What Does "Ivermectin Testing" Mean in a Laboratory?
Ivermectin is a macrocyclic-lactone anthelmintic — a mixture of ≥ 90 % avermectin H₂B1a and < 10 % avermectin H₂B1b derived from Streptomyces avermitilis — used in humans (onchocerciasis, strongyloidiasis, lymphatic filariasis, scabies) and widely in livestock and companion animals. "Ivermectin testing" in an analytical laboratory is not one assay but three distinct services, each answering a different question and run on a different matrix to a different standard. They are often confused, so the first job of a test report is to name which one was performed:
- Biological-fluid quantification — measuring ivermectin in plasma/whole blood to support pharmacokinetic (PK) studies, therapeutic drug monitoring (TDM) and dose-response research (the LC-MS/MS method validated to FDA/EMA bioanalytical guidance).
- Veterinary-residue testing — measuring ivermectin (and the other avermectin-class drugs) in food of animal origin (muscle, liver, fat, milk) against the maximum residue limits (MRLs), per GB 31658.16-2021 / GB 31650 in China.
- Drug-substance / drug-product assay — pharmacopeia identity, assay and impurity testing of the API or the formulated tablet/injectable.
The competitor literature that dominates the SERP — the PRINCIPLE COVID-19 trial, Mayo Clinic patient information, breast-cancer Phase I/II trials, cancer-FAQ pages — is clinical-trial and patient-education content, not analytical-laboratory testing. This article covers the three analytical-laboratory services, because those are what a testing laboratory is asked to perform.
Why Ivermectin Is Hard to Measure
Three chemical properties of ivermectin shape every analytical method, and a lab that ignores them produces unreliable numbers:
- High lipophilicity and heavy tissue distribution — ivermectin partitions into fat and skin, so plasma/whole-blood concentrations are low (peaks of ~36–119 ng/mL after a 400 µg/kg oral dose); sensitive detection (sub-ng/mL LLOQ) is required, and plasma concentrations run ~40 % higher than whole blood because of high binding to serum albumin.
- No native UV/fluorescence usable at trace levels — older HPLC methods needed ~1000 µL of sample and fluorescent derivatisation (trifluoroacetic anhydride / N-methylimidazole) to reach sensitivity. Modern methods avoid derivatisation by using MS/MS detection of the [M+NH₄]⁺ ammonium adduct (m/z 892.5 → 307.1), which is the most intense and reliable ion under ESI-positive.
- Matrix effects — phospholipids, haemoglobin and co-extracted lipids suppress or enhance the ion signal, so a clean sample-preparation (Hybrid SPE phospholipid removal, or SPE) plus a stable-isotope internal standard (ivermectin-D₂) are essential; the normalised matrix factor must be ~1.0 across six donor sources.
Biological-Fluid Quantification (PK / TDM)
For clinical pharmacokinetic studies and therapeutic drug monitoring, ivermectin is quantified in plasma and whole blood by LC-MS/MS validated to FDA Guidance for Industry (Bioanalytical Method Validation, 2018) and EMA (2012). A representative validated method (Kaewkhao 2024):
- Sample — 100 µL plasma or whole blood (small volume enables PK sampling).
- Extraction — automated Hybrid-SPE phospholipid removal with acetonitrile:water (90:10) + ivermectin-D₂ internal standard; recovery 96–123 %, normalised matrix factor ~1.0.
- LC — Poroshell 120 EC-C18 50 × 3.0 mm, 2.7 µm; mobile phase acetonitrile : 2 mM ammonium formate + 0.5 % formic acid (90:10); 5 min runtime.
- MS/MS — ESI+, [M+NH₄]⁺, SRM 892.5 → 307.1 (ivermectin) and 894.5 → 309.1 (ivermectin-D₂).
- Range — 0.970–384 ng/mL in both matrices; LLOQ 0.970 ng/mL; intra/inter-assay precision < 15 % (< 20 % at LLOQ); no carryover; stability through 5 freeze/thaw cycles and ≥ 55 h autosampler at 10 °C.
The pass criteria — linearity r² > 0.99, accuracy 85–115 %, precision ≤ 15 %, six-donor matrix effect within 0.85–1.15, incurred-sample-reanalysis (ISR) ≥ 67 % within 20 % — are the FDA/EMA bioanalytical acceptance standards, and a compliant PK report documents all of them, not just a single concentration.
Veterinary-Residue Testing in Food of Animal Origin
Because ivermectin is used in food-producing animals, the second service measures residues in edible tissues and milk against the MRL. In China the framework is:
- MRL standard — GB 31650-2019 (食品中兽药最大残留限量) and its supplement GB 31650.1-2022. For ivermectin the marker residue is 22,23-dihydroavermectin B1a, ADI 0–10 µg/kg bw. Typical MRLs (GB 31650-2019): bovine muscle 100, fat 100, liver 500, kidney 1000, milk 100 µg/kg.
- Method standard — GB 31658.16-2021 (动物性食品中阿维菌素类药物残留量的测定) covers the whole avermectin class — abamectin, ivermectin, doramectin, eprinomectin — by HPLC with fluorescence detection (after derivatisation) and by LC-MS/MS (no derivatisation, [M+NH₄]⁺). Aquatic products follow GB 29695-2013.
- Sample prep — acetonitrile extraction, alkaline-alumina or C18 SPE clean-up; for the fluorescence route, trifluoroacetic-anhydride/N-methylimidazole derivatisation.
The choice between HPLC-fluorescence and LC-MS/MS is driven by required sensitivity and the number of analogues to resolve: LC-MS/MS is now the dominant technique (since ~2010) because it does not need baseline chromatographic separation of the analogues and reaches lower LOQs, while HPLC-fluorescence remains valid where an MS is unavailable. The matrix effect remains the principal difficulty, controlled by matrix-matched calibration or isotope internal standard.
This service sits alongside other veterinary-residue work — see our pesticide residue testing and milk testing / testing of milk and dairy products.
Drug-Substance and Drug-Product Assay
The third service is pharmacopeia testing of the ivermectin active pharmaceutical ingredient (API) and the formulated product (tablet, injectable, pour-on). The pharmacopeia monograph governs identity, assay and impurity:
- Identity — by HPLC retention-time match against a reference standard, IR, and/or MS.
- Assay — HPLC (UV or fluorescence after derivatisation) quantifying the B1a + B1b content against reference standard; the USP and Ph. Eur. monographs specify the B1a:B1b ratio and the acceptance range.
- Related substances / impurities — HPLC impurity profile, including the B1a-8,9-Z-isomer and degradation products, against pharmacopeia thresholds.
- Dissolution / content uniformity — for tablets; release profile in specified media.
This is a release-and-stability service, distinct from the trace-residue and PK services: it asks "is this batch what it claims to be, at the labelled potency, free of degradants", whereas the residue service asks "is the food below the MRL" and the PK service asks "what is the patient's plasma level".
What Belongs on the Report — and Which Matrix Was Tested
The single most common reporting failure is conflating the three services. A compliant ivermectin test report must state:
- Which service — biological-fluid quantification, veterinary-residue, or drug-substance/product assay.
- Matrix — plasma vs whole blood (biological); muscle / liver / fat / milk / aquatic product (residue); API / tablet / injectable (product).
- Standard — FDA/EMA bioanalytical guidance (biological); GB 31658.16-2021 / GB 31650-2019 (residue); USP/Ph. Eur./ChP monograph (product).
- Method — LC-MS/MS or HPLC-fluorescence; extraction; internal standard; LLOQ; range.
- Validation / acceptance — for biological, the FDA/EMA parameters; for residue, recovery, repeatability, LOQ ≤ MRL; for product, the pharmacopeia acceptance criteria.
- Result vs the applicable limit — concentration vs reference range (TDM), concentration vs MRL (residue), assay % vs pharmacopeia range (product).
FAQ
What are the three types of ivermectin testing a laboratory performs?
Biological-fluid quantification (plasma/whole blood, LC-MS/MS to FDA/EMA bioanalytical guidance — for PK studies and TDM); veterinary-residue testing (food of animal origin, GB 31658.16-2021 / GB 31650, against MRLs); and drug-substance/product assay (API/tablet/injectable, pharmacopeia identity/assay/impurity). The matrix and the standard are different for each.
Why is LC-MS/MS the preferred method for ivermectin?
Because ivermectin has no usable native fluorescence at trace levels and partitions heavily into tissue (low plasma levels), older HPLC methods needed large sample volumes and chemical derivatisation. LC-MS/MS detects the [M+NH₄]⁺ adduct (m/z 892.5 → 307.1) with sub-ng/mL sensitivity, does not need derivatisation or baseline separation of the avermectin analogues, and — with a stable-isotope internal standard (ivermectin-D₂) — controls the matrix effect. It has been the dominant technique for both PK and residue work since ~2010.
What is the MRL for ivermectin in food of animal origin in China?
Per GB 31650-2019, for the marker residue 22,23-dihydroavermectin B1a (ADI 0–10 µg/kg bw): bovine muscle 100, fat 100, liver 500, kidney 1000, milk 100 µg/kg. Other species and tissues have their own limits; GB 31650.1-2022 supplements and revises some.
Why are ivermectin concentrations higher in plasma than in whole blood?
Because ivermectin is highly bound to human serum albumin, so it concentrates in the plasma fraction; reported plasma concentrations are up to ~40 % higher than whole blood. A PK method must state which matrix was calibrated and not freely convert between them.
Can a single LC-MS/MS method cover both PK and residue testing?
The chromatography and MS detection are similar (same [M+NH₄]⁺ transition), but the sample preparation, calibration range, internal standard, validation criteria and acceptance limits are different (sub-ng/mL biological vs µg/kg food; FDA/EMA bioanalytical vs GB 31658.16 method validation). A method validated for plasma is not automatically valid for liver extract, so each matrix requires its own validated method.
Our Testing Capabilities
As an ISO/IEC 17025-accredited third-party laboratory, Beijing ZKGX Research provides the three ivermectin testing services:
- Biological-fluid quantification — LC-MS/MS for ivermectin in plasma and whole blood to FDA (2018) / EMA (2012) bioanalytical guidance, with ivermectin-D₂ internal standard, Hybrid-SPE extraction, sub-ng/mL LLOQ, full validation (linearity, accuracy, precision, matrix effect across six donors, carryover, stability) and incurred-sample reanalysis, for pharmacokinetic studies and therapeutic drug monitoring.
- Veterinary-residue testing — GB 31658.16-2021 (HPLC-fluorescence and LC-MS/MS) and GB 29695-2013 (aquatic products) for ivermectin and the avermectin class in muscle, liver, fat, milk and aquatic products, against the GB 31650-2019 / GB 31650.1-2022 MRLs.
- Drug-substance / product assay — pharmacopeia identity, assay and related-substances testing of the ivermectin API, tablets, injectables and pour-on formulations (USP / Ph. Eur. / ChP monographs).
Sample types include human/animal plasma and whole blood, edible animal tissues (muscle, liver, fat, kidney), milk and dairy products, aquatic products, the API and formulated dosage forms. If you have a specific matrix, target limit (PK range / MRL / pharmacopeia spec), or compliance target (FDA / EMA / GB / NMPA / CAC / EU), contact the laboratory to confirm the correct standard, method and reporting format before testing.