Aflatoxins Explained: Trigger Conditions, B/G/M Classification, and Reliable Quantitation (Isotope Internal Standards / Reference Standards / Matrix Reference Materials and QC ｜Tables 1–3)

1.Real-world concern: Why are aflatoxins repeatedly flagged?

In “dry goods” such as peanuts, corn, tree nuts, and spices, a common instinct is: if you see mold, throw it away; if you don’t, it’s safe. The trouble with aflatoxins is precisely that they are not simply “a layer of mold on the surface,” but rather a food-chain risk that can be amplified by high temperature, high humidity, and storage conditions.

The World Health Organization (WHO) clearly notes that mold growth can occur on the surface of foods as well as inside foods; moreover, many mycotoxins are chemically stable, and subsequent processing does not necessarily “eliminate” the risk. Therefore, control points should be moved upstream to drying, storage, sorting, and testing.

The link between aflatoxins and health risk is also clear: WHO indicates that high-dose exposure can cause acute poisoning (typically dominated by liver injury), while long-term low-dose exposure is associated with an increased risk of liver cancer.

2.Basic concepts: What exactly are aflatoxins?

2.1 Definition: They are not “mold,” but “toxins” produced by molds

Aflatoxins are a class of mycotoxins—secondary metabolites produced by certain Aspergillus species under suitable conditions. WHO notes that aflatoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, among others.

2.2 Commonly affected commodities

WHO lists high-frequency affected crops including:

(1). Cereals/grains: corn (maize), sorghum, wheat, rice, etc.

(2). Oilseeds/legumes: peanuts, soybeans, sunflower seeds, cottonseed, etc.

(3). Spices and nuts: spices such as chili, black pepper, turmeric, ginger, etc.; tree nuts such as pistachios, almonds, walnuts, coconut, Brazil nuts, etc.

2.3 Why does the “dairy chain” also get involved?

Aflatoxins are a group of mycotoxins (Aflatoxins, AFs). Among them, Aflatoxin B1 (AFB1) is commonly found in moldy grains/oilseeds and their feed materials. When lactating animals (especially dairy cows) consume feed contaminated with AFB1, AFB1 is metabolized in vivo into Aflatoxin M1 (AFM1), which can be excreted in milk—so it may be detected in milk and dairy products.

WHO’s mycotoxin fact sheets explicitly state that when animals ingest contaminated feed, the toxin can also appear in milk in the form of AFM1.

The U.S. FDA’s Compliance Policy Guide likewise explains that AFM1 is a metabolite formed during normal physiological metabolism after animals ingest AFB1. Because this metabolite may occur in dairy products, exposure of dairy cows to AFB1-contaminated feed should be minimized as much as possible.

2.4 Key boundary: Mold present ≠ toxin present; no visible mold ≠ toxin absent

Aflatoxins are chemical substances produced by molds under specific conditions. The presence or absence of visible mold does not directly equate to whether toxins are present:

(1). Visible mold spots/odor suggest increased risk of mold activity, but do not necessarily mean high toxin levels have already formed. Whether toxins are produced depends on species/strain, temperature–humidity conditions, and time.

(2). Conversely, the absence of obvious visible mold does not guarantee “no toxin.” Toxins may form early and persist through later stages; contamination is often heterogeneous, with “localized high” levels and uneven overall distribution.

(3). Risk control should not rely only on “looks good or not.” A more reliable pathway is to move controls upstream: drying and storage management + sorting to remove abnormal kernels/particles + testing data to lock in batch risk. For nuts/peanuts/corn products suspected of mold issues, discarding is the safer option.

3.Classification: What do the letters B, G, and M mean?

Aflatoxins (AFs) are not a single molecule, but a family of structurally related toxins. In food and feed monitoring—and in regulations—the most common and frequently cited members include AFB1, AFB2, AFG1, and AFG2 (often found in high-risk raw materials and products such as grains, nuts, and spices), as well as the key dairy-chain indicator AFM1.

Here, B/G come from the blue/green fluorescence these compounds exhibit under UV light, while M refers to metabolites associated with Milk; 1/2 denote closely related congeners within the same series (typically, “type 1 is more important”).

Aflatoxin family quick reference

Family member	Typical occurrence	Why it matters
AFB1 (Aflatoxin B1)	High-risk raw materials and products such as peanuts/corn/tree nuts/spices; can also enter the feed chain	The most carcinogenic in the family and the most extensively studied; risk assessment and regulation often center on it or list it separately
AFB2, AFG1, AFG2	Often co-occur with AFB1 in similar foods; proportions vary with raw materials and storage/transport conditions	In most regulatory and testing contexts, “total aflatoxins / total AFs” commonly refers to B1 + B2 + G1 + G2 (excluding M1)
AFM1 (Aflatoxin M1)	Milk and dairy products: derived from in vivo metabolism after animals ingest AFB1-contaminated feed	A transfer/exposure indicator for the dairy chain; typically has a separate limit and monitoring requirement

Notes:

1. B: from Blue fluorescence — these aflatoxins show blue fluorescence under UV light.

2. G: from Green (green/yellow-green) fluorescence — these aflatoxins show green (yellow-green) fluorescence under UV light.

3. M: from Milk — AFM1 is a metabolite formed (via metabolic hydroxylation) after animals ingest AFB1; it can be excreted in milk and is therefore monitored as a key indicator in milk and dairy products.

4. Numbers 1 / 2: different congeners within the same series; B2/G2 can generally be considered dihydro derivatives of B1/G1. Risk assessment and regulation tend to focus more on B1 (most evidence, highest toxicity/carcinogenicity). Many regulations also specify both a “B1 single-compound” limit and a “total aflatoxins (B1+B2+G1+G2)” limit.

4.Risks and common misconceptions: acute liver injury, chronic carcinogenicity, and why heating is not reliable

Key point	Core conclusion	Evidence
1. Acute risk (high dose)	High-dose exposure can cause acute aflatoxicosis, which can be life-threatening. The primary target organ is the liver, commonly presenting as acute liver injury and, in severe cases, progressing to liver failure.	WHO notes that high doses of aflatoxins can cause acute poisoning and may be fatal, typically manifested through liver damage.
2. Chronic risk (long-term)	Manage aflatoxins as genotoxic carcinogens: avoid reducing risk to a simple “safe threshold.” A more operational approach is to cap the upper end of exposure in the food chain using regulatory maximum levels/action levels (ML/AL), and to characterize risk using representative exposure assessment (e.g., MOE or cancer risk estimates), with AFB1 treated as the key member for control. In populations with hepatitis B virus (HBV) infection, liver cancer risk is more sensitive, requiring stricter exposure control.	EFSA states that aflatoxins are genotoxic, and AFB1 can cause hepatocellular carcinoma; EFSA applies BMDL and uses the MOE approach for risk characterization, and cites carcinogenic potency values from JECFA (2016) for risk estimation; in some cases, an MOE below 10,000 may raise a health concern. Codex documents also specify that risk characterization follows JECFA 83 (2016) methods, and apply a higher risk factor for cancer risk estimation in HBV-exposed populations.
3. Heating ≠ a solution	Aflatoxins are relatively stable in foods and are difficult to reliably and completely eliminate under typical cooking temperatures. Some processing/sorting steps can reduce levels, but this is not the same as “heating makes it safe.” Therefore, control points should be moved upstream to raw materials and storage (mold prevention, moisture control, removal of abnormal kernels/particles), and batch risk should be locked in using testing data.	WHO notes that most mycotoxins are chemically stable and can survive food processing.

5.Whole-chain aflatoxin risk control quick reference: trigger conditions → critical control points → actionable steps

Stage	When risk is most likely to be amplified (trigger conditions)	Key control point	Specific actions	Why it works
Field / pre-harvest	Drought/heat stress; frequent insect damage/mechanical injury; delayed harvest / high over-maturity fraction	Reduce conditions favoring infection and toxin production	Control insects and reduce mechanical damage; mitigate drought/high-temperature stress where feasible; harvest at optimal maturity and avoid delays	Lowering the “starting risk” makes downstream loss of control less likely
Post-harvest drying	Slow drying; incomplete drying; re-wetting due to rain/humidity rebound	“Dry thoroughly as quickly as possible” + prevent re-wetting	Dry promptly and sufficiently; protect from rain and re-wetting; keep wet lots separated from dry lots	Drying and staying dry suppress mold growth and toxin production
Storage & transport	High humidity/condensation; high temperature; poor ventilation; insects/rodents	Temperature–humidity / ventilation / pest management	Control moisture and prevent condensation; ventilate to reduce dampness; after reaching “safe moisture/water activity,” use hermetic storage to prevent re-wetting; control insects/rodents; minimize breakage and powdering. Example (peanuts, Codex): when water activity aw < 0.7 and moisture < 9%, toxigenic Aspergillus struggles to grow/produce toxins; therefore, dry to this range first, then “seal/hermetic-store.”	Storage is often where risk continues to be “amplified.” First drive moisture below the growth/toxin-production threshold, then use hermetic storage to lock out re-wetting
Processing & sorting	A small number of moldy/insect-damaged/discolored kernels mixed into a large batch	Remove high-risk particles	Manual picking/color sorting; remove broken, moldy, discolored kernels; dehulling/blanching/roasting as supportive “reduction” measures	“Removing contaminated kernels” is usually more reliable than expecting processing to fully eliminate toxins
Dairy chain	Dairy cattle feed contaminated with AFB1	Control AFB1 in feed → control AFM1 in milk	Avoid/remove contaminated feed; monitor AFM1 in milk when necessary	AFM1 arises from in vivo metabolism; the critical control point is the feed
Testing & limits	High-risk raw materials/batches; unknown origin or anomalous batches	Lock risk with data	Screen high-risk lots; manage against local regulatory limits; isolate and dispose/handle non-compliant lots; sample according to standards. Note: aflatoxin contamination is often highly heterogeneous—sampling representativeness is itself a control point. Prioritize “composite sampling + thorough homogenization, then subsampling/aliquoting” to reduce small-sample randomness. (AFM1 example in dairy: FDA states that liquid milk samples may use a “≥10-pound composite sample from ≥10 units/portions,” and bulk lots should be thoroughly mixed before sampling.)	Converts “invisible risk” into a manageable metric. Composite sampling and homogenization reduce the chance that a localized hotspot dominates the result, improving the stability of batch decisions

6.Product navigation table | Locate by research task: aflatoxin reference standards / internal standards / QC materials (Tables 1–3)

Research task / experimental need	Suggested table to check first	Logic for choosing the table
Establish/validate an LC–MS/MS quantitation method for aflatoxins in foods, feed, nuts, spices (need to correct “matrix effects / recovery / drift”)	Table 1	First assemble the isotope internal standard set (¹³C₁₇-B1/B2/G1/G2/M1) so isotope dilution (IDMS) can correct matrix effects and sample-prep losses in a unified way, yielding more stable performance and easier validation.
LC–MS/MS method exists, but results fluctuate / recovery is unstable; want to troubleshoot whether error comes from prep vs instrument	Table 1 → Table 3	Use internal standards to anchor instrument/ionization variability first (Table 1), then use matrix QC materials to verify whole-process repeatability across extraction–cleanup–injection (Table 3), making it easier to localize the problematic step.
Multi-toxin “simultaneous screening/joint assay” for B1/B2/G1/G2/M1/M2; need standards for retention time and ion-pair confirmation	Table 2	Standards/standard solutions establish retention time, MRM transitions, and response factors; multi-analyte assays require high-purity standards and working solutions for identification + quantitation and system suitability.
Prepare external calibration or matrix-matched curves; do spike-recovery; calculate LOD/LOQ (routine analysis/testing)	Table 2	Table 2 includes high-purity standards (≥98% HPLC) and ready-to-use working solutions (acetonitrile), suitable for quickly building/maintaining calibration curves and performing spike recovery while reducing errors from in-house solution preparation.
Detection/method validation for AFM1 in milk and dairy (low levels; high accuracy requirement)	Table 2 → Table 1	Build the calibration and identification first using AFM1 standards/solutions (Table 2), then apply U-¹³C₁₇-M1 as an isotope internal standard (Table 1) for more robust quantitation at low concentrations and in complex matrices.
Routine laboratory QC (QC charts), proficiency testing/method comparisons: need matrix-based reference materials to test the whole workflow	Table 3	Matrix QC materials (wheat/corn) truly cover extraction, cleanup, and matrix effects across the full workflow; they reflect real-sample behavior better than neat standard solutions.
Evaluate how different sample-prep workflows (IAC immunoaffinity columns, QuEChERS, SPE, etc.) affect recovery/cleanup	Table 3 → Table 2	Compare processes first using matrix QC materials (Table 3), then use standards/working solutions for spike-recovery and checks of linearity/accuracy (Table 2), so you control both “real matrix differences” and the “quantitation reference.”
Stability/repeatability assessment near low levels (close to detection limits)	Table 3	Near LOD, signals are weak and noise contributes more; small shifts in matrix effects, injection, or ion-source condition can be amplified into result variability (values jumping up/down, larger bias). Low-level AFM1 QC materials enable long-term monitoring of within-day/between-day precision, system drift, background interference, and carryover, and verify that low-end QC requirements remain consistently met.
Toxicology/mechanism studies on aflatoxins (cell exposure, metabolic activation, DNA adducts), need convenient stock-solution preparation	Table 2	High-concentration AFB1 DMSO solution (10 mM) is better for preparing concentration gradients and reducing weighing errors; it also serves as a reproducible stock source for exposure/mechanistic studies.
Need quantitation results with stronger inter-lab comparability / traceability	Table 1 → Table 2	Isotope internal standards (Table 1) provide a more traceable correction framework; standards/working solutions (Table 2) provide calibration and identification anchors. The combination better matches high-stringency method development and review/audit expectations.

Table 1 | Isotope-labeled Internal Standards (¹³C₁₇) | For IDMS/LC–MS/MS Quantitative Correction

Category	CAS No.	Aladdin Cat. No.	Name	Specification / Purity	Key Features & Applications
Isotope internal standard | ¹³C₁₇ labeled (IDMS/LC–MS/MS)	2707441-97-0	A299684	U-[¹³C₁₇]-Aflatoxin M1	0.5 μg/mL in acetonitrile	Isotope dilution internal standard: used to correct matrix effects, recovery, and instrument drift in LC–MS/MS quantitation of AFM1 (especially in milk/milk powder, etc.); used for method validation and within-/between-batch QC.
Isotope internal standard | ¹³C₁₇ labeled (IDMS/LC–MS/MS)	1217449-45-0	A299679	Aflatoxin B1-¹³C₁₇ (isotope)	0.5 μg/mL in acetonitrile	Isotope internal standard: for isotope-dilution quantitation of AFB1 (grains, nuts, feed, etc.); corrects matrix effects/recovery and improves inter-laboratory comparability.
Isotope internal standard | ¹³C₁₇ labeled (IDMS/LC–MS/MS)	1217470-98-8	A299681	Aflatoxin B2-¹³C₁₇ (isotope)	0.5 μg/mL in acetonitrile	Isotope internal standard: corrects extraction and ionization differences in AFB2 quantitation; suitable for internal-standard panel setup and method validation in multi-toxin assays.
Isotope internal standard | ¹³C₁₇ labeled (IDMS/LC–MS/MS)	1217444-07-9	A299682	Aflatoxin G1-¹³C₁₇ (isotope)	0.5 μg/mL in acetonitrile	Isotope internal standard: for isotope-dilution quantitation of AFG1, improving accuracy and comparability in complex matrices (grains/nuts/spices, etc.).
Isotope internal standard | ¹³C₁₇ labeled (IDMS/LC–MS/MS)	1217462-49-1	A299683	Aflatoxin G2-¹³C₁₇ (isotope)	0.5 μg/mL in acetonitrile	Isotope internal standard: for correction and method validation in AFG2 quantitation; suitable for multi-toxin assays with one-to-one internal standard–analyte matching.

Table 2 | Analytical Reference Standards / Standard Solutions (Neat Standard + Working Solution) | For Calibration Curves, Identification & Quantitation, and System Suitability

Category	CAS No.	Aladdin Cat. No.	Name	Specification / Purity	Key Features & Applications
Analytical reference standard | Natural toxin (≥98% HPLC)	1165-39-5	A139993	Aflatoxin G1	≥98% (HPLC)	High-purity standard: for preparing stock solutions, method development and confirmation; commonly used for full-panel quantitation in multi-mycotoxin monitoring together with B1/B2/G2.
Analytical standard solution | Working solution (acetonitrile, quantitative calibration)	1165-39-5	A298918	Aflatoxin G1 Standard Solution	25 μg/mL in acetonitrile	Ready-to-use working solution: for external-standard/matrix-matched calibration curves, spike recovery, and LOD/LOQ evaluation; suitable for quantitative calibration of G1 in multi-toxin assays.
Analytical reference standard | Natural toxin (≥98% HPLC)	7220-81-7	A140008	Aflatoxin B2	≥98% (HPLC)	High-purity standard: for establishing multi-toxin detection/screening methods; for preparing stock solutions and confirming retention time/ion transitions; used for simultaneous quantitation of the B-toxin profile.
Analytical standard solution | Working solution (acetonitrile, quantitative calibration)	7220-81-7	A298916	Aflatoxin B2 Standard Solution	25 μg/mL in acetonitrile	Ready-to-use working solution: for external-standard/matrix-matched calibration of B2, spike recovery, and linearity evaluation; reduces concentration errors and lot-to-lot variation from in-house solution preparation.
Analytical reference standard | Natural toxin (≥98% HPLC)	7241-98-7	A140002	Aflatoxin G2	≥98% (HPLC)	High-purity standard: for qualitative/quantitative analysis and method validation in simultaneous B/G series assays; used to assess chromatographic separation (isomers/close-eluting components) and spectral features.
Analytical standard solution | Working solution (acetonitrile, quantitative calibration)	7241-98-7	A298917	Aflatoxin G2 Standard Solution	25 μg/mL in acetonitrile	Ready-to-use working solution: for G2 calibration curves, spike recovery, and system suitability testing; convenient for routine quantitation in simultaneous B/G series analyses.
Analytical reference standard | Natural toxin (≥98% HPLC)	6795-23-9	A139552	Aflatoxin M1	≥98% (HPLC)	High-purity standard: for preparing stock solutions and confirming retention time/ion transitions; for establishing and validating LC–MS/MS methods (or, after derivatization/cleanup, chromatographic methods) for M1 in foods/dairy.
Analytical standard solution | Working solution (acetonitrile, quantitative calibration)	6795-23-9	A139553	Aflatoxin M1 Standard Solution	Analytical standard, 0.5 μg/mL in acetonitrile	Low-concentration working solution: for routine M1 calibration curves, system suitability tests, and between-batch drift monitoring; commonly used as a working standard in dairy/liquid-sample methods.
Analytical reference standard | Natural toxin (≥98% HPLC)	6885-57-0	A139558	Aflatoxin M2	≥98% (HPLC)	High-purity standard: for establishing screening methods for metabolites/co-occurring toxins; used for identification/quantitation, spike recovery, and interference assessment in multi-toxin analyses.
Toxicology/mechanism research solution | High-concentration B1 in DMSO	1162-65-8	A1499966	Aflatoxin B1	Moligand™, 10 mM in DMSO	Convenient for toxicology exposure and metabolic activation mechanism studies (e.g., CYP-mediated activation, DNA adduct research); can also serve as a high-concentration stock for spiking and response establishment during method development.
Analytical reference standard | Natural toxin (≥98% HPLC)	1162-65-8	A1511427	Aflatoxin B1	≥98%	High-purity AFB1 standard: for preparing stock solutions, building calibration curves, and confirming system suitability (retention time/ion transitions/response) in foods/feed/grains/nuts/spices; also a key component standard for “total aflatoxins (B1+B2+G1+G2)” multi-analyte assays.

Table 3 | Matrix / QC Materials (QC / Reference Materials) | For Method Validation, Routine Laboratory QC, and Low-Level Monitoring

Category	CAS No.	Aladdin Cat. No.	Name	Specification / Purity	Key Features & Applications
QC material | Matrix reference (wheat)	1165-39-5	A299714	Aflatoxin QC Material, from Wheat	19.15 ppb (±9.27 ppb)	Matrix QC material: for evaluating accuracy and precision of wheat/grain testing methods, spike recovery, and batch-release QC (control charts); suitable for validating overall performance of sample preparation (cleanup/extraction).
QC material | Matrix reference (corn, B1)	1162-65-8	A777915	Aflatoxin QC Material, from Corn	Moligand™, 3.73 ± 0.75 μg/kg	Corn matrix QC (B1): for routine QC and method comparison/validation of B1 in corn and related products; can be used to assess cleanup and recovery performance of immunoaffinity cleanup/SPE in complex matrices.
QC material | Matrix reference (corn)	1165-39-5	A299710	Aflatoxin QC Material, from Corn	130.1 ppb (±27.97 ppb)	Corn matrix QC material: for assessing linear range at higher contamination levels, dilution strategies, and risks of saturation in sample preparation; used for internal laboratory QC and method stability tracking.
QC material | Low-level M1 (ppb)	6795-23-9	A299698	Aflatoxin M1 QC Material	0.5 ppb (±0.02 ppb)	Low-level QC material: for long-term stability and repeatability assessment near the method detection/quantitation range; used to monitor the reliability of the full workflow (extraction–cleanup–instrument analysis) at low concentrations.

For more related articles, please see below:

Stable Isotopes: Adding an “Invisible Barcode” to Molecules to Make Quantification, Tracing, and Source Attribution More Reliable (with Selection Guide and Product Tables 1–3)

Analysis of Titration Solution: Definition, Standards, and Use