Determination of Aflatoxin B₁, a Group 1 Carcinogen, in Oil-Bearing Crops by the Wayeal LCMS-TQ9200 LC-MS/MS System

Aflatoxin B₁ is a highly toxic secondary metabolite produced primarily by Aspergillus flavus and Aspergillus parasiticus. Among the aflatoxin family, it exhibits the strongest toxicity and the widest distribution. It exerts its toxic effects by inhibiting intracellular DNA and RNA synthesis and interfering with protein metabolism, with a highly targeted damaging effect on the liver. Compared with other mycotoxins such as ochratoxin and patulin, aflatoxin B₁ is extremely toxic and has a well-established carcinogenicity. It has been classified as a Group 1 carcinogen by the World Health Organization (WHO). It is frequently detected in oil-bearing crops such as peanuts, corn, nuts, and vegetable oils, making it a priority contaminant for food safety control worldwide.

Contamination with aflatoxin B₁ typically occurs when oil-bearing crops are exposed to high temperature and high humidity conditions during planting, harvesting, or storage, which promote fungal growth. Moreover, due to its stable chemical properties, aflatoxin B₁ cannot be destroyed by conventional cooking temperatures, leading to recurring residue issues in agricultural products and processed foods.

Short-term ingestion of a high dose can cause acute poisoning, presenting with symptoms such as severe abdominal pain, vomiting, jaundice, and liver failure, which may be fatal in severe cases. Long-term low-dose exposure leads to accumulation in the liver, inducing chronic liver injury such as hepatic fibrosis and cirrhosis, and may even trigger primary liver cancer. Populations with lower immunity, including children, the elderly, and individuals with pre-existing liver diseases, are at a higher risk of aflatoxin B₁ poisoning.

In China, the national standard GB 2761-2017 (National Food Safety Standard – Maximum Levels of Mycotoxins in Foods) specifies the maximum residue limits for aflatoxin B₁ in various types of food. Additionally, GB 5009.22-2016 (National Food Safety Standard – Determination of Aflatoxins B and G in Foods) provides official analytical methods for the determination of aflatoxin B₁.

This application note describes the establishment of an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of aflatoxin B₁ using the Wayeal LCMS-TQ9200 LC-MS/MS system.

Keywords: Triple quadrupole; Aflatoxin B₁; Isotope dilution liquid chromatography-tandem mass spectrometry.

1. Instrument and Reagents

1.1 Instrument Configuration

Table 1 Instrument Configuration List

1.2 Reagents and Standards

Table 2 Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Vortex mixer

High-speed centrifuge

Analytical balance

Centrifuge

Solid-phase extraction (SPE) manifold (with vacuum pump)

Nitrogen evaporator

Shaker

2. Experiment Method

2.1 Solution Preparation

2.1.1 5 mmol/L Ammonium Acetate Solution: Weigh 0.39g of ammonium acetate, dissolve it in water, and dilute to 1000mL. Mix well.

2.1.1 Methanol-Acetonitrile Solution: Add 100mL of acetonitrile to 100mL of methanol, then mix well.

2.2 Sample Pretreatment

2.2.1 Sample Extraction

Pass the wheat flour through a 2mm aperture test sieve. Weigh 5g of the sample (accurate to 0.01g) into a 50mL centrifuge tube. Add 100μL of isotope internal standard working solution (100ng/mL), mix by vortexing, and allow to stand for 30 min. Add 20mL of methanol-water solution (70+30, v/v), vortex to mix, and shake on an orbital shaker for 20min. Centrifuge at 6000r/min for 10min. Collect the supernatant for subsequent use.

2.2.2 Sample Purification

Accurately transfer 4 mL of the supernatant into a container, add 23 mL of 1% Triton X-100 in PBS, and mix well.

After the original liquid in the immunoaffinity column has drained completely, transfer the above sample solution into a 50 mL syringe barrel. Adjust the flow rate so that the sample solution passes through the column steadily at a rate of 1–3 mL/min. After the sample solution has completely drained, add 2 × 10 mL of water into the syringe barrel and wash the immunoaffinity column at a steady flow rate. After the water has drained, dry the column using a vacuum pump. 

Disconnect the vacuum system. Place a 10 mL graduated test tube under the immunoaffinity column, remove the 50 mL syringe barrel, and add 2 × 1 mL of methanol to elute the column at a controlled flow rate of 1–3 mL/min. Then dry the column again using a vacuum pump. Collect all of the eluate into the test tube. Gently evaporate the eluate to near dryness under a stream of nitrogen at 50 °C. Add 1 mL of the initial mobile phase, vortex for 30 seconds to dissolve the residue. Filter through a 0.22 μm membrane filter, and collect the filtrate into an autosampler vial for subsequent injection.

2.3 Experiment Condition

2.3.1 Liquid Chromatography Method

Column: C18, 1.7 μm, 2.1 × 50 mm

Mobile Phase Phase: A: Methanol-acetonitrile solution; Phase B: 5mmol/L ammonium acetate solution

Flow Rate: 0.3 mL/min

Column Temperature: 40 °C

Injection Volume: 3µL

2.3.2 Mass Spectrometry Method

Table 3 Compound Mass Spectrometry Parameters

Note: The asterisk (*) indicates the quantitative ion.

3. Experiment Result

3.1 Standard Chromatogram

The determination of aflatoxin B₁ and the isotope internal standard 13C17-aflatoxin B₁ was completed within 5 minutes. As illustrated in Figure 1, both analytes showed excellent peak shapes and satisfactory responses, fulfilling the requirements for the experimental analysis.

Fig 1 Chromatograms of Aflatoxin B₁ and the Isotope Internal Standard 13C17​-Aflatoxin B₁

3.2 Linear Range

An appropriate volume of the standard stock solution and mixed isotope internal standard working solution were accurately transferred and diluted with the initial mobile phase to prepare a series of standard working solutions with aflatoxin B₁ concentrations of 50, 20, 10, 5, 2, 1, and 0.5ng/mL. Each solution contained the isotope internal standard at a concentration of 2ng/mL. A calibration curve was constructed using these standards. Over the linear range of 0.5–50ng/mL, and after correction using 13C 17-aflatoxin B₁ as the internal standard, the deviations between the measured and nominal aflatoxin B₁ concentrations were within the maximum permissible deviation. The correlation coefficient (R) was greater than 0.999, indicating excellent linearity.

Fig 2 Standard Curve of Aflatoxin B₁

3.3 Repeatability

Standard solutions at three concentrations (1, 10, and 20 ng/mL) were injected six times consecutively. The results are shown in the table below. The relative standard deviations (RSDs) of the retention times and sample amounts for aflatoxin B₁ at low, medium, and high concentrations were all within 5%, meeting the requirements of the experiment.

Table 4 Repeatability Test for Aflatoxin B₁ at Low, Medium, and High Concentrations

3.4 Spike Recovery

Aflatoxin B₁ standard solution was spiked into the sample to achieve a final concentration of 5ng/mL. The spiked sample was then analyzed by LC-MS/MS. The average result from six consecutive injections was 5.147ng/mL, with an RSD of 1.954%. The calculated recovery rate was 102.94%, which meets the requirements of the experiment.

3.5 Blank Residue

After continuously injecting the 20 ng/mL standard solution, a blank sample was subsequently injected to evaluate carryover. As shown in Figure 3, no carryover was detected in the blank sample.

Fig 3 Blank Chromatogram of the Compound

3.6 Sample Test

Aflatoxin B₁ was not detected in Sample A (Fig 4).

Fig 4 Chromatogram of Aflatoxin B₁ in Sample A

4. Conclusioon

In this study, an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of aflatoxin B₁ was established using the Wayeal LCMS-TQ9200 liquid chromatography-tandem mass spectrometry system. The obtained data demonstrate that all chromatographic peaks exhibit good peak shapes without tailing. The sensitivity meets the experimental requirements, and the correlation coefficient (R) is greater than 0.999, satisfying the linearity criteria. The repeatability at low, medium, and high concentrations is within 5%, and no system carryover is observed after high-concentration sample injection. These results indicate that the method, when equipped with the Wayeal liquid chromatography-tandem mass spectrometry system, fulfills the requirements for routine qualitative and quantitative analysis of target samples.