The Principle of HPLC Detection of Malachite Green in Feed

Malachite green, also known as aniline green, Victoria green or China green, is a green crystal with metallic luster. Malachite green chemical functional group triphenylbenzene gas (CH4) is a carcinogen, and it is easy to remain in fish and in the situation, there is the risk of mutagenic, teratogenic and carcinogenic, so China, United States, Canada Many countries, such as Canada and the European Union, have stopped using them in economic fish (except ornamental fish) and human food fish. Fishmeal is an important source of animal egg whitening. The fishmeal containing malachite green and its metabolites can be added to the feed to pass through the food chain to human health.
Hawach uses the NY/T 1756-2009 standard to measure the malachite green in feed by Hawach high performance liquid chromatography (HPLC). This standard is applicable to compound feed, concentrated feed, additive premix feed, malachite in fish meal. Determination of green and colorless malachite green content. The limit of quantitation of liquid chromatography for malachite green and colorless malachite green was 10 μg/kg; the limit of quantitation for liquid chromatography-tandem mass spectrometry was 1.0 μg/kg. The method is briefly introduced as follows:
1. Principle: The malachite green and colorless malachite green in the sample were extracted with acetonitrile-sodium acetate buffer solution. The extract was extracted by dichloromethane liquid, and then purified by solid phase extraction column, and connected with lead dioxide column. The cyano column was separated by high performance liquid chromatography, detected by ultraviolet visible light detector, and quantified by external standard method.
2. Analysis steps
(1)Extract about 5g (accurate to 0.001g) of the weighed feed sample, place it in a 100ml centrifuge tube, add 1.5ml hydroxylamine hydrochloride solution, 2ml p-toluenesulfonic acid solution, 5ml sodium acetate buffer solution and 20ml acetonitrile, add about 2g. Acidic alumina, shaken for 2 min, sonicated for 30 min, centrifuged at 4 000 r/min for 5 min. The supernatant was transferred to a 250 ml separatory funnel, and 20 ml of acetonitrile was again added to the centrifuge tube, shaken for 2 min, centrifuged at 4000 r/min for 5 min, and the supernatant was combined into a separatory funnel.
(2)Liquid-liquid extraction Add 50 ml of sodium chloride solution, 50 ml of dichloromethane, shake and let stand. After collecting the lower liquid with an evaporation flask, 20 ml of dichloromethane was again added to the separatory funnel, and the mixture was shaken, allowed to stand for separation, and the lower liquid was collected in the same evaporation flask. The collected liquid was rotary evaporated under reduced pressure at 35 ° C (note: the temperature should not be directly increased by 35 ° C at the beginning to avoid boiling) to near dryness, and 5 ml of acetonitrile was added to dissolve the residue for use.
2.3 Purification
(1)Activation of the solid phase extraction column The two columns were connected in series with the neutral alumina column on top and the PRS column in the lower order. The two columns were pre-washed with 5 ml of acetonitrile before use.
(2)Loading The sample of 2.3.1 was slowly transferred to a neutral alumina column and the column was passed under vacuum (maintaining a flow rate of 1 ml/min). The evaporation flask was washed twice with 5 ml of acetonitrile, and the washings were transferred to a column, and the column was washed with 5 ml of acetonitrile.
(3)Elution Collection The neutral alumina column was discarded and eluted with 2 ml of acetonitrile-sodium acetate buffer solution and 1 ml of hydroxylamine hydrochloride solution. The eluate was collected and passed through a membrane (pore size 0.45 μm).
2.4 Sample determination
(1)Chromatographic conditions
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: 1.0 ml/min.
Column temperature: room temperature.
Injection volume: 50 μl.
Detection wavelength: 600 nm.
(2)Quantitative determination Adjust the operating parameters of the instrument according to the instructions of the high performance liquid chromatograph. The standard working solution and sample solution of malachite green and colorless malachite green were injected into the liquid chromatograph to obtain the peak area response value, and the external standard method was used for quantification.