What laboratory assays and sensitivity thresholds are used to detect ivermectin in biological samples?
Executive summary
Modern laboratories detect ivermectin in blood, plasma, tissue and environmental matrices using a suite of chromatographic and immunoassay techniques; tandem mass spectrometry methods (LC–MS/MS or UHPLC–MS/MS) provide the greatest combination of sensitivity and specificity while fluorescence‑derivatized HPLC and ELISA remain useful for screening or specific matrices (plasma, feeds, liver) [1] [2] [3] [4]. Reported limits of detection and quantification vary widely with matrix and method — from sub‑nanogram per milliliter performance for some immunoassays and optimized LC–MS/MS workflows to microgram‑per‑milliliter levels for older HPLC procedures — so choice of assay depends on required sensitivity, throughput, and whether confirmatory identification is needed [4] [1] [5].
1. LC–MS/MS / UHPLC–MS/MS: the confirmatory gold standard
Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS or UHPLC–MS/MS) is the method favored for small‑volume human pharmacokinetics and regulatory residue confirmation because it combines low-volume extraction, multiple‑reaction‑monitoring identification and quantitation with low ng/mL or sub‑ng/mL limits in many validated protocols [1] [6] [3]. Validation papers report limits of detection and quantification in the low ng/mL range — for example an optimized LC–MS/MS assay set a LOD at 0.485 ng/mL and an LLOQ at 0.970 ng/mL in plasma/whole blood with acceptable precision (<15%) [1] — while UHPLC–MS/MS approaches for tissues achieved LOQs appropriate for monitoring withdrawal periods (μg/kg to low μg/kg) and used SRM transitions for confirmatory identification [3].
2. HPLC with fluorescence or UV detection: established, sometimes less sensitive
High‑performance liquid chromatography with fluorescence detection (HPLC‑FL or LC‑FL) has a long pedigree for ivermectin assays and can reach useful linear ranges (for example 0.2–400 ng/mL in one validated plasma method) but typically requires derivatization and larger sample volumes and can be slower in throughput compared with automated LC–MS/MS methods [2] [7]. Some HPLC methods report detection limits in the hundreds of ng/mL to μg/mL range (for example a stability‑indicating RP‑HPLC reported detection limits ≈0.3 μg/mL) making them suitable for product QC or feed/tissue analysis but less ideal for trace human PK work [5] [7].
3. Immunoassays (ELISA): sensitive screening, cross‑reactivity caveats
Enzyme‑linked immunosorbent assays have been developed as sensitive screening tools with reported LODs down to 0.1 ng/mL and useful coefficients of variation in the 0.3–10 ng/mL working ranges, which makes ELISA attractive for high‑throughput screening of biological fluids or tissue extracts [4] [8]. These assays, however, are subject to antibody cross‑reactivity with related avermectins and generally require confirmation by chromatographic‑mass spectrometric methods when regulatory or forensic certainty is needed [9] [8].
4. GC‑MS and other chromatographic variants: confirmatory and niche uses
Gas chromatography–mass spectrometry (GC‑MS) after derivatization has been used historically as a confirmatory technique and remains in the literature for tissue residue work, while newer chromatographic variants such as micellar liquid chromatography or VAMS (volumetric absorptive microsampling) coupled with UPLC–MS/MS address sample convenience and environmental or field sampling needs [9] [10] [6]. These approaches extend the range of feasible matrices but each brings different LOD/LOQ characteristics and sample preparation demands [6] [10].
5. Sample preparation and matrix dependence: the practical limiter of sensitivity
Reported sensitivity thresholds depend as much on sample extraction (SPE, QuEChERS, protein precipitation, derivatization) and matrix effects as on the detector; for instance QuEChERS plus UHPLC–MS/MS achieved tissue LOQs useful for withdrawal monitoring while VAMS with protein precipitation and UPLC–MS/MS enabled microsampling with MRM transitions tuned to ivermectin ions [3] [6]. Method papers explicitly warn that LODs and LLOQs must be interpreted by matrix — plasma, whole blood, fat, liver, feed and environmental samples show very different background and recovery characteristics [1] [11] [3].
6. Practical recommendation and limitations of the literature
For trace human or preclinical pharmacokinetic analysis, validated LC–MS/MS or UHPLC–MS/MS methods are recommended for their combination of sub‑ng/mL to low‑ng/mL quantitation and confirmatory ion transitions [1] [3]; ELISA and fluorescence‑derivatized HPLC remain valuable for screening and higher‑concentration matrices but either lack specificity or demand larger samples and derivatization [4] [2] [7]. The literature shows meaningful variability in published LOD/LOQ figures depending on matrix and lab conditions and highlights throughput and run‑time tradeoffs (longer HPLC runs versus faster UPLC‑MS/MS), so any laboratory decision should be guided by the target matrix, required sensitivity and whether confirmatory identification is legally or clinically required [1] [5] [3].