What does a positive mitragynine (Enzyme Immunoassay - EIA) result with a negative nitragynine (Liquid Chromatography-Tandem Mass Spectrometry - LC-MS/MS) result indicate?

Positive Mitragynine EIA with Negative LC-MS/MS: Interpretation

A positive mitragynine enzyme immunoassay (EIA) with a negative liquid chromatography-tandem mass spectrometry (LC-MS/MS) result most likely represents a false-positive screening test, and the patient should be considered negative for kratom/mitragynine use.

Understanding the Testing Discrepancy

Confirmatory Testing Hierarchy

• LC-MS/MS is the gold standard confirmatory method and should be considered the definitive result when screening and confirmatory tests disagree 1.
• Screening immunoassays (EIA) are designed for high sensitivity but can produce false-positive results due to cross-reactivity with structurally similar compounds 1, 2.
• When screening and confirmatory results are discordant, the confirmatory test result takes precedence in clinical and forensic interpretation 1.

Mechanism of False-Positive EIA Results

• Enzyme immunoassays rely on antibody recognition which can cross-react with substances that share structural similarities with the target analyte 1.
• Different assays have varying sensitivities and specificities for detecting specific drug isoforms or metabolites 1, 2.
• Cross-reactive molecules that cause false-positive screening results are typically identified and excluded by the higher specificity of LC-MS/MS 1, 3.

Clinical Interpretation Algorithm

Step 1: Verify Confirmatory Test Quality

• Ensure the LC-MS/MS method was validated for mitragynine detection and can identify all relevant diastereomers (mitragynine, speciogynine, speciociliatine, mitraciliatine) 4.
• Confirm adequate sample quality and proper specimen handling, as LC-MS/MS requires appropriate sample preparation and processing 1.

Step 2: Consider Timing and Pharmacokinetics

• Mitragynine has a terminal half-life of approximately 23 hours (range 3-24 hours depending on the study) with time to maximum concentration around 0.83-1.5 hours after oral administration 5, 6.
• Only 0.14% of mitragynine is excreted unchanged in urine, with most appearing as metabolites 5, 7.
• If significant time has elapsed between use and testing, metabolites rather than parent compound may be present 5, 8.

Step 3: Assess for Metabolite Detection

• The most abundant urinary metabolite is 9-O-demethylmitragynine, produced by CYP2C19, CYP3A4, and CYP2D6 8.
• Other major metabolites include 16-carboxymitragynine and 7-hydroxymitragynine (produced exclusively by CYP3A4) 8.
• Verify whether the LC-MS/MS panel includes detection of mitragynine metabolites, not just the parent compound 4, 8.

Common Pitfalls and Caveats

Assay-Specific Limitations

• Not all LC-MS/MS methods separate or detect all four mitragynine diastereomers (mitragynine, mitraciliatine, speciogynine, speciociliatine), which may lead to underestimation of total kratom alkaloids 4.
• Some commercial assays may fail to detect all mitragynine isoforms or fragments, particularly in specialized applications 2.
• The presence of all diastereomers rather than individual substances indicates intake of kratom plant material rather than isolated compounds 4.

When to Consider Repeat Testing

• If clinical suspicion remains high despite negative LC-MS/MS, consider testing with a different LC-MS/MS method or laboratory 3, 2.
• Request specific metabolite testing (9-O-demethylmitragynine, 16-carboxymitragynine) if only parent compound was measured 8.
• Verify that the confirmatory method has adequate sensitivity, as mitragynine concentrations in chronic users can range from 0.3-1.8 μM 5, 6.

Clinical Context Considerations

• False-positive EIA results do not indicate kratom use and should not be used for clinical decision-making or legal/employment consequences 1.
• Document the discrepancy clearly, noting that LC-MS/MS is the definitive test 1, 2.
• Consider whether the patient is taking medications metabolized by CYP2C19, CYP2D6, or CYP3A4 that might theoretically interfere with testing, though this is unlikely to cause false-positive screening 8.