A method for detecting phenidone content in aquatic products

Abstract

The present invention discloses a method for detecting the content of phenidone in aquatic products, comprising the following steps: (1) dissolving phenidone in methanol to prepare standard solutions of different concentrations; (2) mixing the sample with acetonitrile, grinding and centrifuging to obtain a first supernatant; (3) placing the first supernatant in a flask containing PSA, C 18 and MgSO4 in a QuECherS purification tube, oscillated and centrifuged to obtain a second supernatant; (4) filtering the second supernatant through an organic filter membrane to obtain a sample solution; (5) respectively sending the standard solution and the sample solution to a high performance liquid chromatography tandem mass spectrometer for detection, and then comparing the obtained detection results to perform qualitative and quantitative detection. The present invention has simple pretreatment, accurate quantitative determination, and high sensitivity. The detection limit of phenidone is 5.0 μg / kg, and it can quickly and highly sensitively perform qualitative and quantitative detection of the phenidone content in aquatic products.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product inspection, and particularly relates to a method for detecting the phenidone content in aquatic products. Background Art

[0002] Phenidone is also known as phenidate, pyranil, and pyratol. Its chemical name is 1-phenyl-3-pyrazolidinone. It is a white needle-shaped crystal or crystalline powder with a chemical formula of C9H 10 N2O has a molecular weight of 162.19. Phenidone has strong reducing properties, which can prevent food from oxidative spoilage, but excessive levels can still cause serious food safety issues. Currently, there are no detection methods or limit standards for phenidone in aquatic products. Summary of the Invention

[0003] The present invention aims to overcome the defects of the prior art and provide a method for detecting the content of phenidone in aquatic products.

[0004] The technical solutions of the present invention are as follows:

[0005] A method for detecting the content of phenidone in aquatic products comprises the following steps:

[0006] (1) Dissolve phenidone in methanol to prepare standard solutions of different concentrations;

[0007] (2) mixing the sample with acetonitrile, grinding, and centrifuging to obtain a first supernatant;

[0008] (3) Place the first supernatant in a solution containing PSA, C 18 and MgSO4 in a QuECherS purification tube, shaken and centrifuged to obtain a second supernatant;

[0009] (4) filtering the second supernatant through an organic filter membrane to obtain a sample solution;

[0010] (5) The above-mentioned standard solution and the sample solution are respectively sent to a high performance liquid chromatography tandem mass spectrometer for detection, and then the obtained detection results are compared. If the retention time of the mass chromatographic peak in the sample solution is compared with that of the standard solution, the variation range is within ±2.5%, and the deviation of the relative abundance of the two daughter ions of phenidone in the sample solution and the relative abundance of the standard solution with the same concentration in the sample solution does not exceed the range of the following table, it can be determined that the sample contains phenidone, and the qualitative detection is completed;

[0011] Relative ion abundance >50％ >20％～50％ >10％～20％ ≤10％ Allowable relative deviation ±20％ ±25％ ±30％ ±50％ .

[0012] In a preferred embodiment of the present invention, the method further comprises step (6): quantitatively detecting the content of phenidone in the sample by using an external standard method.

[0013] Further preferably, the step (6) is: using the external standard method, a standard curve is drawn according to the detection results of standard solutions of different concentrations, the coordinates of the standard curve are the concentrations of the standard solution, and the ordinate is the chromatographic peak area of ​​the phenidone ion in the standard solution. The detection results of the sample solution are substituted into the corresponding formula of the standard curve to quantitatively detect the phenidone content in the sample.

[0014] In a preferred embodiment of the present invention, the purity of phenidone used to prepare the standard solution is greater than 98%.

[0015] In a preferred embodiment of the present invention, the PSA, C 18 and MgSO4 contents are 150 mg, 150 mg and 900 mg respectively.

[0016] In a preferred embodiment of the present invention, the centrifugal conditions in step (2) and step (3) are both: 4000 rpm, 5 min.

[0017] In a preferred embodiment of the present invention, the pore size of the organic filter membrane is 0.22 μm.

[0018] In a preferred embodiment of the present invention, the HPLC conditions of the HPLC tandem mass spectrometer are as follows: chromatographic column: WATERS C18 1.7 μm, 100×2.1 mm column or equivalent column; flow rate: 0.3 mL / min; injection volume: 1 μL; mobile phase A is 5 mmol / L ammonium acetate containing 0.1% formic acid, mobile phase B is acetonitrile, and gradient elution.

[0019] Further preferably, the conditions of the gradient elution are shown in the following table:

[0020] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 80 20 2.00 80 20 3.00 30 70 5.00 30 70 5.5 80 20 8.00 80 20 .

[0021] More preferably, the mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometer are as follows: ion source: electrospray ion source; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring (MRM); electrospray voltage: 4000 V; capillary voltage: 65 V; auxiliary gas pressure: 35 psi; backflush gas: 11 L / h.

[0022] The beneficial effects of the present invention are: the present invention has simple pretreatment, accurate quantification, high sensitivity, a detection limit of phenidone of 5.0 μg / kg, and can quickly and highly sensitively perform qualitative and quantitative detection of the phenidone content in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is the MRM spectrum of 10 ng / mL phenidone in Example 1 of the present invention.

[0024] Figure 2 This is the MRM diagram of phenidone in the blank eel sample in Example 1 of the present invention.

[0025] Figure 3 This is the MRM chart of the blank eel sample in Example 1 of the present invention spiked with 2.5 μg / kg of phenidone. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0027] Example 1

[0028] 1. Instruments and Reagents

[0029] 1. Instruments: Agilent 1290 / 6470 HPLC-tandem mass spectrometer (with ESI source); chromatographic column: WATERS C18 1.7 μm, 100 × 2.1 mm; BS223S electronic balance (Beijing Sartorius); XLJ-IIB low-speed centrifuge (Shanghai Anting Scientific Instrument Factory); TGL-16G high-speed centrifuge (Shanghai Anting Scientific Instrument Factory); XK80-A vortex mixer (Jiangsu Xinkang Medical Instrument Co., Ltd.); TG-20 tissue grinder (Skills Group (Xiamen) Co., Ltd.); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0030] 2. Reagents and consumables: Methanol (chromatographic grade); acetonitrile (chromatographic grade); ammonium acetate (chromatographic grade); formic acid (chromatographic grade); water (grade 1); disposable pipette (1 mL); disposable syringe (1 mL); 5 mL plastic centrifuge tube; 50 mL plastic centrifuge tube; 0.22 μm organic filter membrane; sample bottle (2 mL).

[0031] 15mL QuECherS purification tube: contains 150mg PSA, 150mg C 18 , and 900mgMgSO4

[0032] 3. Standard substances and standard solutions

[0033] Standard substance: phenidone, purity greater than 98%;

[0034] Preparation of standard stock solution: Phenidone standard stock solution: Accurately weigh 10.0 mg of phenidone standard into a 5 mL beaker, dissolve it with a small amount of methanol, transfer it to a 50 mL brown volumetric flask, and dilute to 50 mL with methanol, with a concentration of 200 mg / L.

[0035] Preparation of standard intermediate solution: Pipette 0.5 mL of phenidone standard stock solution into a 100 mL brown volumetric flask, dilute to volume with methanol, mix well, and the concentration is 1.00 μg / mL.

[0036] 2. Test methods

[0037] Accurately weigh 2.00 g of sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 10 mL of acetonitrile, grind in a tissue grinder, centrifuge at 4000 rpm for 5 min, transfer the supernatant to a 15 mL QuECherS purification tube, shake at high speed for 5 min, centrifuge at 4000 rpm for 5 min, take the supernatant and pass it through a 0.22 μm organic filter membrane for determination.

[0038] 3. Instrument parameters and measurement conditions

[0039] 1. HPLC Conditions: Column: WATERS C18 1.7 μm, 100 x 2.1 mm or equivalent; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Mobile phase A: 5 mmol / L ammonium acetate (containing 0.1% formic acid), Mobile phase B: acetonitrile, Gradient elution. Specific gradient elution conditions are detailed in Table 1.

[0040] Table 1 Gradient elution conditions

[0041] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 80 20 2.00 80 20 3.00 30 70 5.00 30 70 5.5 80 20 8.00 80 20

[0042] 2. Mass spectrometry conditions: Ion source: electrospray ion source; Scan mode: positive ion scan; Detection mode: multiple reaction monitoring (MRM); Electrospray voltage: 4000V; Capillary voltage: 65V; Auxiliary gas pressure: 35psi; Backflush gas: 11L / h. Detailed information on phenidone ions is shown in Table 2 below, and the MRM spectrum of phenidone is shown in Figures 1 to 3 (The order is 10 ng / mL phenidone standard solution, blank eel sample, and blank eel sample with phenidone added at a level of 2.5 μg / kg).

[0043] Table 2 Precursor ion and fragment ion information

[0044]

[0045] 4. Result determination

[0046] 1. Qualitative determination: Measure the sample and standard solution according to the above conditions. If the retention time of the mass chromatographic peak in the sample is consistent with that of the standard solution (within the range of ±2.5%); the relative abundance of the two daughter ions of the target compound in the sample is consistent with the relative abundance of the standard solution with equivalent concentration, and the relative abundance deviation does not exceed the requirements of Table 4, then it can be determined that the target compound is present in the sample.

[0047] Table 4 Maximum allowable deviation of relative abundance of qualifier ions

[0048]

[0049] 2. Quantitative Determination: Use blank eel solution to create a standard solution curve at concentrations of 1 ng / mL, 2.0 ng / mL, 4.0 ng / mL, 10 ng / mL, and 20 ng / mL. Quantitative determination is performed using the external standard method. Draw a standard curve using the concentration of the standard solution as the abscissa and the chromatographic peak area of ​​the phenidone quantification ion as the ordinate.

[0050]

[0051] Where: X is the residual amount of the component to be measured in the sample, μg / kg; c is the concentration of the component to be measured obtained from the standard working curve, ng / mL; V is the final volume of the sample solution, mL; m is the sample weight, g.

[0052] V. Conclusion

[0053] 1. Linear range, detection limit and quantification limit: According to the above method, the detection limit (LOD) is the target concentration corresponding to a signal-to-noise ratio of not less than 3 times (S / N ≥ 3) and meeting the accuracy requirements of the method, and the quantification limit (LOQ) is the target concentration corresponding to S / N ≥ 10. Phenidone shows a good linear relationship in the range of 1.0 to 20 ng / mL (R 2 >0.999), LOD was 1.5 μg / kg, and LOQ was 5.0 μg / kg (see Table 5 for details).

[0054] Table 5 Linear range, detection limit and lower limit of quantification

[0055]

[0056] 2. Recovery and relative standard deviation: A spike recovery experiment was carried out using blank eel as the matrix at spike levels of 2.5 μg / kg, 5.0 μg / kg, and 50 μg / kg. Each spike concentration was measured in parallel six times, and the recovery and relative standard deviation (RSD) within the batch were calculated.

[0057] Table 6 Recovery and relative standard deviation

[0058]

[0059] 3. Conclusion The data showed that the linear range, recovery range and precision of the method met the requirements of GB / T 27404-2008 (see Table 7 for details). The limit of detection (LOD) was 1.5 μg / kg and the limit of quantification (LOQ) was 5.0 μg / kg. The method can be used to detect trace residues of phenidone in aquatic products.

[0060] Table 7 Comparison of the results of this embodiment with the requirements of GB / T 27404-2008

[0061] project Results of this Example GB / T 27404-2008 requirements <![CDATA[Calibration curve correlation coefficient R 2 > 0.9996 ≥0.98 Recovery rate range 83.4％～115％ 60％～120％ Precision 5.85％～10.84％ ＜15％

[0062] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for detecting the content of phenidone in aquatic products, characterized in that: The steps include: (1) Dissolve phenidone in methanol to prepare standard solutions of different concentrations; (2) After mixing the sample with acetonitrile, grinding and centrifuging, a first supernatant is obtained; (3) Place the first supernatant above in a 18 and MgSO4 in a QuECherS purification tube, shaken and centrifuged to obtain a second supernatant; (4) Filtering the second supernatant through an organic filter membrane to obtain a sample solution; (5) The above-mentioned standard solution and the sample solution are respectively sent to a high performance liquid chromatography tandem mass spectrometer for detection, and then the obtained detection results are compared. If the retention time of the mass chromatographic peak in the sample solution is within ±2.5% compared with that of the standard solution, and the deviation of the relative abundance of the two daughter ions of phenidone in the sample solution and the relative abundance of the standard solution with the same concentration in the sample solution does not exceed the range of the table below, it can be determined that the sample contains phenidone and the qualitative detection is completed; (6) quantitatively detecting the content of phenidone in the sample using an external standard method; The HPLC conditions for the HPLC-MS / MS were as follows: chromatographic column: WATERS C18 1.7 μm, 100 × 2.1 mm column; flow rate: 0.3 mL / min; injection volume: 1 μL; mobile phase A: 5 mmol / L ammonium acetate containing 0.1% formic acid; mobile phase B: acetonitrile; gradient elution; the gradient elution conditions are shown in the following table: Ion source: electrospray ion source; scanning mode: positive ion scanning; detection mode: multiple reaction monitoring; electrospray voltage: 4000 V; capillary voltage: 65 V; auxiliary gas pressure: 35 psi; backflush gas: 11 L / h.

2. The detection method according to claim 1, wherein: The step (6) is: using the external standard method, a standard curve is drawn according to the detection results of standard solutions of different concentrations, the coordinates of the standard curve are the concentrations of the standard solutions, and the ordinate is the chromatographic peak area of ​​the phenidone ion in the standard solutions. The detection results of the sample solution are substituted into the corresponding formula of the standard curve to quantitatively detect the phenidone content in the sample.

3. The detection method according to claim 1 or 2, wherein: The purity of the phenidone used to prepare the standard solution is greater than 98%.

4. The detection method according to claim 1 or 2, wherein: The PSA, C 18 and MgSO4 contents are 150 mg, 150 mg and 900 mg respectively.

5. The detection method according to claim 1 or 2, wherein: The centrifugal conditions in step (2) and step (3) are both: 4000 rpm, 5 min.

6. The detection method according to claim 1 or 2, wherein: The pore size of the organic filter membrane is 0.22 μm.

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