Which specialized laboratory tests can specifically identify ivermectin and its metabolites?

1. Mass spectrometry with liquid chromatography: the workhorse for detection

The principal, widely reported approach to detect ivermectin and its metabolites combines chromatographic separation (HPLC or UHPLC) with mass spectrometric detection (MS or tandem MS); papers identifying human ivermectin metabolites used ultra‑high performance liquid chromatography coupled with high‑resolution mass spectrometry to map M1–M13 metabolites after microsomal or human dosing experiments ^{[1] [3]}, and optimized LC‑MS/MS methods have been validated specifically for ivermectin quantification in plasma and whole blood with high sensitivity and throughput suitable for pharmacokinetic studies ^[2].

2. Tandem MS (LC‑MS/MS) for targeted quantitation and confirmation

For routine quantitative and confirmatory testing, tandem mass spectrometry (LC‑MS/MS) operating in multiple reaction monitoring or selected reaction monitoring modes permits highly selective detection of the ivermectin parent compound and chosen metabolite transitions, with validated methods reporting low limits of detection and reproducible recoveries used in both clinical and veterinary residue surveillance ^{[2] [4]}.

3. High‑resolution MS and exact‑mass platforms for metabolite discovery

When the goal is structural identification or discovery of unknown metabolites, high‑resolution platforms (e.g., UHPLC‑Q‑TOF, Orbitrap, FT‑ICR) provide accurate mass and MS/MS fragmentation patterns that distinguish demethylated, hydroxylated, deglycosylated and other phase I products reported in human microsome and hepatocyte studies ^{[1] [4] [5]}, and several groups have highlighted UHPLC‑HRMS as necessary to capture the diversity of M1–M13 species ^[3].

4. NMR and orthogonal chemistry for definitive structural assignment

Mass spectrometry patterns alone can propose structures, but unambiguous assignment of novel metabolites has required nuclear magnetic resonance (NMR) — including 1H‑NMR — to verify the molecular structures of many hydroxylated or demethylated ivermectin metabolites in both older metabolic studies and in recent human work where purified material was available ^{[1] [6] [3]}.

5. Matrix‑specific methods and practical assays (plasma, whole blood, tissues, environmental matrices)

Different matrices drive different validated assays: clinical studies favor streamlined LC‑MS/MS workflows with automated extraction for plasma and whole blood (high throughput, stability data reported) ^[2], food and environmental monitoring commonly use HPLC with fluorescence detection or UHPLC‑MS/MS calibrated with doramectin or ivermectin standards for soil/feed/water and tissue residue work ^{[7] [4]}, and residue labs use multiple identification points (precursor and product ion transitions) to meet confirmatory criteria ^[4].

6. How labs specificially distinguish metabolites: standards, fragments and enzymology

Specific identification of metabolites relies on three pillars: matching retention time and characteristic precursor→product ion transitions to authentic standards or library spectra, accurate‑mass MS/MS fragment interpretation to assign demethylation or hydroxylation sites, and orthogonal confirmation (synthesis or NMR) when possible; metabolic pathway work that mapped CYP3A4 as the main enzyme also supports assignment by reproducing metabolites in microsomes and comparing products to in vivo blood samples ^{[3] [8] [6]}.

7. Limitations, gaps and practical considerations

Laboratories should note limits: not all phase II conjugates are well characterized in humans yet, some metabolite identifications have been based on parent mass and fragment matching without full NMR confirmation, and sensitive high‑resolution methods require reference standards or synthesized metabolites for absolute confirmation and quantitation — shortcomings flagged in pharmacokinetic and mosquito‑effect studies ^{[8] [3] [9]}.

8. Bottom line: which tests to use for specific identification

For specific identification of ivermectin and its metabolites use (a) UHPLC‑HRMS (Q‑TOF/Orbitrap) for discovery and structural proposals, (b) LC‑MS/MS (tandem MS) with validated transitions for targeted confirmation and quantification in biological or environmental samples, and (c) NMR (1H‑NMR or full NMR suite) to verify structures when purified metabolite or synthetic standards are available — all approaches documented across human metabolism and residue literature ^{[1] [2] [3] [4]}.