1.Technical Background

Bongkrekic acid (BA) is a kind of mitochondrial toxin produced by Pseudomonas cocovenenans subtype Farino fermentans that can cause food poisoning. Its structure is shown in the figure below. It is a fat-soluble acidic compound, mainly produced in fermented cereal products, spoiled coconut food and fresh white fungus products. It has stronger biological toxicity than cyanide. Ingestion of contaminated food can cause poisoning and even death in severe cases.

Currently, the common methods and the methods specified by the national standard are solid-phase extraction column purification and liquid-liquid extraction purification methods, which still has some problems such as complicated operation, large consumption of organic reagents, high interference, and low recovery rate. Pribolab Technology R&D Center developed a multi-functional purification column "one-step" clean method, which is used with high performance liquid phase chromatography with diode array detection (HPLC-DAD) or liquid chromatography tandem mass spectrometry, for the detection of oryzae residues in food. This new method has simpler pretreatment, faster, and environmentally friendly. It is sensitive with higher recovery and better purification effect, suitable for the determination of rice yeast acid in various food substrates. 

2.experimental method

   2.1 Reagent consumables

Pribolab® 500μg/mL Bongkrekic acid standard substance (acetonitrile)

Pribolab®·ODS·C18 Chromatographic column

PriboFast® MFC 335 cleanup column

PriboFast glass fiber filter paper

Acetonitrile-water:84-16,V/V

   2.2 Sample preparation and extraction

The dry sample is crushed and sieved, and the wet sample is fully homogenized. Weigh 25.0g of the sample into an Erlenmeyer flask, add 100 mL of acetonitrile -water, mix them well, and ultrasound extract for 30 minutes, and finally centrifuge at 5000 r/min for 5 minutes or filter with glass fiber filter paper.

2.3 Purification of the sample

Take 8mL of supernatant and add it to the transparent glass tube, and let the liquid pass through the PriboFast® 335 column to complete the purification. Take the purified solution and filter through membrane. Now it’s ready for liquid phase or liquid quality analysis.

 3. Determination 

4  experimental result 

4.1 Chromatogram

Figure 1 Liquid chromatogram of the Bongkrekic acid

Figure 2 TIC chromatogram of the Bongkrekic acid

   4.2 Result analysis

The structural formula of Mycolic acid contains 3 carboxyl groups (-COOH), which is strong in acidity and is easy to lose protons and become negatively charged. Its retention factor and selectivity are greatly affected by pH. When the mobile phase is acidic, ionization can be avoided and consequently symmetrical chromatographic peaks are obtained. Therefore, the liquid chromatography uses methanol + 1% acetic acid water (80:20,V/V) as the mobile phase, and the separation effect is great. The method detection limit is 0.2μg/g. In the liquid quality test, 0.1% formic acid water-acetonitrile is used in the mobile phase. The same acidic environment inhibits the ionization of orycholic acid and leave it in molecular form. The retention in the reverse stationary phase is enhanced, while the contact with the stationary phase is reduced. The combination of the remaining silanol groups in the silanol which helps to improve the peak shape, and the detection limit of the method is 0.1μg/kg.

In this experiment, samples of food sour soup, rice noodles, and white fungus that are often contaminated by rice yeast acid were selected for the standard addition experiment. the samples were spiked 2 mg/kg Bongkrekic acid, and each addition was made in 6 parallel. The results are shown in the table below.

5.Conclusion 

   In this experiment, a multifunctional purification column "one step "method was established to purify Bongkrekic acid in food. It has the characteristics of simple and efficient pretreatment and high recovery rate. It is suitable for HPLC and Lc/MS/MS method to detect Bongkrekic acid. It is of great significance to the monitoring of food-realted risks and the making of relevant standards.

Procurement guidelines for experimental consumables

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