New LC–MS/MS Method Tracks 18 Anthelmintic Residues in Meat and Seafood

Every year, livestock and farmed fish around the world receive billions of doses of anthelmintics, the drugs that rid animals of parasitic worms. These compounds keep herds healthy and aquaculture operations profitable, but they leave a quieter legacy in the food on our plates: trace residues embedded in muscle, milk, eggs, and fat. Regulators set maximum residue limits, or MRLs, to keep exposure within safe bounds, yet enforcement depends entirely on whether laboratories can actually measure the drugs in question. In Korea, that chain has had a weak link. For widely used substances such as niclosamide, only qualitative screening methods existed, meaning inspectors could confirm a drug’s presence but not reliably quantify how much of it remained in a steak or a fillet of flatfish.

A team at Korea’s National Institute of Food and Drug Safety Evaluation, led by Ye-Bin Jang and corresponding author Mi-Ok Kim, has now closed that gap. Writing in Food Science of Animal Resources, the researchers describe an optimized liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that simultaneously quantifies 18 structurally diverse anthelmintic and antiparasitic compounds across nine animal-derived food matrices: beef, pork, chicken, eggs, milk, porcine fat, flatfish, eel, and shrimp. Beyond niclosamide, the method brings rarely monitored compounds such as derquantel, bithionol, and nitroxinil into quantitative reach, expanding both the chemical and the biological scope of routine residue surveillance.

The analytical challenge is formidable because the target compounds span six major chemical classes with very different physicochemical personalities. Benzimidazoles such as triclabendazole attack larval and adult parasites alike; salicylanilides including closantel, rafoxanide, and niclosamide also knock down ectoparasites like ticks; tetrahydropyrimidines such as morantel and pyrantel act as nicotinic receptor agonists; imidazothiazoles like levamisole target roundworms; and the macrocyclic lactones—abamectin, ivermectin, doramectin, eprinomectin, and moxidectin—are large, extremely lipophilic molecules that cling stubbornly to fatty tissue. Add praziquantel and nitroxinil to the list, and a single run must coax compounds ranging from small polar bases to sprawling lipid-like structures through the same column and ion source.

Sample preparation began with the QuEChERS approach—Quick, Easy, Cheap, Effective, Rugged, and Safe—which uses salt-induced phase separation to pull analytes from homogenized tissue into acetonitrile. The team compared three salt formulations: the unbuffered Original method with magnesium sulfate and sodium chloride, the citrate-buffered EN 15662 version, and the acetate-buffered AOAC variant. The verdict was decisive. In flatfish, the buffered methods produced calibration curves for abamectin with coefficients of determination of just 0.2304 and 0.0385—essentially useless—while the Original salts delivered R² values above 0.99 for all 18 compounds. The researchers attribute the failure of buffered systems to citrate and acetate ions altering salting-out efficiency and promoting co-extraction of lipids, proteins, and phospholipids, which then suppress ionization of the lipophilic macrocyclic lactones during electrospray.

Extraction used acetonitrile acidified with 0.1% formic acid, a combination known to improve recovery and precision for veterinary drugs in animal tissue. Clean-up followed with dispersive solid-phase extraction, and here the sorbent recipe mattered enormously. C18 alone left chlorfluazuron, levamisole, triclabendazole, and keto-triclabendazole with substandard linearity; PSA alone failed for chlorfluazuron and triclabendazole. Only the pairing of 400 milligrams of PSA with 400 milligrams of C18, plus 1,200 milligrams of magnesium sulfate, achieved R² values of at least 0.99 across the entire panel. PSA’s amino groups mop up organic acids and sugars, while C18 adsorbs nonpolar fats and fat-soluble interferences—a synergy the team standardized by purifying a fixed 8-milliliter extract volume for every matrix.

On the instrument side, chromatographic separation ran on a Waters Xbridge C18 column with a gradient of water containing 5 millimolar ammonium formate and 0.1% formic acid against acetonitrile with 0.1% formic acid. Retention times spanned from 1.9 minutes for morantel to 6.4 minutes for ivermectin. Detection used a Shimadzu LCMS-8060 triple quadrupole in multiple reaction monitoring mode, switching between positive and negative electrospray ionization, with precursor ions, product ions, and collision energies optimized by direct infusion of standards. The most intense transition served for quantification while secondary transitions confirmed identity.

Matrix effects proved to be the method’s central analytical adversary. Co-extracted lipids and proteins distort electrospray ionization, and the team measured signal changes of at least 20% for most analytes. Ion suppression dominated, ranging down to −96.08%, with abamectin, levamisole, pyrantel, and triclabendazole suppressed in every single matrix. Moxidectin, by contrast, showed ion enhancement in several foods, including chicken, milk, and all three fishery products. Rather than turning to isotope-labeled internal standards—impractical and costly for an 18-compound panel—the researchers built matrix-matched calibration curves in blank extracts of each food, so standards and samples ionize under identical chemical conditions and systematic signal bias cancels out at the quantification stage.

Validated under CODEX guideline CAC/GL-71, the method met every performance criterion. Matrix-matched calibration curves exceeded R² of 0.99 for all compounds in all nine matrices. Limits of quantification ranged from 0.0033 to 0.0054 milligrams per kilogram, all below half of the established MRLs. Intraday recovery and precision across the matrices fell between 72.0 and 119.1% with coefficients of variation no higher than 18.2%, comfortably inside CODEX windows, and interday testing on three consecutive days with two analysts held recoveries between 72.0 and 118.7% with CVs at or below 19.0%.

To test real-world utility, the team analyzed 60 market samples—30 livestock and 30 fishery products chosen for high Korean consumption. None of the 18 target analytes was detected, a reassuring snapshot of the domestic supply, though the authors stress that continued surveillance remains essential. The method’s true significance lies in what it enables going forward: quantitative enforcement for drugs like niclosamide that previously escaped numeric scrutiny, a single validated workflow spanning meat, dairy, eggs, and seafood, and a technical foundation for setting and revising safety standards. As veterinary drug use intensifies globally and resistant parasites push dosing upward, tools like this one turn regulatory limits from paper promises into measurable, enforceable protections for the food supply.

Subject of Research: Development and validation of a QuEChERS-based LC–MS/MS method for quantifying multiclass veterinary anthelmintic residues in animal-derived foods

Article Title: Optimized LC–MS/MS method for simultaneous determination of multiclass veterinary anthelmintic residues in animal-derived matrices

Article References: Optimized LC–MS/MS method for simultaneous determination of multiclass veterinary anthelmintic residues in animal-derived matrices. (n.d.). https://doi.org/10.1007/s44463-026-00098-1

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00098-1

Keywords: anthelmintics, LC-MS/MS, veterinary drug residues, QuEChERS, food safety, maximum residue limits, matrix-matched calibration, livestock products, fishery products, niclosamide, mass spectrometry, residue monitoring