Cyclopiazonic acid HPLC detection scheme for rice wine

First, the background introduction

Yellow wine is China's national specialty, also known as rice wine, which plays an important role in the world's three major wines (yellow wine, wine and beer). The winemaking technology is unique and has become a typical representative and model of the Eastern brewing industry. In recent years, studies have found that rice wine will inevitably produce arc azo acid during the fermentation process, which requires the detection of arc azo acid.

Cyclopiazonic acid (CPA) is a metabolite of Penicillium genus of the genus Aspergillus and Penicillium, aliased with cyclopiazonic acid. CPA is a kind of nerve poison, which can cause liver, kidney and gastrointestinal damage, weight loss, weakness, loss of appetite, diarrhea, dehydration, depression, angulation, convulsions, etc., can affect the absorption of calcium, reduce The quality of the eggshell affects the hatching of the breeder and can be left in the meat and egg milk. It has been reported that a large number of penicillium bacteria have been isolated from food and feed, and dozens of mycotoxins have been identified, the most of which is CPA. It is widely found in food crops such as peanuts and soybeans, often with aflatoxins. Therefore, CPA poisoning is often masked by aflatoxin poisoning.

At present, the detection methods of cyclopiazonic acid include thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and the like. Among them, Zambonin, Li Yepeng and others used 50μm CW/TPR as the extraction fiber, and analyzed the determination of arc azo acid in cheese by HPLC-UV/DAD. The detection limit of this method was 7ng/mL. However, the relevant national standards for the detection of arc azolic acid have not yet been formulated, and there is no solution for the detection of arc azo acid in rice wine. For this reason, Puruibang has introduced a multifunctional function for purifying concentrated arc azo acid. Purify the column. The rapid purification column is a kind of solid phase affinity column which utilizes multi-functional adsorption packing to adsorb and remove interference peaks in mycotoxin detection, and is suitable for HPLC liquid phase detection of arcs in cereals, beans, peanut nuts and yellow wine. The sample of nitrogen acid is purified, the purification step is simple and fast, and the recovery rate is high.

Second, multi-functional purification column - high performance liquid chromatography

1. Required equipment and supplies

High Performance Liquid Chromatograph and Column: Mycotoxin-specific column (PRC-18, Pribolab).

Multi-functional purification column: PriboFast ^® 230 cyclopiazonic acid multi-purpose purification column (M2300, 5ml, 25 / box, Pribolab).

High-speed homogenizer: up to 22000rpm (EQ-WR-1L, Pribolab).

Glass fiber filter paper: PriboFast ^® glass fiber filter paper (GMF-110, Pribolab)

Standard: Arc azo acid / cyclopiazonic acid (STD #8022, Pribolab)

2 . Sample preparation

2.1. Measure 25 ml of rice wine and place in a 250 ml homogenizer or a 250 ml Erlenmeyer flask. 70 ml of methanol and 10 ml of 6% sodium hydrogencarbonate water were added in that order. Stir at high speed for 2 minutes (homogenizer) or shake on a shaker for 1 hour.

2.2. The extract was passed through Whatman No. 4 filter paper or glass fiber filter paper, and then 50 ml of the filtrate was pipetted into a 250 ml separatory funnel. Add 100 ml of hexane (degreased extract) and mix gently to avoid emulsification.

2.3. After separating the two layers, carefully collect another liquid from the aqueous layer by adding a 50 ml solution of 10% potassium chloride. Discard the n-hexane. After acidification with 2 ml of 6N hydrochloric acid, 50 ml of chloroform was added, and the mixture was gently mixed, and the lower organic layer was collected in a 250 ml Erlenmeyer flask.

2.4. The 50 ml of chloroform added was extracted by a repeated method, and the chloroform extract was combined twice in a conical flask. 50 g of anhydrous sodium sulfate (sodium sulfate) was added and allowed to stand for 1 hour.

2.5. The extract was filtered and collected into a 200 ml rotary evaporation flask and evaporated to dryness at 40 °C.

3. Multi-functional purification column purification

3.1. Purification column activation: The PriboFast 230 cyclopiazonic acid multi-purpose purification column was passed through a column of 15 ml of chloroform to control the maximum flow rate of 3 ml/min. Do not allow the column to dry.

3.2. Enrichment: The dried extract in the rotary evaporator bottle was dissolved in 10 ml of chloroform and passed through the column at a maximum flow rate of 2 ml/min.

3.3 Washing: Do not let the column dry up. After the entire solution has passed through the column, the column is washed. After washing the column with 10 ml of chloroform:acetone (1 + 1) and 10 ml of chloroform:methanol (95 + 5), washing was continued with 10 ml of diethyl ether. Discard the washing solution.

3.4. Elution of CPA: elute with 10 ml of chloroform:methanol (75:25), control the maximum flow rate to 2 ml/min, and collect the eluate using a suitable container (for example, 20 ml conical tube, preferably Silicon alkylate).

3.5, blow dry: nitrogen or dry evaporate in a vacuum bath at 40 ° C.

3.6. Reconstitution: Dissolve the residue with 1 ml of mobile phase. The specimen was lysed by vortex mixing for 30 seconds and ultrasonic.

4. Preparation of standard solution and standard addition solution

4.1, CPA storage solution: 100 μg / ml dissolved in methanol.

4.2. Working standard: Pipette 100 μL of 100 μg/ml CPA standard solution (10 μg CPA) into an amber automatic sampling bottle and dissolve in 0.9 ml of mobile phase. The corresponding concentration is 10 μg/ml.

5. HPLC conditions