Method for Detecting Malachite Green in Feed by HPLC

Malachite green, also known as aniline green, is a green crystal with a metallic luster. The chemical functional group triphenyl methane (CH4) of malachite green is a carcinogen, and it is easy to remain in fish and the environment, and there are risks of mutagenesis, teratogenicity, and carcinogenicity. Therefore, many countries such as China, the United States, Canada, and the European Union prohibit its use in economic fish (except ornamental fish) and human food fish. Fish meal is an important source of animal protein in feed, and fish meal containing malachite green and its metabolites can threaten human health through the food chain when added to feed.

According to NY/T 1756-2009 standard, high performance liquid chromatography (HPLC) was used to determine malachite green in feed. This standard is applicable to the determination of malachite green and colorless malachite green content in compound feed, concentrated feed, additive premixed feed, and fishmeal. The limits of quantification of malachite green and colorless malachite green by liquid chromatography were both 10 μg/kg; the limits of quantification of liquid chromatography-tandem mass spectrometry were both 1.0 μg/kg.

The method is briefly introduced as follows:


The malachite green and colorless malachite green in the sample were extracted with acetonitrile-sodium acetate buffer solution, the extract was subjected to liquid-liquid extraction with dichloromethane, and then purified by a solid-phase extraction column, and a cyano column connected to a lead dioxide column was used to Separation by high-performance liquid chromatography, detection by an ultraviolet-visible light detector, and quantification by external standard method.

Analysis steps


Weigh about 5g of the feed sample (accurate to 0.001g), put it in a 100ml centrifuge tube, add 1.5ml of hydroxylamine hydrochloride solution, 2ml of the p-toluenesulfonic acid solution, 5ml of sodium acetate buffer solution and 20ml of acetonitrile, add about 2g of acidic oxidation Aluminum, shake for 2 minutes, sonicate for 30 minutes, and centrifuge at 4 000 r/min for 5 minutes. Transfer the supernatant to a 250 ml separatory funnel, add 20 ml of acetonitrile to the centrifuge tube again, shake for 2 min, centrifuge at 4000 r/min for 5 min, and combine the supernatant into the separatory funnel.

Liquid-liquid extraction

Add 50ml of sodium chloride solution and 50ml of dichloromethane, swirl, and let stand to separate layers. After collecting the lower layer liquid with an evaporating flask, add 20ml of dichloromethane again to the separatory funnel, shake it, let stand to separate layers, and collect the lower layer liquid in the same evaporating flask. Rotate the collected solution under reduced pressure at 35°C (note: do not raise the temperature directly to 35°C at the beginning to avoid bumping) until nearly dry, add 5 ml of acetonitrile to dissolve the residue, and set aside.


Activation of SPE cartridges

Connect the two columns in series in the order that the neutral alumina column is on the top and the PRS column is on the bottom, and pre-wash the two columns with 5ml of acetonitrile before use.

Sample loading

Slowly transfer the sample solution in 2.3.1 to the neutral alumina column, and pass through the column under a vacuum (keep the flow rate at 1 ml/min). Wash the evaporating flask twice with 5 ml of acetonitrile, transfer all the washings to the column, and finally wash the small column with 5 ml of acetonitrile.

Elution collection

Discard the neutral alumina column, eluted with 2 ml of acetonitrile-sodium acetate buffer solution and 1 ml of hydroxylamine hydrochloride methanol solution, collect the eluate, pass through a membrane (pore size 0.45 μm), and measure on the machine.

Sample determination

Chromatographic conditions

Chromatographic column: cyano column, 250 mm × 4.6 mm, post-column lead oxide column: 35 mm × 4.6 mm. Mobile phase: acetonitrile + sodium acetate buffer solution = 60 + 40 (v + v);
Flow rate: 1.0ml/min.
Column temperature: room temperature.
Injection volume: 50 μl.
Detection wavelength: 600 nm.

Quantitative determination

Adjust the operating parameters of the instrument according to the instructions of high-performance liquid chromatography. Inject malachite green and colorless malachite green standard working solutions and sample solutions into the liquid chromatograph to obtain the response value of the chromatographic peak area, which is quantified by the external standard method.