CN104807906A - Method for detecting piperazine residue in poultry with high efficiency - Google Patents

Abstract

The invention relates to the field of detection of veterinary drug residues, in particular to a method for efficiently detecting piperazine residues in poultry meat. In the method, after the poultry meat is extracted and purified, it is detected by ultra-high performance liquid chromatography-tandem mass spectrometry. Compared with other detection methods in terms of recovery rate, precision and sensitivity, the present invention has the advantages of simplicity, speed, accuracy and sensitivity.

Description

A method for efficiently detecting piperazine residues in poultry meat

technical field

The invention relates to the field of detection of veterinary drug residues, in particular to a method for efficiently detecting piperazine residues in poultry meat.

Background technique

At present, the detection methods of piperazine residues at home and abroad mainly include gravimetric analysis, colorimetry, non-aqueous titration, gas chromatography, high performance liquid chromatography and high performance liquid chromatography-tandem mass spectrometry. Gravimetric methods, colorimetric methods and non-aqueous titration methods are not suitable for the detection of piperazine in biological samples and have low sensitivity. Piperazine has no luminescent group and cannot be directly detected by ultraviolet detectors and fluorescence detectors. It needs a derivatization reaction to carry a luminescent group. The test steps are cumbersome, and the derivatized products are unstable, resulting in large errors in the obtained data. So far, the application of high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to pig tissue, bovine tissue, egg, although the detection method of piperazine residue in honey has been reported, but Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) studies for the detection of piperazine residues in porcine tissues, bovine tissues, eggs, honey, and poultry meat have not been reported.

Contents of the invention

In order to detect and confirm the residues of piperazine in poultry meat and meet the EU standards, the present invention provides an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which can quickly and efficiently detect the residual piperazine in poultry meat. Residues of piperazine.

The technical solution adopted in the present invention is: a method for efficiently detecting piperazine residues in poultry meat, which is to extract and purify the poultry meat, and detect it by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Using 0.1% formic acid aqueous solution-methanol as the mobile phase, the flow rate is 0.2 mL/min, gradient elution, and the injection volume is 5.0 μL. Electrospray positive ion mode ionization (ESI ⁺ ), multiple reaction monitoring mode scanning (MRM), and external standard method were used for quantitative analysis.

The poultry meat extraction and purification is to use the mixed solution of 5.0% trichloroacetic acid aqueous solution and acetonitrile as the extractant to treat poultry meat samples to obtain an extract, add acetonitrile-saturated n-hexane to the extract to remove fat, and use solid phase extraction method purify. Specifically, the following operations can be adopted: after homogenizing the poultry meat sample, add 5.0% trichloroacetic acid aqueous solution and acetonitrile mixed solution (volume ratio 3:1) to shake, mix and extract directly, centrifuge, and transfer the supernatant. Repeat the extraction 1-2 times, and combine the supernatants to obtain the extract.

After adding acetonitrile-saturated n-hexane to the extract to remove fat, pass through a mixed cationic solid-phase extraction column (Strata-X-C), first activate and balance the SPE column with methanol, ultrapure water, and 2.0% formic acid aqueous solution, and load the sample After the extract was drained at a constant speed, it was rinsed with 2.0% aqueous formic acid, ultrapure water, and methanol in sequence. After the column was dried, it was finally eluted with 10.0% ammoniacal acetonitrile. Finally, the eluent was blown dry with nitrogen, and the residue was dissolved by vortexing with 2.0mL of 20.0% methanol aqueous solution before being used on the machine, and 2.0mL of the complex solution was transferred to a centrifuge tube, centrifuged at room temperature for 10min, and the supernatant was passed through a 0.22μm filter membrane, and the filtrate was For UPLC-MS/MS analysis.

Compared with other detection methods in three aspects of recovery rate, precision and sensitivity, the method has the advantages of simplicity, speed, accuracy and sensitivity.

Description of drawings

Fig. 1 Electrospray primary mass spectrometry scan diagram of piperazine.

Figure 2 Electrospray MS/MS scanning diagram of piperazine.

Figure 3 Matrix-added standard curve for piperazine in chicken muscle.

Figure 4 Total ion chromatogram (TIC) and extracted ion chromatogram (XIC) of blank chicken muscle.

Figure 5 The total ion chromatogram (TIC) and extracted ion chromatogram (XIC) of the blank chicken muscle sample added with 1 μg/kg (LOQ) standard.

Detailed ways

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

The present invention will be described in further detail below in conjunction with specific examples and with reference to data. These examples are only for illustration of the present invention and do not limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The sources and trade names of the reagents used, as well as those whose components must be listed, are indicated when they appear for the first time, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

The solution percentage concentration involved in the present invention is the mass percentage concentration unless otherwise specified.

1. Breeding and Sample Collection of Experimental Poultry

The day-old meat poultry sold to various live poultry farmer’s markets and supermarkets were selected respectively, that is, 16-week-old Jinghai yellow chicken, 10-week-old Yangzhou goose and 8-week-old Cherry Valley duck, 12 each, half male and half female, Raised in single cages, fed full-price feed without any drugs, and had free access to water. After 2 weeks of feeding, the breast muscles on both sides were taken as blank muscle samples when slaughtered, stored at -35°C, and the samples were minced to make them homogeneous before use.

2. Extraction and purification steps of the present invention

1) Homogenize the poultry meat with a pulverizer;

2) Accurately weigh (2.0±0.02)g homogenized blank poultry meat sample and place it in a 50.0mL polypropylene centrifuge tube with a cover;

3) Add 6.0mL of 5.0% trichloroacetic acid aqueous solution and 2.0mL of acetonitrile, vortex and mix for 2min, and extract with water bath ultrasonic oscillation for 10min;

4) Centrifuge at 12000×g, 4°C for 10 minutes, and transfer the supernatant to another 50.0mL centrifuge tube;

5) Repeat the extraction (6.0mL of 5.0% trichloroacetic acid aqueous solution and 2.0mL of acetonitrile to extract the precipitate again, vortex for 2min, ultrasonic oscillation for 10min), centrifuge at 12000×g, 4°C for 10min, and combine the supernatants twice;

6) Add 10.0 mL of n-hexane saturated with acetonitrile to the supernatant, vortex for 3 minutes, centrifuge at 6000×g, 4°C for 5 minutes, discard the upper layer of n-hexane, and repeat degreasing once to finally obtain the extract;

7) Activate the mixed cation solid-phase extraction cartridge (Strata-X-C) with 3.0mL methanol, 3.0mL ultrapure water, and 3.0mL 2.0% formic acid aqueous solution in turn;

8) Add the extract to the activated solid-phase extraction column, and drain at a uniform speed;

9) Rinse with 3.0mL 2.0% formic acid aqueous solution, 3.0mL ultrapure water and 3.0mL methanol in sequence;

10) After the solid phase extraction column is dried, elute with 10.0mL 10.0% ammonia water acetonitrile into a 10mL centrifuge tube;

11) Place the centrifuge tube in a dry bath nitrogen blower, and dry it with nitrogen at 40°C;

12) Take 2.0mL 20.0% methanol aqueous solution and vortex to dissolve the residue, and transfer to a 2.0mL stoppered centrifuge tube;

13) Centrifuge at 12000×g for 10 min at room temperature, filter the supernatant through a 0.22 μm filter membrane, and use the filtrate for UPLC-MS/MS determination.

3 Test conditions

3.1 Liquid chromatography conditions

Column: Waters ACQUITY HSS T3, 1.8μm, 100.0mm×2.1mm id; guard column: ACQUITY BEH C ₁₈ , 1.7μm, 2.1×5mm id; mobile phase: solution A is 0.1% (volume fraction) formic acid aqueous solution, solution B is methanol, gradient elution program is shown in Table 1; flow rate: 0.2mL/min; column temperature: 35.0℃; sample chamber temperature: 4℃; injection volume: 5.0μL; six-way valve: 0～1.3min→mass spectrometer (B position), 1.3～4min→waste liquid (A position), 4～8min→mass spectrometer (B position) bits).

Table 1 Gradient elution program

      

3.2 Mass Spectrometry Conditions

The molecular mass of piperazine is 86.1, and the mass-to-charge ratio of its parent ion should be [M+H] ⁺ (M is the molecular weight) by using the ESI (+) scanning method. According to the precursor ion scan chart and the product ion scan chart (see Figure 1 and Figure 2), the ions corresponding to the two peaks with the highest abundance and no interference are selected as the product ions, and the base peak is used as the quant ion. Finally, optimize the declustering voltage (DP) and collision energy (CE) to achieve the best abundance value.

Other mass spectrometry conditions are as follows, ion source: electrospray ionization source (ESI); ionization mode: positive ion mode; scanning mode: multiple reaction monitoring (MRM); electrospray ionization voltage (IS): 5500V; ion source temperature (TEM) : 550℃; Ion source spray gas (Gas1): 50psi; Auxiliary heating gas (Gas2): 50psi; Curtain gas (CUR): 35psi; Collision gas (CAD): 8psi; Collision cell outlet voltage (CXP): 12V; Input voltage (EP): 10V; dwell time of each quantitative ion pair: 100.0ms, dwell time of each qualitative ion pair: 100.0ms. The molecular weight and mass spectrum parameters of piperazine are shown in Table 2.

Table 2 Molecular weight and mass spectrometry parameters of piperazine

      

Note: ^* quantitative transition

4 Quantitative and qualitative methods

4.1 Drawing of standard curve

Accurately weigh 2.0 g of homogenized blank poultry samples into ten 50.0 mL polypropylene centrifuge tubes with lids. After sample pretreatment, prepare matrix extracts and combine them together as blank matrix extracts.

Dilute the standard stock solution with the blank matrix extract to prepare standard working solutions with different concentration levels, the concentrations are 1.0, 5.0, 10.0, 50.0, 100.0, 200.0ng/mL (corresponding to the concentration of piperazine in the blank muscle sample is respectively 1.0, 5.0, 10.0, 50.0, 100.0, 200.0μg/kg, that is, the linear range is 0.01MRL ~ 2MRL), under the above optimized ultra-high performance liquid chromatography and mass spectrometry conditions, UPLC-MS/MS detection is carried out. The determination of each concentration point was repeated 3 times, and the average value was taken. Take the concentration of the matrix standard working solution as the abscissa (x), and the peak area of the quantitative ion (m/z 87.3>44.1) as the ordinate (y), draw a standard curve, and use this curve as the quantitative curve of the sample to be measured, And find out its regression equation and determination coefficient.

Taking Jinghai yellow chicken muscle as an example, the linear regression equation, coefficient of determination and linear range are shown in Table 3, and the standard curve is shown in Figure 3. If the concentration of the sample to be tested exceeds the linear range, the sample to be analyzed should be diluted, and the obtained result is multiplied by the dilution factor to obtain the original sample concentration.

Table 3 Linear regression equation, coefficient of determination and linear range of piperazine in chicken muscle

      

As can be seen from Table 3 and accompanying drawing 3, under the conditions of the present invention, in standard substance concentration is in the scope of 1.0～200.0 μ g/kg, the peak area (y) of the quantifier ion (m/z 87.3>44.1) of piperazine and its concentration (x) is linearly correlated, and the linear relationship is good. The linearity of piperazine in muscle of Yangzhou goose and cherry valley duck was consistent with it.

4.2 Determination of recovery rate and precision

Accurately weigh 2.0 g of the homogenized blank poultry sample into a 50.0 mL stoppered centrifuge tube, add 100.0 μL each of three different concentrations of standard working solutions of 1.0, 2.0, and 4.0 μg/mL (corresponding to the blank muscle sample added Concentrations are 50.0, 100.0, 200.0 μg/kg (i.e. 1/2MRL, 1MRL and 2MRL), vortexed and mixed and then left to stand for 10min, each concentration level is set to 6 parallels, after extraction and purification methods, in the above optimization Under good liquid chromatography and mass spectrometry conditions (3.1, 3.2), perform UPLC-MS/MS analysis. The total ion chromatogram (TIC) and extracted ion chromatogram (XIC) of blank chicken muscle and blank chicken muscle sample added with 1 μg/kg (LOQ) standard are shown in Figure 4 and Figure 5, respectively.

Intra-day precision measurement: Use the same standard curve and the same instrument to repeatedly measure samples with concentrations of 50.0, 100.0, and 200.0 μg/kg at different times on the same day to obtain the intra-day (intra-batch) precision.

Determination of inter-day precision: Repeat the measurement of samples with the above concentrations 6 times with different standard curves and the same instrument on different days within a week to obtain the inter-day (batch-to-batch) precision.

Substitute the peak area of the quantifier ion (m/z 87.3>44.1) obtained by adding the sample into the standard curve to obtain the concentration, and compare it with the concentration of the actually added analyte to obtain the recovery rate of addition.

Under these conditions, the addition recovery and precision of piperazine in poultry meat extracted by the method of the present invention are shown in Table 4.

Table 4 The recovery and precision of piperazine addition in poultry meat (n=6)

      

      

Note: a. Maximum residue limit

4.3 Determination of detection limit and quantification limit

Six parallel blank poultry samples were taken for the addition recovery test. After extraction and purification, they were analyzed under UPLC-MS/MS conditions, the baseline noise was measured, and the average value was calculated. Calculate the signal-to-noise ratio of each confirmed product ion at the lowest quantitative level of actual confirmation, and the product ion signal-to-noise ratio is greater than or equal to 10 (S/N≥10), and the sample is positive according to the European Commission’s 2002/657/EC resolution Confirmation conditions, the corresponding added concentration was defined as the limit of quantitation (LOQs). The limit of detection (LODs) is the lowest concentration of the sample that is actually added and meets the positive confirmation conditions of the sample, and is determined by the signal-to-noise ratio of the product ion being greater than or equal to 3 (S/N≥3).

According to the addition and recovery test of 6 parallel blank poultry meat samples, the quantification limit of piperazine in chicken muscle, duck muscle and goose muscle is 1.0 μg/kg, and the detection limit is 0.3, 0.4 and 0.3 μg/kg respectively

4.4 Determination of determination limit and detection capacity

Select 20 blank poultry meat samples (2.0g), add 100.0μL 2.0μg/mL piperazine standard working solution, vortex mix, after extraction and purification, carry out UPLC-MS/MS analysis, obtain the standard deviation (SD ). The calculation formulas of CCα and CCβ are CCα=MRLs+1.64×SD (α=5%) and CCβ=CCα+1.64×SD (β=5%), respectively.

According to the recovery test of 20 parallel blank poultry meat samples added with MRL concentration levels, the determination limits (CCα) of piperazine in chicken muscle, duck muscle and goose muscle were 103.0, 104.2 and 102.1 μg/kg, respectively, and the detection capacity (CCβ) were respectively They were 106.0, 108.4 and 104.2 μg/kg. It can fully meet the requirements of drug residue analysis.

Claims (5)

1. A method for efficient detection of piperazine residues in poultry meat, characterized in that, after the poultry meat is extracted and purified, it is detected with ultra-high performance liquid chromatography-tandem mass spectrometry. 2. method according to claim 1, it is characterized in that, the condition of ultra high performance liquid chromatography-tandem mass spectrometry is: with 0.1% formic acid aqueous solution-methanol as mobile phase, flow velocity is 0.2mL/min, gradient elution, carry out The sample volume was 5.0 μL; electrospray positive ion mode was used for ionization, multiple reaction monitoring mode was used for scanning, and external standard method was used for quantitative analysis. 3. The method according to claim 1, wherein the extraction and purification of poultry meat is as follows: treating the poultry meat sample with a mixed solution of 5.0% trichloroacetic acid aqueous solution and acetonitrile as an extractant to obtain an extract, the extract After adding acetonitrile-saturated n-hexane to remove the fat, it was purified by solid phase extraction. 4. The method according to claim 3, characterized in that, the specific steps of extracting and purifying the poultry meat are: after homogenizing the poultry meat sample, add 5.0% trichloroacetic acid aqueous solution and acetonitrile mixed solution for oscillation and mixing Extract directly, centrifuge, and transfer the supernatant; repeat the extraction 1-2 times, and combine the supernatant to obtain the extract; After adding acetonitrile-saturated n-hexane to the extract to remove the fat, pass the mixed cationic solid phase extraction column Strata-X-C, firstly use methanol, ultrapure water, and 2.0% formic acid aqueous solution to activate and balance the SPE column, and wait for After the extract was drained at a constant speed, it was rinsed with 2.0% formic acid aqueous solution, ultrapure water, and methanol in sequence. After the column was dried, it was finally eluted with 10.0% ammoniacal acetonitrile. Finally, the eluent was blown dry with nitrogen, and the residue was dissolved by vortexing with 2.0mL of 20.0% methanol aqueous solution before being used on the machine, and 2.0mL of the complex solution was transferred to a centrifuge tube, centrifuged at room temperature for 10min, and the supernatant was passed through a 0.22μm filter membrane, and the filtrate was For UPLC-MS/MS analysis. 5. The method according to claim 4, characterized in that, the mixed solution of 5.0% trichloroacetic acid aqueous solution and acetonitrile, wherein the volume ratio of 5.0% trichloroacetic acid aqueous solution and acetonitrile is 3:1.