CN104730157A - Method for detecting residues of malachite green in health products by using UPLC-MS - Google Patents

Abstract

The invention discloses a method for detecting residues of malachite green in health products by using UPLC-MS. The method comprises the following steps: (1) extracting, namely, precisely weighing 5.00g of a health product sample, adding the health product sample into a first centrifugal tube, adding a deuterated malachite green-D5 acetonitrile solution which serves as an interior label standard solution, adding acetonitrile, extracting, centrifuging and adding liquid supernatant into a colorimetric tube; additionally adding acetonitrile into a second centrifugal tube, washing a cutter head, adding washing liquid into the first centrifugal tube, pounding precipitate, carrying out ultrasonic oscillation, centrifuging, combining liquid supernatant into the colorimetric tube and making up to the constant volume by using acetonitrile; (2) purifying, namely taking the solution prepared in the step (1), adding the solution into an activated neutral alumina column, collecting the overall outflow liquid, evaporating, making up to the constant volume of the residue liquid by using acetonitrile, carrying out ultrasonic oscillation, adding an ammonium acetate aqueous solution, carrying out ultrasonic oscillation again and filtering by using a filtration membrane; and (3) detecting, namely detecting filtrate by using UPLC-MS. According to the method, quick and accurate effects are achieved, the reproducibility is high, and the consumption of organic reagents is reduced.

Description

UPLC-MS is adopted to detect the residual method of health products Malachite Green

Technical field

The present invention relates to a kind of UPLC-MS that adopts and detect the residual method of health products Malachite Green

Background technology

Malachite green, as dyestuff, was used as the parasite of improvement fish or fish-egg, fungi or bacteriological infection originally; Chemical functional group triphenylmethane in malachite green can be carcinogenic, and a lot of country forbids, but still has fisherman to use when preventing and treating infection of marine fishes fungi; In May, 2002, malachite green was listed in " veterinary drug of food animal forbidding and compound inventory thereof " by the Ministry of Agriculture, was important food security qualitative items.

The detection of exploitation malachite green and its residues can reduction of expenditure, Improve Efficiency, there is no the report that ultra high efficiency liquid chromatography mass spectrometric combined instrument detects malachite green vestigial method at present.

Summary of the invention

The object of this invention is to provide a kind of favorable reproducibility, accuracy rate is high, and the little analytical cycle of reagent consumption is short, adopts UPLC-MS to detect the residual method of health products Malachite Green.

Technical scheme of the present invention is summarized as follows:

Adopt UPLC-MS to detect the residual method of health products Malachite Green, comprise the steps:

(1) extract: precision takes 5.00g health products sample in 50mL first centrifuge tube, to add 200 μ L concentration be the deuterated malachite green-D5 acetonitrile solution of 1ug/ml is interior mark standard solution, add 9-11mL acetonitrile, supersonic oscillations extract 1.5-3min, 20-40s is extracted in 7500-8500r/min homogenate, the centrifugal 4-6min of 3500-4500r/min, supernatant is transferred in 25mL color comparison tube; Separately get 50mL second centrifuge tube and add 9-11mL acetonitrile, washing homogenate cutter head 10s, cleansing solution moves in the first centrifuge tube, smash the precipitation in the first centrifuge tube to pieces with glass rod, eddy blending machine vibrates 20-40s, supersonic oscillations 4-6min, the centrifugal 4-6min of 3500-4500r/min, supernatant is incorporated in 25mL color comparison tube, is settled to 25mL with acetonitrile, shakes up for subsequent use;

(2) purify: pipette solution that 5.00mL step (1) obtains and add on the neutral alumina column that activated, efflux is received with concentrated bottle graft, with 4mL acetonitrile wash neutral alumina column, collect whole efflux, 40-50 DEG C of rotary evaporation is to the raffinate being less than 1mL, and raffinate acetonitrile is settled to 1mL, sonic oscillation 4-6min, add 1.0mL5mmol/L ammonium acetate solution, sonic oscillation 1min, through 0.2 μm of membrane filtration;

(3) measure: get the filtrate that step (2) obtains and carry out UPLC-MS mensuration,

Draw malachite green acetonitrile standard solution that 1 μ L concentration is 1ug/ml respectively, acetonitrile standard solution that 1 μ L concentration is the deuterated malachite green-D5 of 1ug/ml, analyze under following chromatographic condition, obtain chromatogram;

UPLC condition is:

A) chromatographic column: AcquityBEHC18 post, 50mm × 2.1mm, granularity 1.8 μm;

B) mobile phase: A-acetonitrile; B-5mmol/L ammonium acetate solution;

Time	Flow velocity (ml/mim)	A%	B%
				Initial	0.3	50	50
0.5	0.3	50	50
				3.5	0.3	85	15
3.6	0.3	98	2
				4.5	0.3	99	1
4.6	0.3	50	50
				5	0.3	50	50

C) column temperature: 35 DEG C;

D) sample size: 1 μ L.

The advantage of the inventive method:

Adopt UPLC-MS to detect malachite green vestigial methods analyst sample, can accomplish to detect fast and accurately residual.While raw material and finished product security implementation are more effectively controlled, shorten analysis time, reduce organic reagent consumption.Favorable reproducibility, accuracy rate is high;

Accompanying drawing explanation

Fig. 1 is deuterated malachite green-D5 chromatogram;

Fig. 2 is malachite green chromatogram.

Embodiment

The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.Below in conjunction with specific embodiment, the invention will be further described.

Embodiment 1

Adopt UPLC-MS to detect the residual method of health products Malachite Green, comprise the steps:

(1) extract: precision takes 5.00g health products sample in 50mL first centrifuge tube, to add 200 μ L concentration be the deuterated malachite green-D5 acetonitrile solution of 1ug/ml is interior mark standard solution, add 10mL acetonitrile, supersonic oscillations extract 2min, 30s is extracted in 8000r/min homogenate, the centrifugal 5min of 4000r/min, supernatant is transferred in 25mL color comparison tube; Separately get 50mL second centrifuge tube and add 10mL acetonitrile, washing homogenate cutter head 10s, cleansing solution moves in the first centrifuge tube, smash the precipitation in the first centrifuge tube to pieces with glass rod, eddy blending machine vibrates 30s, supersonic oscillations 5min, the centrifugal 5min of 4000r/min, supernatant is incorporated in 25mL color comparison tube, is settled to 25mL with acetonitrile, shakes up for subsequent use;

(2) purify: pipette solution that 5.00mL step (1) obtains and add on the neutral alumina column that activated, efflux is received with concentrated bottle graft, with 4mL acetonitrile wash neutral alumina column, collect whole efflux, 45 DEG C of rotary evaporations are to the raffinate being less than 1mL, and raffinate acetonitrile is settled to 1mL, sonic oscillation 5min, add 1.0mL5mmol/L ammonium acetate solution, sonic oscillation 1min, through 0.2 μm of membrane filtration;

(3) measure: get the filtrate that step (2) obtains and carry out UPLC-MS mensuration,

Draw malachite green acetonitrile standard solution that 1 μ L concentration is 1ug/ml respectively, acetonitrile standard solution that 1 μ L concentration is the deuterated malachite green-D5 of 1ug/ml, analyze under following chromatographic condition, obtain chromatogram 1 and Fig. 2;

UPLC condition is:

A) chromatographic column: AcquityBEHC18 post, 50mm × 2.1mm, granularity 1.8 μm;

B) mobile phase: A-acetonitrile; B-5mmol/L ammonium acetate solution;

Table 1:

Time	Flow velocity (ml/mim)	A%	B%
				Initial	0.3	50	50
0.5	0.3	50	50
				3.5	0.3	85	15
3.6	0.3	98	2
				4.5	0.3	99	1
4.6	0.3	50	50
				5	0.3	50	50

C) column temperature: 35 DEG C;

D) sample size: 1 μ L.

Mass Spectrometry Conditions

According to test substance mass number characteristic sum with reference to ion pair, tuning instrument condition obtains the mass number information of two kinds of materials as table 2:

Table 2

Embodiment 2

Adopt UPLC-MS to detect the residual method of health products Malachite Green, comprise the steps:

(1) extract: precision takes 5.00g health products sample in 50mL first centrifuge tube, to add 200 μ L concentration be the deuterated malachite green-D5 acetonitrile solution of 1ug/ml is interior mark standard solution, add 9mL acetonitrile, supersonic oscillations extract 1.5min, 40s is extracted in 7500r/min homogenate, the centrifugal 6min of 3500r/min, supernatant is transferred in 25mL color comparison tube; Separately get 50mL second centrifuge tube and add 9mL acetonitrile, washing homogenate cutter head 10s, cleansing solution moves in the first centrifuge tube, smash the precipitation in the first centrifuge tube to pieces with glass rod, eddy blending machine vibrates 20s, supersonic oscillations 4min, the centrifugal 6min of 3500r/min, supernatant is incorporated in 25mL color comparison tube, is settled to 25mL with acetonitrile, shakes up for subsequent use;

(2) purify: pipette solution that 5.00mL step (1) obtains and add on the neutral alumina column that activated, efflux is received with concentrated bottle graft, with 4mL acetonitrile wash neutral alumina column, collect whole efflux, 40 DEG C of rotary evaporations are to the raffinate being less than 1mL, and raffinate acetonitrile is settled to 1mL, sonic oscillation 4min, add 1.0mL5mmol/L ammonium acetate solution, sonic oscillation 1min, through 0.2 μm of membrane filtration;

(3) measure: get the filtrate that step (2) obtains and carry out UPLC-MS mensuration;

Draw malachite green acetonitrile standard solution that 1 μ L concentration is 1ug/ml respectively, acetonitrile standard solution that 1 μ L concentration is the deuterated malachite green-D5 of 1ug/ml, analyze under following chromatographic condition;

UPLC condition is with embodiment 1, and Mass Spectrometry Conditions is with embodiment 1.

Embodiment 3

Adopt UPLC-MS to detect the residual method of health products Malachite Green, comprise the steps:

(1) extract: precision takes 5.00g health products sample in 50mL first centrifuge tube, to add 200 μ L concentration be the deuterated malachite green-D5 acetonitrile solution of 1ug/ml is interior mark standard solution, add 11mL acetonitrile, supersonic oscillations extract 3min, 20s is extracted in 8500r/min homogenate, the centrifugal 4min of 4500r/min, supernatant is transferred in 25mL color comparison tube; Separately get 50mL second centrifuge tube and add 11mL acetonitrile, washing homogenate cutter head 10s, cleansing solution moves in the first centrifuge tube, smash the precipitation in the first centrifuge tube to pieces with glass rod, eddy blending machine vibrates 40s, supersonic oscillations 6min, the centrifugal 4min of 4500r/min, supernatant is incorporated in 25mL color comparison tube, is settled to 25mL with acetonitrile, shakes up for subsequent use;

(2) purify: pipette solution that 5.00mL step (1) obtains and add on the neutral alumina column that activated, efflux is received with concentrated bottle graft, with 4mL acetonitrile wash neutral alumina column, collect whole efflux, 50 DEG C of rotary evaporations are to the raffinate being less than 1mL, and raffinate acetonitrile is settled to 1mL, sonic oscillation 6min, add 1.0mL5mmol/L ammonium acetate solution, sonic oscillation 1min, through 0.2 μm of membrane filtration;

(3) measure: get the filtrate that step (2) obtains and carry out UPLC-MS mensuration;

Draw malachite green acetonitrile standard solution that 1 μ L concentration is 1ug/ml respectively, deuterated malachite green that 1 μ L concentration is 1ug/ml

The acetonitrile standard solution of-D5, analyzes under following chromatographic condition;

UPLC condition is with embodiment 1, and Mass Spectrometry Conditions is with embodiment 1.

In order to verify accuracy of the present invention, we adopt certain product 3 lot numbers not detecting malachite green vestigial that market is sold, and add malachite green, and measure sample by method of the present invention and the conventional HPLC method adopted, its data are in table 3.

Table 3:

Empirical tests, method of the present invention fast, accurate, organic reagent consumption is few, and accuracy rate is high.

Claims (1)

1. adopt UPLC-MS to detect the residual method of health products Malachite Green, it is characterized in that comprising the steps:

(1) extract: precision takes 5.00g health products sample in 50mL first centrifuge tube, to add 200 μ L concentration be the deuterated malachite green-D5 acetonitrile solution of 1ug/ml is interior mark standard solution, add 9-11mL acetonitrile, supersonic oscillations extract 1.5-3min, 20-40s is extracted in 7500-8500r/min homogenate, the centrifugal 4-6min of 3500-4500r/min, supernatant is transferred in 25mL color comparison tube; Separately get 50mL second centrifuge tube and add 9-11mL acetonitrile, washing homogenate cutter head 10s, cleansing solution moves in the first centrifuge tube, smash the precipitation in the first centrifuge tube to pieces with glass rod, eddy blending machine vibrates 20-40s, supersonic oscillations 4-6min, the centrifugal 4-6min of 3500-4500r/min, supernatant is incorporated in 25mL color comparison tube, is settled to 25mL with acetonitrile, shakes up for subsequent use;

(2) purify: pipette solution that 5.00mL step (1) obtains and add on the neutral alumina column that activated, efflux is received with concentrated bottle graft, with 4mL acetonitrile wash neutral alumina column, collect whole efflux, 40-50 DEG C of rotary evaporation is to the raffinate being less than 1mL, and raffinate acetonitrile is settled to 1mL, sonic oscillation 4-6min, add 1.0mL5mmol/L ammonium acetate solution, sonic oscillation 1min, through 0.2 μm of membrane filtration;

(3) measure: get the filtrate that step (2) obtains and carry out UPLC-MS mensuration,

Draw malachite green acetonitrile standard solution that 1 μ L concentration is 1ug/ml respectively, acetonitrile standard solution that 1 μ L concentration is the deuterated malachite green-D5 of 1ug/ml, analyze under following chromatographic condition, obtain chromatogram;

UPLC condition is:

A) chromatographic column: AcquityBEHC18 post, 50mm × 2.1mm, granularity 1.8 μm;

B) mobile phase: A-acetonitrile; B-5mmol/L ammonium acetate solution;

Time Flow velocity (ml/mim) A% B% Initial 0.3 50 50 0.5 0.3 50 50 3.5 0.3 85 15 3.6 0.3 98 2 4.5 0.3 99 1 4.6 0.3 50 50 5 0.3 50 50

C) column temperature: 35 DEG C;

D) sample size: 1 μ L.