Abstract: Organophosphorus pesticides are organic compounds containing organic phosphorus pesticides used to prevent and control plant diseases, insects, and pests. This kind of pesticide has many varieties, high efficacy, wide use, easy to decompose, and generally does not accumulate in humans and animals. It is an extremely important compound in pesticides.
The method for simultaneous detection of organophosphorus pesticide residues in Chinese medicinal materials by high performance liquid chromatography-tandem mass spectrometry was studied. A CAPCELLPAKMGC18 reverse phase column was used, acetonitrile was used as the extraction solution, and Carb / PSA column was used as the purification column, and liquid chromatography tandem mass spectrometry was used for determination. The linear range of the method is 10 ~ 500g / L. The 11 organophosphorus pesticides are linear in this range, and the correlation coefficient is 0.9961 ~ 9999. In the concentration range of 10 ~ 100g / kg, the recovery rate of the standard addition is between 70% ~ 110%, the relative standard deviation is 1.4% ~ 11%, and the minimum detection limit is 2 ~ 20g / kg, which meets the requirements of residue detection and analysis.
The extensive use of organophosphorus pesticides will accumulate and function in crops, pose a threat to humans and animals, and will also have a negative impact on the environment. At present, the analysis and research of organophosphorus pesticide residues in Chinese medicinal materials are few [1 ~ 3], and the most used detection method is gas chromatography [4], and gas-mass spectrometry [5] is also used, but less liquid is used. Mass spectrometry usage. Due to the complex composition of most Chinese herbal medicines, for the analysis of trace residues in complex samples, the anti-interference ability of gas chromatography and gas-mass combination is poor, which requires high sample preparation technology, while high performance liquid chromatography and tandem mass spectrometry can perform secondary Mass spectrometry analysis has strong anti-interference ability and low requirements for pretreatment. By selecting monitoring ions for accurate qualitative and quantitative analysis of some samples with complex composition and dirty background, it is more suitable for the detection and analysis of pesticide residues in Chinese medicinal materials. In this study, solid phase extraction purification combined with high performance liquid chromatography. Tandem mass spectrometry was used to establish the residues of 11 organophosphorus pesticides in Chinese herbal medicines such as gardenia, Scrophulariaceae, plantain, licorice and honeysuckle.
Detection method 1. Experimental part 1.1. Instrument and material liquid chromatography triple quadrupole mass spectrometer; powder machine; tabletop, centrifuge (maximum speed 4500r / min); ultrasonic cleaner; nitrogen dryer; rotary evaporation Instrument; vortex mixer; solid phase extraction, take instrument. Methanol and acetonitrile are chromatographically pure; toluene, acetone, anhydrous sulfuric acid, sodium are analytically pure; formic acid: purity> 98%; activated carbon; Carb / PSA double-layer solid phase extraction column (500mgCarb / 500mgPSA / 6mL). Standard products: dichlorvos; diltiazem; parathion; diazophos; methylpyrimidine; methylglufosin; malathion; chlorpyrifos; chlorpyrifos; fenthion; ethamphos. Prepare standard stock solution (100mg / L) with acetone. Experimental herbs: Gardenia, Scrophulariaceae, plantain, licorice and honeysuckle.
1.2. Sample extraction Weigh 5.0g of Chinese medicinal material sample after crushing and passing through a 045mm sieve in a 50mL plastic centrifuge tube, add 30mL of acetonitrile, add 5g of anhydrous sodium sulfate, cover with a lid, shake vigorously by hand to mix evenly, ultrasonically Centrifuge at 3500r / min for 30min at 30min, take 15mL of supernatant in a 100mL pear-shaped bottle, and rotate to dry under 35.
1.3. Purification of samples Add 05g of activated carbon to the Carb / PSA column, activate the small column with 4mL acetonitrile toluene (volume ratio 3: 1) in advance, dissolve it in three times with 10mL acetonitrile toluene (volume ratio 3: 1) Pass the column, collect the effluent, elute with 20mL of acetonitrile toluene (volume ratio 3: 1), collect the effluent, combine the effluent in a flat-bottomed burner, and evaporate to 35mL under 35. Dissolve and measure after passing through 0.22.m filter membrane.
1.4. Liquid chromatography conditions Chromatography column: CAPCELLPAKMGC18 column (100mm.2.0mm.id 3.m); column temperature: room temperature; flow rate: 200L / min; injection volume: 10L; mobile phase: V (0.1% formic acid in water) : V (methanol) = 50: 50, the proportion of methanol in 0 ~ 3min increases linearly from 50% to 95%, and then maintains for 4.5min, 7.5 ~ 8min, linearly returns to the initial flow ratio, and then equilibrates for 2min.
1.5. Mass spectrometry conditions Ion source: Electrospray ionization source (ESI); Scan mode: Positive ion mode; Detection method: Selective reaction detection (SRM); Electrospray voltage: 4000V; Capillary temperature: 350; Collision gas: High purity Argon; see Table 1 for ion monitoring and collision energy. Table 1. ESIMS / MS monitoring ions and corresponding collision energy organophosphorus pesticide precursor ions 1.6. Linear relationship inspection experiment Accurately absorb the appropriate amount of each pesticide standard stock solution, and use the blank extract to dilute it into a series of different in the range of 10 ~ 500g / L The concentration gradient is measured under the selected optimal liquid-mass separation conditions, with the peak area of ​​each organophosphorus pesticide in the sample as the ordinate and the content (g / L) as the abscissa, and a standard curve is drawn.
1.7. Recovery rate and precision experiment Weigh 5.0g each of 5 kinds of Chinese medicinal materials, carry out blank sample addition experiment at three concentrations of 10, 50 and 100g / kg respectively, and operate according to the method in 1.3 respectively, repeat 5 for each concentration Times, the average recovery and precision were measured.
2. Results and discussion 2.1. The mobile phase used in liquid chromatography-mass spectrometry can not add various surfactants, hardly volatile salts and other substances that have an inhibitory effect on the response of mass spectrometry. Methanol, acetonitrile, mixtures of different ratios and buffers of some volatile salts. This study investigated the mobile phase system of methanol aqueous solution. During the experiment, the appropriate amount of ammonium acetate and formic acid were added. The experimental results showed that the addition of 0.1% formic acid to the methanol aqueous solution helped the ionization of organophosphorus pesticides and the response value was high. At the same time, in order to further improve the peak shape and shorten the analysis time, gradient elution was used. The 11 organophosphorus pesticides all peaked within 7 minutes, and the peak shape was good. See Figure 1 for details.
2.2. Mass spectrometry conditions In ESI positive ion mode, all substances have a good response, and their excimer ion peaks can be observed at the same time. When aligning molecular ions for secondary mass spectrometry optimization, the fragment ions and their respective collision energies are obtained. The fragment ions are selected as the qualitative and quantitative basis (see Table 1), which ensures the sensitivity and accuracy.
2.3. The sample extractant organophosphorus pesticide has a certain polarity, and different solvents are reported in different literatures. This experiment investigated the five extraction solvents of ethyl acetate, acetonitrile, acetone, dichloromethane and n-hexane respectively for 11 organic solvents. The extraction efficiency of phosphorus pesticides showed that the extraction efficiency of acetonitrile and dichloromethane was higher, but the co-extracted extracts of dichloromethane were more, and the interference of impurities was large. Therefore, acetonitrile was selected as the extraction solvent in the experiment.
2.4. Purification method experiments respectively selected activated carbon, C18, SAX, Carb / PSA solid phase extraction column purification, the results show that the purification effect of Carb / PSA solid phase extraction column is ideal, and can ensure the recovery rate of 11 organophosphorus pesticides Above 70%. For samples with high pigment content, it is better to add a certain amount of activated carbon to the small column for decolorization.
2.5. The linear relationship and the minimum detection limit are operated according to the method 1.6. The results show that 11 kinds of organophosphorus pesticides in 5 kinds of Chinese medicinal materials have a good linear relationship in the concentration range of 10 ~ 500g / L. Gardenia, plantain, Scrophulariaceae, The linear correlation coefficients of 11 organophosphorus pesticides in licorice and honeysuckle were 0.9961-0.9999. Standard samples were added to blank samples for the detection limit experiment. Based on the signal-to-noise ratio greater than 3, the minimum detection limit of the method was established. The minimum detection limit of 11 organophosphorus pesticides in five Chinese medicinal materials was 2-20g / kg, see Table 2 for details.
2.6. Recovery rate and precision are operated according to the method 1.7. The average recovery rate and precision are measured. The results show that: within the concentration range of 10 ~ 100g / kg, the average recovery rate of 11 organophosphorus pesticides in gardenia is 84.0% ~ 104.7 %, The relative standard deviation is 1.4% ~ 9.4%; the average recovery rate of organophosphorus pesticides in plantain is 71.6% ~ 87.8%, the relative standard deviation is 2.7% ~ 11%; the average recovery rate of organophosphorus pesticides in Scrophulariaceae is 73 % ~ 110%, the relative standard deviation is 1.8% ~ 7.8%; the average recovery rate of organophosphorus pesticides in licorice is 70% ~ 86.5%, the relative standard deviation is 3.3% ~ 9.5%; the average recovery rate of organic phosphorus pesticides in honeysuckle is 72.5 % ~ 96.8%, the relative average deviation is 2.2% ~ 9.1%.
2.7. Sample testing 28 samples of gardenia, plantain, Scrophulariaceae, licorice and honeysuckle purchased from 6 medicinal herbs stores were tested using the method established in this article. None of the organophosphorus pesticides were detected. This method is used for the detection of organophosphorus pesticide residues in burdock for export. In 2009, a total of 284 burdock samples were tested, and no organophosphorus pesticides were detected.
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