Liquid chromatography-tandem mass spectrometry detection method for pentachlorophenol in marine products

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method for pentachlorophenol in marine products. The liquid chromatography-tandem mass spectrometry detection method comprises the following steps: (1) preparing a sample; (2) extracting; (3) purifying; and (4) liquid chromatography-tandem mass spectrometry detection. According to the method, the PCP in the marine products is detected by adopting a UPLC-MS / MS method, extraction conditions and Captiva EMR-Lipid small column purification conditions are optimized, an isotope dilution internal standard method is used for quantification, the problem of low sample recovery rate is remarkably improved, the linear range of the method is 1-1000 [mu] g / L, the detection limit is 1 [mu] g / kg, the three-gradient standard adding recovery rate and the relative standard deviation are 86%-94% and 3.72%-11.24% respectively (n = 6), the determination result is accurate and reliable, and the method is suitable for industrial production. The method can be used for efficiently detecting pentachlorophenol and sodium salt thereof in marine products.

Description

Technical Field

[0001] The invention relates to a method for detecting pentachlorophenol in seafood, in particular to a method for detecting pentachlorophenol in seafood by liquid chromatography tandem mass spectrometry. Background Art

[0002] Pentachlorophenol (PCP) and its soluble sodium pentachlorophenol (NaPCP) have been commercially produced and released into the environment as active ingredients of herbicides, molluscicides, wood preservatives, fungicides and bactericides in recent decades, and have toxic effects on ecosystems. PCP and its sodium salt are widely used in China to eliminate snails that are the host of parasitic schistosomiasis in rice fields and ponds. Because of its relatively high hydrophobicity, environmental persistence and long half-life in water, PCP can accumulate in organisms through the food chain and is not easily biodegradable. In addition, PCP has a high vapor pressure and is easy to evaporate from the surface of treated wood, leading to human inhalation. Acute inhalation of excessive PCP can lead to circulatory systemic effects and even death accompanied by heart failure, posing a serious threat to human life. PCP is designated as a possible carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC).

[0003] PCP enters the ocean through wastewater and river inputs. It has been detected to varying degrees in marine plants and animals such as algae, annelids, mollusks, crustaceans, and sediments. In recent years, the content of pentachlorophenol in seafood has been an important target of aquatic safety monitoring, so it is necessary to detect pentachlorophenol in seafood. At present, the detection methods of pentachlorophenol and its sodium salt mainly include gas chromatography (GC), liquid chromatography (LC) or gas chromatography-mass spectrometry (GC-MS). Among them, GC and GC-MS require derivatization of samples, which is relatively cumbersome; LC has poor qualitative properties and low sensitivity. The sample pretreatment methods for PCP in animal-derived foods mainly use liquid-liquid extraction and solid-phase extraction, but the above methods are cumbersome and require large amounts of reagents. Summary of the invention

[0004] The invention aims to solve the above problems existing in the prior art pentachlorophenol detection method and provides a liquid chromatography tandem mass spectrometry detection method for pentachlorophenol in seafood which is simple to operate, highly efficient, and has accurate and reliable measurement results.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: A liquid chromatography tandem mass spectrometry detection method for pentachlorophenol in seafood of the present invention comprises the following steps: (1) Sample preparation Take the edible part of the seafood, homogenize it and weigh 2.0±0.02g of the sample into a 50mL centrifuge tube. When the seafood is fish, the edible part is the dorsal muscle part; when the seafood is shrimp, shellfish and crab, the edible part is the muscle tissue; after homogenization, it can be frozen at -18℃.

[0006] (2) Extraction Add 100 μL of 100 μg / L pentachlorophenol- 13 To prepare the C6 isotope internal standard working solution, add 2.0 g of anhydrous magnesium sulfate and 10 mL of 1% acetic acid acetonitrile solution, vortex and oscillate for extraction, perform ultrasonic extraction, centrifuge and obtain the supernatant, extract the residue once again with 1% acetic acid acetonitrile solution, combine the extracts in a rotary evaporator, evaporate to dryness, add 2 mL of 75% acetonitrile aqueous solution, vortex and redissolve to obtain a redissolved solution for purification. The present invention adopts the isotope dilution internal standard method for quantification to improve the problem of low sample recovery rate; since the sample contains some water, anhydrous magnesium sulfate is added to absorb the water to increase the extraction efficiency of the extractant, and the PCP part in the sample exists in a bound form, so the present invention further adopts ultrasound-assisted acid hydrolysis on the extracted solution to increase the extraction efficiency of the extractant; the inventor found through experiments that the good protein precipitation effect of acetonitrile makes the main interfering substances in the extract liquid weak polar compounds such as lipids, and adding 1% glacial acetic acid can make the acidified acetonitrile clearer and cleaner, therefore, the present invention finally determines that 1% acidified acetonitrile is the best extractant of the present invention, and 10mL is the best extraction amount.

[0007] (3) Purification Transfer all the reconstituted solution through the Captiva EMR-Lipid column, filter and collect all the filtrate, filter the filtrate to obtain the machine solution. Seafood contains a large amount of protein, fat and other long-chain substances, which may be extracted together, affecting the test results and may shorten the service life of the instrument. In order to better study the recovery rate of sodium pentachlorophenol, the inventors used 4 types of P / N Captiva EMR-Lipid, Florisil SPE, Si / PSA, OasisMAX solid phase extraction columns and CNWBOND PSA dispersed solid phase extraction agents to compare the recovery rates. It was found that the recovery rate of Captiva EMR-Lipid was 93%, which was the best effect. The Captiva EMR-Lipid extraction column also has the characteristics of no activation and rapid filtration, which saves time and improves purification efficiency. Therefore, the Captiva EMR-Lipid column is selected for purification in the present invention.

[0008] (4) Liquid chromatography tandem mass spectrometry detection The solution was drawn with an injection needle and loaded into the machine according to the set chromatography-mass spectrometry conditions for UPLC-MS / MS analysis. The peak area of ​​pentachlorophenol obtained was substituted into the standard working curve to calculate the concentration of pentachlorophenol.

[0009] Preferably, in step (2), vortex oscillation extraction is performed for 5-7 min; ultrasonic extraction is performed for 5-15 min; centrifugation is performed at 6000-8000 r / min for 5-7 min; and rotary evaporation is performed at 35-40°C.

[0010] Preferably, in step (3), the filtrate is filtered using a 0.22 μm organic filter membrane.

[0011] Preferably, in step (4), the chromatographic conditions are: using a Waters Acquity UPLC BEH C18 column with a column size of 100 mm×2.1 mm and 1.7 μm; instrument room temperature: 20° C.; chromatographic column temperature: 40° C.; mobile phase A is 5 mmol / L ammonium acetate solution, and mobile phase B is methanol; the gradient elution program is as follows: initial 40% B; 0-6.5 min, 40%-100% B; 6.5-9.0 min, 100% B-100% B; 9.0-9.5 min, 100% B-40% B; 9.5-12 min, 40%-40% B; injection volume: 10 μL; flow rate: 0.2 mL / min. PCP is highly hydrophobic, and most research reports usually use C18 chromatographic columns as analytical columns. However, different brands of C18 chromatographic columns have different production processes such as silica purity, bonding and hybridization technology, resulting in different retention performance and selectivity, which in turn affects the sensitivity of the target in mass spectrometry analysis. The inventors found that different brands of C18 chromatographic columns have different retention abilities for PCP, and the later the PCP elution time, the stronger its mass spectrometric response. The inventors found through experiments that the Waters Acquity UPLC BEH C18 chromatographic column has the strongest retention ability and the highest mass spectrometric response when analyzing PCP, so the Waters Acquity UPLC BEH C18 column was selected; pentachlorophenol is a halogenated phenol substance, an acidic compound, and stable in nature. Compared with the acetonitrile mobile phase system, the PCP elution time is later in the methanol mobile phase system and the mass spectrometric response value is higher. In order to effectively improve the peak shape of the target compound, prevent the occurrence of peak drift, and stabilize the elution time, a buffer salt (ammonium acetate) is added to the aqueous phase; the electrospray ion source (ESI) has good sensitivity in the mobile phase containing ammonium acetate, so methanol is finally determined to be the organic phase and 5 mmol / L ammonium acetate is the aqueous phase.

[0012] Preferably, the mass spectrometry conditions are: multiple reaction monitoring mode; capillary voltage: 2.5 kV; ion source temperature: 150°C; desolvation gas temperature: 500°C; cone gas flow: 200 L / h; desolvation gas flow: 1000 L / h.

[0013] Preferably, the mass spectrometry conditions are: multiple reaction monitoring mode; capillary voltage: 2.5 kV; ion source temperature: 150°C; desolvation gas temperature: 500°C; cone gas flow: 200 L / h; desolvation gas flow: 1000 L / h.

[0014] Preferably, in step (4), the standard curve is obtained by the following method: accurately pipette pentachlorophenol standard solution, dissolve and dilute it with methanol to form a 1 mg / L standard stock solution, dilute it with different blank matrices to form 0.5, 1, 2, 5, 10, and 20 ng / ml matrix-matched standard working solutions, measure according to the conditions of step (4), draw a standard working curve with the injection mass concentration as the x-axis and the response value as the y-axis, and quantify by external standard method.

[0015] Therefore, the present invention has the following beneficial effects: the UPLC-MS / MS method is used to detect PCP in seafood, the extraction conditions and Captiva EMR-Lipid column purification conditions are optimized, the isotope dilution internal standard method is used for quantification, and the problem of low sample recovery rate is significantly improved. The linear range of the method is 1-1000 μg / L, the detection limit is 1 μg / kg, and the three gradient spike recovery rates and relative standard deviations are (n=6) 86%-94% and 3.72%-11.24%, respectively. The determination results are accurate and reliable, and the method can be used for the efficient detection of pentachlorophenol and its sodium salt in seafood. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison chart of the response intensities of 8 pentachlorophenol ion pairs.

[0017] Figure 2 This is a comparison chart of PCP recovery rates using different purification methods.

[0018] Figure 3 This is a comparison chart of the extraction recovery rates of different extraction agents. DETAILED DESCRIPTION

[0019] 1. Instruments and Reagents AcquityTM ultra-high performance liquid chromatograph (UPLC); Quattro PremierTM tandem quadrupole mass spectrometer (Waters, USA); Centrifuge 5810 high-speed centrifuge (Eppendorf); MS2 vortex mixer (IK, Germany); Captiva EMR-Lipid solid phase extraction column (300 mg / 3 mL, Agilent, USA); 12-channel solid phase extraction device (Supelco, USA); CNWBOND PSA bulk (100 g, per bottle, Anpu Laboratory Technology Co., Ltd.); CNWBOND Florisil SPE Cartridge (1 g, 6 ml, Anpu Laboratory Technology Co., Ltd.); CNWBONDSi / PSA SPE GLASS Cartridge (500mg / 500mg, 6ml, Anpu Laboratory Technology Co., Ltd.); rotary evaporator (Ningbo Tianheng Instrument Factory); ultrasonic cleaner (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.); the experimental water is the first-class water specified in GB / T6682; methanol, acetonitrile, ammonium acetate, and acetic acid are all (HPLC grade); anhydrous magnesium sulfate (analytical grade), 13 C6-pentachlorophenol standard (CAS: 85380-74-1); purity ≥ 97%, TRC Company; pentachlorophenol standard solution (GBW (E) 080475), China National Institute of Metrology.

[0020] 2. Optimization of UPLC-MS / MS analysis method 2.1 Determination of mass spectrometry conditions Use a needle pump to inject 13 C6-PCP and PCP-Na were subjected to full mass spectrometry scan. The results showed 13 C6-PCP and PCP-Na have high responses under negative source conditions, and can form ion peak clusters containing four isotope peaks (m / z 262.7, m / z 264.7, m / z 266.7 and m / z 268.7), respectively, with an abundance ratio of nearly 2:5:2:1. Further secondary mass spectrometry fragmentation analysis of the four isotope ion peaks revealed that the main fragment ions of the above ions are [Cl] － , and there are two isotopic peaks at m / z 35.0 and m / z 37.0 (except for the m / z 262.7 ion), so [M-Na] is selected － >[Cl] － As the ion pairs to be optimized in MRM mode, the collision energy of all eight ion pairs was optimized (e.g. Figure 1The results show that the optimal collision energy conditions for the eight ion pairs are all 15 eV, the abundance of the m / z264.7>35.0 ion pair is the highest, followed by the m / z262.7>35.0 ion pair, and four identification points are obtained with two different parent ions corresponding to the daughter ions, which meets the requirements of EU 2006 / 657 / EC resolution on LC- 13 Qualitative requirements for C6-PCP MS / MS analysis methods.

[0021] 2.2 Selection of solid phase extraction column The recovery rates were compared using four types of P / N solid phase extraction columns: Captiva EMR–Lipid (C), Florisil SPE (A), Si / PSA (B), OasisMAX (E) and CNWBOND PSA (D) dispersed solid phase extraction agent. The test results are shown in Figure 2. Figure 2 As shown in the figure, the recovery rate of Captiva EMR-Lipid was 93%, which was the best.

[0022] 2.3 Selection of extraction solvent and extraction amount Six organic extraction solvents were selected: n-hexane, acetonitrile, ethyl acetate, n-hexane: glacial acetic acid (V / V) (99:1), acetonitrile: glacial acetic acid (V / V) (99:1), ethyl acetate: glacial acetic acid (V / V) (99:1), and the selected extraction solvents were used for extraction under the same conditions in triplicate, and then the extraction recovery of different extraction agents was evaluated. The experimental results are shown in Figure 2. Figure 3 As shown. As can be seen from the figure, the extraction effects of n-hexane, 1% acidified n-hexane, ethyl acetate, and 1% acidified ethyl acetate are not ideal, the acetonitrile recovery rate is between 85% and 94%, and the 1% acidified acetonitrile recovery rate is between 83% and 95%, and the extraction rates of both are significantly higher than the other four extractants. The good protein precipitation effect of acetonitrile makes the main interfering substances in the extract liquid weakly polar compounds such as lipids. Adding 1% glacial acetic acid can make the acidified acetonitrile clearer and cleaner. The extraction recoveries of 1% acidified acetonitrile of 10ml, 15ml and 20ml are 95%, 91%, and 86% respectively. It can be seen that the extraction amount of 10ml of 1% acidified acetonitrile is the most significant, so 1% acidified acetonitrile is determined to be the best extractant of the present invention, and 10ml is the best extraction amount.

[0023] 3. Validation of the Method There are many types of seafood and the matrix is ​​relatively complex. The matrix effect is an important factor affecting the liquid chromatography-tandem mass spectrometry analysis method. The detection signal is usually suppressed or enhanced, especially in electrospray ionization (ESI) mass spectrometry, which will affect the stability and robustness of the analysis method. This has an adverse effect on the sensitivity, precision and authenticity of chromatography-tandem mass spectrometry.

[0024] The present invention is verified from multiple aspects such as ME, linearity, LOQ, authenticity and precision. LOQ is used as the lowest point of the calibration curve to evaluate the linearity of the method, which can provide reliable recovery and repeatability. At the same time, the signal-to-noise ratio (S / N) of more than 10 times is met. All parallel experiments are repeated for 6 times, and the data are expressed as average values. The matrix effect calculation formula is as follows: matrix effect (%) = (slope of matrix matching standard curve / slope of pure solvent standard curve)-1]×100%. The absolute recovery calculation formula is as follows: absolute recovery (%) = peak area of ​​the drug to be tested after the spiked sample is purified / peak area of ​​the drug to be tested in the pure reagent solution with the same concentration as the spiked sample)]×100%. The authenticity of the method is verified by adding 1, 5 and 20 μg / kg blank fish, shellfish and crab samples to determine the recovery (n=6). Intra-day and inter-day precision were evaluated in sextuplicate by measuring the relative standard deviation (RSD) of recoveries for spiked fish, shellfish, crab, and shrimp samples collected on the same day and on six consecutive days for authentic samples.

[0025] Prepare PCP standard solution with a mass concentration range of 1-1000 μg / L for determination, using isotope internal standard 13 C6-PCP evaluated the linearity, and the correlation coefficients of all PCPs were in the range of 0.995 to 0.999, indicating that it had a good linear relationship within this concentration range and met the detection requirements of the instrument. According to the 10-fold signal-to-noise ratio S / N, the detection limit of PCP was 1 μg / kg. In the present invention, the matrix effect and spike recovery tests at three gradient levels of low, medium and high were conducted on samples of fish, shellfish, shrimp and crab. The recovery rate, regression equation, correlation coefficient, relative standard deviation and quantitative limit are shown in Table 1. The average recovery rates of the three gradient spike recovery tests for fish were 90.5%, 88.5%, and 88%, and the average relative standard deviations were 6.88%, 9.72%, and 6.97%; the average recovery rates of the three gradient spike recovery tests for shellfish were 89.8%, 90.4%, and 90.6%, and the average relative standard deviations were 6.19%, 9.29%, and 8.76%; the average recovery rates of the three gradient spike recovery tests for crabs were 90%, 89%, and 90.5%, and the average relative standard deviations were 8.62%, 7.48%, and 5.83%; the average recovery rates of the three gradient spike recovery tests for shrimps were 93.5%, 91.5%, and 92.5%, and the average relative standard deviations were 6.32%, 6.53%, and 6.21%. The quantitative limit for all seafood is 1μg kg -1, evaluate and calculate the ME of different types of seafood, and the results show that the ME of fish, shellfish, crab and shrimp are -9%--11%, -15%-10%, -17%-13% and -12%-6% respectively. It is generally believed that if the ME is higher than -20% or lower than 20%, the signal enhancement or suppression is acceptable, otherwise there is a strong matrix effect. From the results, the present invention has basically no matrix effect on different types of seafood.

[0026] Table 1 Matrix effect, regression equation, correlation coefficient, recovery rate, relative standard deviation and limit of quantification of pentachlorophenol in 11 kinds of seafood (n=6) Example 1 (1) Sample preparation The samples were large yellow croaker purchased from Zhoushan City. The dorsal muscle part of the large yellow croaker was taken and homogenized before weighing 2.0 g of the sample into a 50 mL centrifuge tube. (2) Extraction Add 100 μL of 100 μg / L pentachlorophenol- 13 C6 isotope internal standard working solution, add 2.0g anhydrous magnesium sulfate and 10mL 1% acetic acid acetonitrile solution, vortex shake extraction for 5min, ultrasonic extraction for 10min, centrifuge at 7000r / min for 5min, take the supernatant, repeat extraction once with 1% acetic acid acetonitrile solution in the residue, combine the extracts in a rotary evaporator, evaporate to dryness at 38℃, add 2mL 75% acetonitrile aqueous solution, vortex re-dissolve, obtain the reconstituted solution, and wait for purification; (3) Purification Pipette all the reconstituted solution through the CaptivaEMR-Lipid column, filter and collect all the filtrate, and filter the filtrate with a 0.22 μm organic filter membrane to obtain the upper solution; (4) Liquid chromatography tandem mass spectrometry detection The sample solution was extracted with an injection needle and loaded according to the set chromatographic-mass spectrometric conditions for UPLC-MS / MS analysis. The peak area of ​​pentachlorophenol obtained was substituted into the standard working curve to calculate the concentration of pentachlorophenol. The chromatographic conditions were as follows: Waters Acquity UPLC BEHC18 column was used, with a column size of 100 mm × 2.1 mm, 1.7 μm; the instrument room temperature was 20°C; the chromatographic column temperature was 40°C; the mobile phase A was 5 mmol / L ammonium acetate solution, and B was methanol; the gradient elution program was as follows: initial 40% B; 0-6.5 min, 40%-100% B; 6.5-9.0 min, 100% B-100% B; 9.0-9.5 min, 100% B-40% B; 9. 5-12min, 40%-40%B; injection volume: 10μL; flow rate: 0.2mL / min; mass spectrometry conditions: multiple reaction monitoring mode; capillary voltage: 2.5kV; ion source temperature: 150℃; desolvation temperature: 500℃; cone gas flow: 200L / h; desolvation gas flow: 1000L / h; the standard curve was obtained by the following method: accurately pipette pentachlorophenol standard solution, dissolve and dilute it with methanol to 1mg / L standard stock solution, dilute it with different blank matrices to 0.5, 1, 2, 5, 10, 20ng / ml matrix matching standard working solution, measure according to the conditions of this step, draw the standard working curve with the injection mass concentration as the x-axis and the response value as the y-axis, and quantify by external standard method. The final measured PCP concentration is 5μg / kg.

[0027] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for detecting pentachlorophenol in seafood by liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: (1) Sample preparation Take the edible part of the seafood, homogenize it and weigh 2.0±0.02g of sample into a 50mL centrifuge tube; (2) Extraction Add 100 μL of 100 μg / L pentachlorophenol- 13 C6 isotope internal standard working solution, add 2.0g anhydrous magnesium sulfate and 10mL 1% acetic acid acetonitrile solution, vortex oscillation extraction, ultrasonic extraction, centrifugation to obtain the supernatant, the residue is extracted once with 1% acetic acid acetonitrile solution, the combined extracts are placed in a rotary evaporator, rotary evaporated to dryness, and 2mL of 75% acetonitrile aqueous solution is added, vortexed to redissolve, and the redissolved solution is to be purified; (3) Purification Transfer all the reconstituted solution through the CaptivaEMR-Lipid column, filter and collect all the filtrates, and filter the filtrates to obtain the upper solution; (4) Liquid chromatography tandem mass spectrometry detection The solution was drawn with an injection needle and loaded into the machine according to the set chromatography-mass spectrometry conditions for UPLC-MS / MS analysis. The peak area of ​​pentachlorophenol obtained was substituted into the standard working curve to calculate the concentration of pentachlorophenol.

2. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (2), vortex oscillation extraction is performed for 5-7 minutes; ultrasonic extraction is performed for 5-15 minutes; centrifugation is performed at 6000-8000 r / min for 5-7 minutes; and rotary evaporation is performed at 35-40°C.

3. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (3), the filtrate is filtered using a 0.22 μm organic filter membrane.

4. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (4), the chromatographic conditions are as follows: using a Waters Acquity UPLC BEHC 18 column with a column size of 100 mm×2.1 mm, 1.7 μm; instrument room temperature: 20°C; chromatographic column temperature: 40°C; mobile phase A is 5 mmol / L ammonium acetate solution, and mobile phase B is methanol; the gradient elution program is as follows: initial 40% B; 0-6.5 min, 40%-100% B; 6.5-9.0 min, 100% B-100% B; 9.0-9.5 min, 100% B-40% B; 9.5-12 min, 40%-40% B; injection volume: 10 μL; flow rate: 0.2 mL / min.

5. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions are: multiple reaction monitoring mode; capillary voltage: 2.5 kV; ion source temperature: 150° C.; desolvation gas temperature: 500° C.; cone gas flow rate: 200 L / h; desolvation gas flow rate: 1000 L / h.

6. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that: The mass spectrometry conditions are: multiple reaction monitoring mode; capillary voltage: 2.5 kV; ion source temperature: 150° C.; desolvation gas temperature: 500° C.; cone gas flow rate: 200 L / h; desolvation gas flow rate: 1000 L / h.

7. The method for detecting pentachlorophenol in marine products by liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: In step (4), the standard curve is obtained by the following method: accurately pipette pentachlorophenol standard solution, dissolve and dilute it with methanol to form a 1 mg / L standard stock solution, dilute it with different blank matrices to form 0.5, 1, 2, 5, 10, and 20 ng / ml matrix-matched standard working solutions, measure according to the conditions of step (4), draw a standard working curve with the injection mass concentration as the x-axis and the response value as the y-axis, and quantify by external standard method.

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