Mycotoxins & Aflatoxins in Herbal Extracts: Control & Testing

Mycotoxins are toxic secondary metabolites produced by certain moulds that colonise botanicals before, during and after harvest. The critical and often misunderstood fact is that they are stable, low-molecular-weight chemicals — not living organisms. Kill the mould and the toxin remains. For herbal extracts this reframes the whole problem: mycotoxin control is not microbial control, and it cannot be solved by sterilisation, irradiation or any end-of-line decontamination step. It is won or lost upstream, then proven by analysis. Reading a mycotoxin result well means understanding which toxins matter, where they come from, how the limit was set, and how the sample was drawn.

What mycotoxins actually are

Mycotoxins are produced chiefly by three genera of fungi — Aspergillus, Penicillium and Fusarium — and a single contaminated lot can carry more than one toxin from more than one mould. Several hundred mycotoxins are known, but only a handful are routinely relevant to herbal raw materials and the extracts made from them, because of their toxicity, their prevalence, and their regulatory weight.

Aflatoxins (B1, B2, G1, G2)

Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus and are the most heavily regulated mycotoxin class. Four are individually quantified — aflatoxin B1, B2, G1 and G2 — named for their blue or green fluorescence under UV light. Aflatoxin B1 is the most potent and is classified by IARC as a Group 1 human carcinogen; this is why limits address both B1 alone and the sum of all four (total aflatoxins). Regulatory results are reported in ppb (parts per billion, µg/kg), reflecting how low the meaningful thresholds are.

Ochratoxin A (OTA)

Ochratoxin A is produced by Aspergillus ochraceus, Aspergillus carbonarius and several Penicillium species (notably Penicillium verrucosum in cooler climates). It is nephrotoxic, is classified by IARC as a Group 2B possible human carcinogen, and is notably persistent — it survives ordinary processing and storage. OTA is a particular concern in dried spices, liquorice root, grape-derived materials and many dried roots and barks, and is regulated separately from the aflatoxins, again in ppb.

Other relevant mycotoxins

• Fumonisins (B1, B2) — produced mainly by Fusarium species on grasses and cereals; relevant where botanical material is grass- or grain-associated.
• Zearalenone (ZEN) and deoxynivalenol (DON) — Fusarium toxins of the trichothecene and related families, more typical of cereal-linked materials than classic roots and leaves.
• T-2 and HT-2 toxins — potent Fusarium trichothecenes, occasionally relevant to field-contaminated botanicals.
• Citrinin — a Penicillium and Aspergillus toxin that often co-occurs with ochratoxin A and is specifically watched in fermented red-yeast materials.

Which botanicals and conditions are high-risk

Mycotoxin risk is not evenly distributed across the botanical world. It concentrates in materials that are dense, slow to dry, harvested from or near the soil, or grown and stored in warm, humid climates. Knowing the risk profile of your material tells you how hard to test and how closely to audit the supply chain behind it.

• Roots, rhizomes and barks — dense, soil-contacting material that dries slowly and traps internal moisture (for example liquorice / Glycyrrhiza glabra, ginger, turmeric, ashwagandha / Withania somnifera) sits at the high-risk end for both aflatoxins and OTA.
• Dried fruits and seeds — figs, certain berries and oil-bearing seeds are classic aflatoxin substrates, especially if drying stalls.
• Spices — chilli, paprika, nutmeg, pepper and ginger are repeatedly flagged at borders for aflatoxins and OTA.
• Leaves, flowers and aerial parts — generally lower risk, but not zero where field drying is poor or rains interrupt harvest.
• Hot, humid origins — tropical and subtropical growing regions raise risk for any material, because warmth and humidity favour the toxigenic moulds.

Why mycotoxins form — field and storage

Mycotoxin formation happens in two distinct windows, and controlling only one leaves the door open. Field (pre-harvest) contamination occurs while the plant is still growing — drought stress, insect damage, lodging and untimely rain let toxigenic moulds establish on the living or maturing crop. Storage (post-harvest) contamination is usually the larger and more controllable risk: it is driven by moisture and time after harvest, when inadequately dried material sits in warm, poorly ventilated conditions and the moulds bloom and produce toxin.

Water activity is the master variable

The single most important driver of post-harvest mould growth and toxin production is water activity (Aw) — the free, available water in the material, on a scale from 0 to 1. Toxigenic moulds generally need an Aw above roughly 0.70 to grow, and active aflatoxin production typically requires an Aw above about 0.83 together with warm temperatures. Drying a botanical promptly and thoroughly to a low, stable Aw — and keeping it there through storage and transport — is the most powerful single lever available. A lot that is dried too slowly, dried to the surface only, or re-wetted by humidity or condensation can generate toxin even if it looked clean at harvest.

Because both windows matter, mycotoxin prevention is a chain: good agricultural practice in the field, rapid and complete drying at harvest, and dry, ventilated, pest-controlled storage thereafter. A single weak link — one rained-on drying floor, one humid container — can contaminate a lot that was otherwise handled well.

Why decontamination does not work

This is the point most often misunderstood, and it deserves to be stated plainly: killing the mould does not remove the toxin. Aflatoxins and ochratoxin A are small, chemically stable molecules. The processes that reduce microbial counts — heat, steam, ethylene oxide, irradiation — act on living organisms and leave the toxin chemically intact. Aflatoxin B1, for instance, is only meaningfully degraded at temperatures well above those used in ordinary drying or extraction. A lot can be rendered microbiologically clean and still fail a mycotoxin limit.

Because the toxin cannot be reliably taken out at the end, the only durable control is to stop it being formed in the first place. Prevention lives upstream — in GACP-aligned cultivation, in disciplined drying, and in dry storage — and the laboratory test exists to verify that prevention worked, not to fix it after the fact. Blending a hot lot down with a clean one to hit an average is likewise not control; it is dilution of a hazard, and is prohibited under most regulatory regimes.

Prevention upstream: GACP, drying, storage

Good Agricultural and Collection Practices (GACP) is the framework that turns prevention into routine. It governs how a botanical is grown, harvested, dried, sorted and stored so that toxigenic moulds get the fewest possible chances to establish and produce toxin. The controls are mostly low-tech and procedural — which is precisely why a supplier’s discipline, not their equipment, is what protects you.

• In the field — crop rotation, pest and insect control, timely harvest, and avoiding damage that opens the plant to fungal entry; harvesting at the right maturity and not leaving cut material on damp ground.
• At drying — rapid drying to a low, uniform water activity using clean drying floors or controlled dryers; avoiding rain interruption, ground contact and stacking that traps internal moisture before the core has dried.
• At sorting — removing visibly mouldy, discoloured, broken or insect-damaged pieces, which carry a disproportionate share of any toxin load.
• In storage — dry, cool, ventilated, pest-controlled stores; moisture barriers and desiccants where needed; FIFO rotation and monitoring to catch re-wetting before mould blooms.
• In transit — sealed, dry containers with condensation control, since a humid sea container can re-wet a perfectly dried lot in transit.

Regulatory limits differ by market

There is no single global mycotoxin limit, and this trips up buyers who assume a pass in one market is a pass everywhere. Limits are set by jurisdiction, by toxin, and often by specific commodity — and they can differ by an order of magnitude between regions. The European Union is generally the strictest; the United States and India set their own thresholds. You must specify and test against the limit of the market you are selling into, not the market you are buying from.

How the major markets differ

• European Union — among the strictest globally, with separate, low limits for aflatoxin B1 and for total aflatoxins (B1+B2+G1+G2), plus dedicated limits for ochratoxin A, set per commodity category under EU contaminants legislation.
• United States — the FDA enforces an action level for total aflatoxins in foods, with no separate B1-only food limit in the same form as the EU; the framework and the numbers are not interchangeable with the EU’s.
• India — the FSSAI sets aflatoxin and ochratoxin A limits for foods and spices under its contaminants standards, which differ again from EU and US figures.

Testing methods

Mycotoxins are present at parts-per-billion levels, so the analytical method must be both sensitive and specific. Three approaches dominate, and they serve different purposes — screening versus confirmation, single-class versus multi-toxin.

HPLC-FLD

High-performance liquid chromatography with fluorescence detection (HPLC-FLD) is a long-established confirmatory method for aflatoxins and ochratoxin A, both of which fluoresce. After clean-up (commonly on an immunoaffinity column), the toxins are separated and quantified at ppb sensitivity. It is robust, accurate and widely accepted by regulators for these specific toxins, and remains a reference approach where a defensible single-class number is required.

LC-MS/MS

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the most powerful and increasingly the preferred method. It can quantify many mycotoxins simultaneously — aflatoxins, ochratoxin A, fumonisins, zearalenone, the trichothecenes and more — in a single run, with high sensitivity and unambiguous confirmation by mass. For complex botanical matrices and multi-toxin specifications, LC-MS/MS is the method that does the most in one pass.

ELISA screening

Enzyme-linked immunosorbent assay (ELISA) is a rapid, lower-cost immunoassay used for screening rather than definitive quantification. It is well suited to high-throughput incoming-goods checks and field triage: fast, simple, and good at flagging suspect lots. Its limitations are cross-reactivity and matrix interference, so a positive or borderline ELISA result should be confirmed by HPLC-FLD or LC-MS/MS before any lot is accepted or rejected on the number alone.

Sampling is where results are won or lost

No analytical method, however sensitive, can correct for a poorly drawn sample — and mycotoxin contamination is notoriously uneven. Aflatoxins in particular cluster in “hot spots”: a few heavily contaminated pieces in an otherwise clean lot. Test the wrong handful and a dangerous lot passes, or a sound lot fails. The largest source of error in a mycotoxin result is almost always the sampling, not the laboratory.

• Take many incremental samples from across the whole lot — top, middle and bottom, multiple drums or bags — not a single grab from one container.
• Combine them into a large aggregate sample, then homogenise and sub-divide down to the test portion using a representative method.
• Follow a recognised sampling plan (EU and Codex specify how many increments and what aggregate mass for mycotoxins) rather than an ad-hoc grab.
• Document the sampling so the result is traceable to a defined, representative draw — an undocumented sample undermines an otherwise valid test.

How to specify and what to demand on a COA

A mycotoxin line on a certificate of analysis (COA) is only as meaningful as the specification behind it and the sampling and method in front of it. Vague entries such as “aflatoxins: pass” or “mycotoxins: complies” tell you nothing actionable. Demand specifics, and write them into your purchase specification before the lot is made.

1. 1Name the toxins and the limits — aflatoxin B1 and total aflatoxins (B1+B2+G1+G2), and ochratoxin A where relevant, each with a numeric limit in ppb tied to your destination market.
2. 2State the form tested — confirm the result is for the finished extract, not only the raw material, since toxins can concentrate during extraction.
3. 3Require the method — HPLC-FLD or LC-MS/MS for the reported result; treat ELISA as a screen to be confirmed, not as the COA value.
4. 4Require numeric results with limits of quantification — actual figures (for example “< 1 ppb”) against the spec limit, not a bare “pass”.
5. 5Confirm representative sampling — a documented, recognised sampling plan behind the tested portion.
6. 6Ask for the testing laboratory and accreditation — an accredited (for example ISO/IEC 17025) lab and the report reference.
7. 7Tie testing to the lot — the result must correspond to the specific lot you are receiving, with manufacture date and lot number, not a generic typical certificate.