3;H exceeded 60% throughout, while background never surpassed 10 cpm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eMethod Validation\u003c/h2\u003e\u003cp\u003eThe LSC method developed for quantifying [\u0026sup3;H] amitraz in sheep biological matrices demonstrated strong analytical performance across all tested sample types. To prepare the matrix matched calibration curve, blood, urine, milk, feces, and tissues (kidney, liver, muscle and fat tissues) were spiked with varying volumes of a 5 \u0026micro;Ci/mL working solution, with concentrations ranging from 0.001 to 1.0 \u0026micro;Ci/mL. Calibration curves for blood, urine, milk, feces, kidney, liver, muscle, and fat showed excellent linearity, with coefficients of determination (R\u0026sup2;) ranging from 0.991 to 0.997 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results indicated that the analytical limits of detection (LODs) and limits of quantification (LOQs) varied significantly when comparing pure solutions to different matrices, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In solution, the LODs ranged from 2.0 \u0026times; 10⁻⁵ to 4.0 \u0026times; 10⁻⁵ \u0026micro;Ci/mL and LOQs from 7.0 \u0026times; 10⁻⁵ to 1.2 \u0026times; 10⁻⁴ \u0026micro;Ci/mL. While in matrix-matched samples, the LOD 0.001 \u0026micro;Ci/mL (all matrices tested, except for blood(0.013 \u0026micro;Ci/mL) and milk (0.014 \u0026micro;Ci/mL). Both blood and milk are probably slightly more complex matrices for the LSC. The LOQ values in matrix-matched conditions, were consistently at 0.001 \u0026micro;Ci/mL across most matrices although the values for blood, milk and urine were 0.309 \u0026micro;Ci/mL, 0.319 \u0026micro;Ci/mL and 0.327 \u0026micro;Ci/mL, respectively. These results demonstrate the need to pay attention to the matrix even with sensitive analytical instrumentation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCalibration curves and corresponding R\u003csup\u003e2\u003c/sup\u003e;LOD and LOQ values for different matrices\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConcentration, \u0026micro;Ci/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlood\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUrine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMilk\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFaeces\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKidney\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMuscle\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eFat\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIntensity (cpm)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e550\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19463\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17552\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e16732\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e14205\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e239182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e217641\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e203513\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e183116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e171522\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e174925\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e182942\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e161287\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1283782\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1128662\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1073652\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e969242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e904997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e963805\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e964801\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e861681\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2913239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2704850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2635069\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2284026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2152232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2241009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2371000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2006749\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSlope\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2890857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2670004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2598045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2258661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2126602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2220983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2337006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1986760\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntercept\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-39451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-45548\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-50543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-36657\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-35835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-36089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-45341\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-30842\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorrelation coefficient\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorrelation coefficient, R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.992\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.991\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOD, analytical, \u0026micro;Ci/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.00003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOQ, analytical, \u0026micro;Ci/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.00009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00012\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOD, sample, \u0026micro;Ci\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLOQ, sample, \u0026micro;Ci\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.327\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor the recovery blood, urine, milk, feces, kidney, muscle, liver, and fat (5 each, 1 g or equivalent) were individually spiked with varying volumes of a 5 \u0026micro;Ci/mL working solution of [\u0026sup3;H] amitraz. The samples were processed and analyzed by LSC. Recovery rates were calculated by comparing measured values to expected concentrations derived from the calibration curves. The values ranged from 71.8% to 103.6%, as detailed in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. These values fall within the acceptable range defined by international guidelines [ (EU) 2021/808; VICH GL49] which recommend recoveries between 70% and 120%.\u003c/p\u003e\u003cp\u003eThe highest recovery (up to 103.6%) was for kidneys and slightly lower (72\u0026ndash;85%) for muscles, which demonstrates matrix-dependent extraction and binding affinity. These outcomes are consistent with previous studies that have demonstrated the robustness and reliability of the liquid scintillation counting (LSC) method for analyzing radiolabeled compounds in complex biological matrices (Chughtai et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zirena Vilca et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; International Atomic Energy Agency, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rogliardo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHarper et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that liquid scintillation counters (LSCs) reliably measure both parent compounds and metabolites across various animal tissues, making them well-suited for comprehensive residue depletion and pharmacokinetic studies. The technique is suitable for monitoring tritiated and carbon-14 labeled substances (Eckert \u0026amp; Ziegler, 2020).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery rates for blood, milk and urine samples, standard deviation and relative standard deviation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNominal Concentration, \u0026micro;Ci/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlood\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMilk\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUrine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBlood\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMilk\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUrine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eBetween matrixes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eEstimated concentration, \u0026micro;Ci/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStandard deviation, \u0026micro;Ci/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRelative standard deviation, %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e102.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e85.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e9.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.033\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e87.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e81.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.050\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e75.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e82.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e77.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.067\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.059\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e78.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e83.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e74.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e5.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.080\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e70.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e80.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e97.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.0133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e16.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery rates for faeces in different concentration levels\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNominal Concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFeces\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEstimated concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5532.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e88.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10983.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e87.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17685.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e62500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54479.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e87.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery rates for muscle, kidney and liver samples, standard deviation and relative standard deviation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNominal Concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMuscle\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKidney\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMuscle\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKidney\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eBetween matrices\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eEstimated concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStandard deviation, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRelative standard deviation, %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e105.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e103.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e84.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e80.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e83.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1063.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e987.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1063.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e79.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e85.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e43.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8974.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11359.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12433.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e71.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e90.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e99.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1770.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e16.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20421.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24086.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25888.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e81.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e96.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e103.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2786.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e11.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery rates for fat tissue samples in different concentration levels\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNominal Concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFat\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEstimated concentration, \u0026micro;Ci/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1174.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e94.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11309.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23963.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e95.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e62500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54104.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e86.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHarper et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported similar recovery performance using LSC although for [\u0026sup1;⁴C]-labeled antiparasitic agents in bovine tissues.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDepletion Kinetics in Biological Matrices\u003c/h3\u003e\n\u003cp\u003eRadioactivity levels in faeces peaked rapidly (mean 0.407 \u0026micro;Ci at 3 h) and then declined steadily to low levels by day 28 (mean 0.018 \u0026micro;Ci). This, consistent with amitraz\u0026rsquo;s partial biliary excretion (Chou et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).Radioactivity in blood reached a maximum at 5 h post-dose (mean 28.46 \u0026micro;Ci) and declined sharply (mean 3.70 \u0026micro;Ci at 24 h), followed by a later increase (mean 68.37 \u0026micro;Ci at 672 h), indicating redistribution rather than continuous elimination. Radioactivity in tissues was highest early, especially in fat (mean 1.36 \u0026micro;Ci), reflecting amitraz\u0026rsquo;s high lipophilicity (Pass and Mogg, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Muscle and organs showed much lower levels, and all tissues demonstrated substantial depletion by day 28. Radioactivity in milk remained low throughout the study, with a maximum mean of 0.249 \u0026micro;Ci at 3 h followed by a rapid decline, suggesting minimal risk of exposure to high levels of amitraz in milk ( do Monte Barretto et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Radioactivity in urine peaked at 3 h (mean 4.66 \u0026micro;Ci) and then declined rapidly, confirming renal excretion as a primary elimination route (de Roode, Joosten, \u0026amp; Behe, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur studies demonstrates the importance of using radiolabelled compounds to understand their absorption, distribution, metabolism, and excretion (ADME). Penner et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) reporting radiolabeling in mapping ADME pathways, particularly in preclinical drug development. Fazaeli et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) also reported the use [⁶\u0026sup2;Zn]-labeled amoxicillin in fish with rapid clearance of the compound Rapid distribution and excretion of radioactivity were evident in blood, urine, feces, and edible tissues, with concentrations declining to trace or undetectable levels by day 28.\u003c/p\u003e\u003cp\u003eThese findings are consistent with previous pharmacokinetic profiles reported by Hugnet et al. (1996), which also documented swift clearance of amitraz and its metabolites in food-producing animals. Notably, fat tissue retained higher residue levels than other matrices perhaps due to amitraz\u0026rsquo;s lipophilic nature (Pass and Mogg, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, even in fat, residue concentrations dropped below quantifiable limits. The low and transient presence of amitraz in milk and muscle tissues suggests minimal risk of consumer exposure through these products. Nanjundappa et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported low transfer rates of amitraz into milk.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe depletion of [\u0026sup3;H] amitraz in sheep has been investigated by analyzing tissues and matrices such as liver, kidney, muscle, fat, blood, urine and faeces, using a validated liquid scintillation counting methods. Rapid distribution and excretion of the formamidine acaricide was observed in blood, urine, feces, and edible tissues so that levels were trace or undetectable levels by day 28. Fat retained the drug the longest. Tritium is often noted for its limitation in depletion studies compared to C-14, due to its apparent short half-life and tendency to be unstable. This was not a problem in this study, showing the possible use of tritium labelled drugs in other relevant studies. An innovative sample preparation technique was used in the absence of a sample oxidizer. Similar studies using other drugs of interested will be implemented following the procedures used in this research, with modifications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e- Zehra H. Musliu (ZHM) \u0026ndash; Conceptualization, Methodology, Supervision, Writing \u0026ndash; Original Draft, Corresponding Author.- Stefan Jovanovski (SJ)\u0026ndash; Investigation, Data Curation, Formal Analysis, Writing \u0026ndash; Review \u0026amp; Editing.- Aleksandar Janevski (AJ)\u0026ndash; Validation, Resources, Laboratory Support.- Elizabeta Dimitrieska Stojkovikj (EDS)\u0026ndash; Project Administration, Sample Collection, Writing \u0026ndash; Review \u0026amp; Editing.- Dea Musliu( DM)\u0026ndash; Visualization, Data Interpretation, Quality Control.- James Jacob Sasanya (JJS)\u0026ndash; Regulatory Insight, Writing \u0026ndash; Review \u0026amp; Editing, Funding Acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the support of the International Atomic Energy Agency (IAEA) under the Coordinated Research Project D52043, \u0026ldquo;Depletion of Veterinary Pharmaceuticals and Radiometric Analysis of their Residues in Animal Matrices.\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePass MA, Mogg TD (1995) Pharmacokinetics and metabolism of amitraz in ponies and sheep. 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DMD. 401867\u0026ndash;1877\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFazaeli SY, Shahhosseini G, Neisi A, Sasanya JJ, Ashtari P, Feizi Zenjanab S (2024) Depletion imaging using radionuclides: A novel method for evaluation of veterinary pharmaceuticals using [⁶\u0026sup2;Zn]-labeled amoxicillin. Radiochim Acta 112:261\u0026ndash;272\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amitraz, [³H] amitraz, sheep, residue depletion, liquid scintillation counting","lastPublishedDoi":"10.21203/rs.3.rs-7792400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7792400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe depletion of amitraz in sheep is investigated following intramuscular administration of the tritium-labelled [\u0026sup3;H] drug. A validated liquid scintillation counting (LSC) method was employed to quantify residues in multiple biological matrices from sheep, including blood, urine, milk, feces, and tissues such as kidney, liver, muscle, and fat. Excellent linearity (R\u0026sup2; \u0026ge; 0.991), low detection limits (2\u0026ndash;4 \u0026times; 10⁻⁵ \u0026micro;Ci/mL) and very good recovery rates ranging from 72% to 104% were determined. The radioactive drug was distributed rapidly in the sheep, peaking in blood and excreta in a few hours. The amitraz residue levels in edible tissues dropped to trace or undetectable levels by day 28 after treatment. Fat tissue retained the residues the longest, while levels in milk and muscle remained low throughout the experiment. The study findings are relevant for regulatory decisions and responsible use of pesticides such as amitraz for food production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Depletion of [³H] Amitraz in Sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 16:00:30","doi":"10.21203/rs.3.rs-7792400/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d44c4348-2e4e-4d4d-940f-350ef6efee1f","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:02:36+00:00","versionOfRecord":{"articleIdentity":"rs-7792400","link":"https://doi.org/10.1007/s10967-025-10670-6","journal":{"identity":"journal-of-radioanalytical-and-nuclear-chemistry","isVorOnly":false,"title":"Journal of Radioanalytical and Nuclear Chemistry"},"publishedOn":"2025-12-24 15:58:10","publishedOnDateReadable":"December 24th, 2025"},"versionCreatedAt":"2025-11-07 16:00:30","video":"","vorDoi":"10.1007/s10967-025-10670-6","vorDoiUrl":"https://doi.org/10.1007/s10967-025-10670-6","workflowStages":[]},"version":"v1","identity":"rs-7792400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7792400","identity":"rs-7792400","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}