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Detection of malachite green and crystal violet residues in fish

Application Introduction

Malachite green, green triphenylmethane dyes and toxic triphenylmethane chemicals, can be used as drugs to kill fungi, bacteria and parasites.

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Introduction

Malachite green, green triphenylmethane dyes and toxic triphenylmethane chemicals, can be used as drugs to kill fungi, bacteria and parasites. China’s government has forbidden the use of malachite green in pollution-free aquaculture due to its carcinogenicity in long-term overuse.

In this paper, the simultaneous detection of malachite green, leuco malachite green, crystal violet and leuco crystal violet in fish was established.

Experimental

Material and Reagents

HPLC purity of acetonitrile; experimental water (ultra-pure water); analytical pure ammonium acetate;

Standard solution mix: solution of malachite green, leuco malachite green, crystal violet, leuco crystal violet in acetonitrile;

Internal standard solution: dissolve 2 ng/mL deuterated malachite green and deuterated leuco malachite green in acetonitrile: 5 mmol/L ammonium acetate solution (1:1, V/V);

Cleanert Alumina N, 1000 mg/3 mL.

Sample Extraction

Transfer 5 g homogenized fish sample, 200 μL 2 ng/mL internal standard solution and 11 mL acetonitrile into a 50 mL centrifuge tube. Then sonicate for 2 min, homogenize at 8000 rpm for 30 s and centrifuge at 4000 rpm for 5 min. Put all the supernatant into a 25 mL colorimetric tube. Wash the homogenate knife head with 11 mL acetonitrile in another centrifuge tube for 10 s and transfer the liquid to the centrifuge tube of sample residue. Pound the residue with glass rod, vortex for 30 s, sonicate for 5 min, then centrifuge at 4000 rpm for 5 min. Blend the two supernatant, reconstitute into 1 mL of acetonitrile and shake for sample preparation.

Sample Preparation

Activate Cleanert Alumina-N with 5 mL acetonitrile. Load 5 mL of extraction onto the cartridge, collect the eluent into a KD concentrate bottle, elute the cartridge with 4 mL acetonitrile, and then collect the eluent into the same KD concentrate bottle. Rotary evaporate under 45°C to less than 1 mL, reconstitute into 1 mL of acetonitrile and sonicate for 5 min. Then add into 1 mL 5 mmol/L ammonium acetate, sonicate for 1 min and filter residue through 0.22 μm nylon filter.

Above steps were operated on Qdaura® Automated SPE Workstation.

Instrumentation

Column: Unisol C18, 3 µm, 100 Å, 3.0 × 50 mm;

Mobile phase: A: 0.1% formic acid; B: Acetonitrile;

Flow rate: 0.3 mL/min;

Column temperature: 30 °C;

Injection: 5 mL;

Ion source: ESI+;

Electrospray voltage:5500 V;

Nebulizer pressure: 50 psi;

Curtain gas pressure: 25 psi;

Auxiliary gas pressure: 50 psi;

Ion source temperature: 500 °C;

Scan mode: MRM.

Table 1 Gradient

Time (min)

A%

B%

0.0

60

40

1.0

60

40

2.0

10

90

6.0

10

90

6.1

60

40

10.0

60

40


Table 2 MS Parameters

Analytes

Q1

Q3

DP/V

CE/V

Malachite green

329.1

313.2

95.1

49.7

208.2

95.1

46.9

Leuco malachite green

331.1

239.3

105.1

42.0

316.3

105.1

30.5

Crystal violet

372.4

251.4

112.1

53.4

358.4

112.1

47.3

Leuco crystal violet

374.2

239.2

114.8

40.2

318.2

114.8

49.1

D5- Malachite green

334.0

318.2

97.0

53.1

213.3

97.0

48.9

D6- Leuco malachite green

337.4

240.2

107.4

40.8

322.4

107.4

31.5

* underlined were quantitative ions

Results and Discussion

Table 3 shows that the recoveries of spiked samples ranged from 90% to 115% and the CVs were under 10% using Cleanert Alumina-N by LC-MS/MS to determinate malachite green and crystal violet in fish. Typical method performance results were well within acceptable criteria. Figure 1 shows a good separation was obtained by using Unisol C18 column to detect malachite green and crystal violet simultaneously.

Table 3 Recoveries of malachite green and crystal in spike samples (2.5 µg/kg)

Analytes

Retention Time/min

Recoveries/%

CV/%

Malachite green

1.65

90.3

4.4

Leuco malachite green

4.17

98.1

1.8

Crystal violet

3.87

113.7

2.7

Leuco crystal violet

1.46

97.0

1.5

Figure 1 Chromatogram of 0.01 µg/mL standard sample mix

Conclusion

In this study, the sample preparation methods of malachite green and crystal violet in fish were established, and the samples were detected by LC-MS / MS. The result shows that the overall recoveries ranged from 90% to 115% and the CVs were lower than 10% for 2.5 mg/kg spiking fish samples, which meets the testing requirements. This study indicates that the method was suitable for detection of malachite green and crystal violet in fish.

Ordering Information

Products

Specification

Cat.No

Cleanert® Alumina-N

1000 mg/3 mL

AL0003-N

Qdaura® Automated SPE Workstation

4 channels, 24 positions

SPE-40

Unisol C18

3 µm, 100 Å, 3.0 × 50 mm

VA930503-0

Nitrogen evaporator

Maximal 15 samples

NV15-G

1.5 mL vials

Screw neck vials,   12 × 32 mm

AV1001-6

Caps and Septa

Screw neck cap,   center hole; red silicone/ white PTEE septa, slitted

AV2200-0

Syringe   Filter (Nylon)

Monofilm,   13 mm, 0.22 μm

AS021320

Disposable   Needle-Free injection systems

2 mL, 100/pk

LZSQ-2ML

 

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