Key words
Tobacco; organophosphorus pesticide residue; large volume without splitting; GC-FPD
Summary
In this paper, GC-FPD combined with large volume splitless injection technique was used to establish a highly efficient and sensitive method for the determination of organophosphorus pesticide residues in tobacco. For the tobacco samples, the modified QuEChERS method was used to extract the organophosphorus pesticide residues in tobacco by deionized water immersion and ethyl acetate acetone mixed solvent, and purified by Carbon-NH2 composite column, and directly analyzed without concentration. Through experiments, it is found that: 1) Using a large volume splitless injection technique, when the injection volume is 30 ul, the detection of each organophosphorus pesticide residue is improved by nearly 25 times compared with the conventional splitless injection of 1 ul; The organic phosphorus pesticide residue standard prepared by the blank tobacco extract purified by Carbon-NH2 composite column can significantly improve the peak shape and sensitivity of each organic phosphorus. In general, the use of GC-FPD combined with large volume splitless injection technology is a very sensitive and efficient detection method for organophosphorus pesticide residues in tobacco, which can greatly reduce the time-consuming time for sample concentration during pretreatment. At the same time, it has high detection sensitivity.
1 Introduction
With the development of science and technology and people's constant attention to health, the detection of pesticide residues in food, consumer goods and the environment has been increasingly valued by more and more countries and testing institutions [1]. With China's accession to the WTO and the International Tobacco Control Convention, the maximum amount of pesticide residues in cigarettes and tobacco is regulated internationally [2]; in addition, a small amount of pesticide residues in tobacco, especially organophosphorus pesticides, High toxicity, which directly affects the health of smokers when smoking cigarettes, it is necessary to establish an effective and feasible test method to determine the residual amount of organophosphorus pesticides in tobacco.
At present, there are many methods for detecting organophosphorus pesticide residues in tobacco at home and abroad, mainly including GC-FPD, GC-MS and GC-MS/MS methods [2-4]. Among them, the GC-FPD method is widely used by many testing institutions because of its simple formulation, high selectivity for organophosphorus detection, and greatly reduced interference in complex matrices. In terms of pre-treatment of tobacco organophosphorus pesticides, the QuEChERS method is a fast, simple, inexpensive, sample preparation method that covers a wide range of applications (including pesticide types and substrate types), and its low-cost and efficient sample processing technology can be used. The broad range of pesticide residues in most food matrices has been widely adopted by several international pesticide residue analysis agencies. However, most of the organophosphorus pesticide residues in the sample have a low concentration. In order to achieve accurate determination, it is usually necessary to concentrate to increase the concentration of the target, and in the process, a part of the low-boiling organic phosphorus pesticide is caused. The loss of volatilization affects the accurate quantification of it; and this process is time consuming, which is not conducive to the improvement of detection efficiency. In this regard, a large-volume injection technique can be used to increase the sensitivity of the organophosphorus pesticide residue detection by increasing the sample injection amount, without performing sample concentration, and achieving a comparison with the conventional injection method to determine the organophosphorus pesticide residue. The same sensitivity, which simplifies the sample preparation step. The temperature-programmed large-volume injection technology is currently the most widely used large-volume injection technology. It can separate the solvent from the liner through the carrier gas at a temperature slightly above the boiling point of the solvent, and then rapidly heat up to vaporize the target. Separate into the column [5]. In this process, some low-boiling pesticide residues are lost with the shunting of the solvent, and eventually result in a low detection sensitivity. To overcome this problem, another large-volume injection technique—the simultaneous solvent-concentration injection technique (SCR) is used to carry all the solvents and targets of the large-volume injection through the pre-column, and then slowly evaporate the solvent to reach the column. The detector is vented, and the target with a slightly higher boiling point than the solvent reaches the detector after the solvent is detected, thereby ensuring that all target components of the boiling range are separated from the column and reach the detector, thus ensuring high detection sensitivity [ 6-7].
In view of this, this paper uses the improved QuEChERS method for extraction, carbon-NH2 composite column purification, GC-FPD combined with large volume splitless injection technology, direct injection without concentration to efficiently and sensitively detect organic phosphorus in tobacco. Residue content.
2. Experimental materials
2.1 Instruments and reagents
2.1.1 Instruments
Trace 1310 GC-FPD Gas Chromatograph (Thermo Fisher Scientific); Triplus RSH 3-in-1 Autosampler (Thermo Scientific); TR-35MS Column (30 m × 0.25 mm × 0.25 μm) (Thermo Fisherscientific, P/N : 260C142P); Large volume splitless kit (includes 5mm x 0.32 mm pre-column, glass two-way and large volume splitless calculation software) (Thermo Fisher scientific, P/N).
2.1.2 Reagents, consumables
Dibromophosphorus, methamidophos, fast-acting phosphorus, phosphine, methyl endophos, terbutaphos, ethion, dimethoate, methyl pyrimidine, methyl parathion, malathion, The standards of chlorpyrifos, parathion and bromide were 100ppm and purchased from the National Standards Center; sodium chloride and anhydrous sodium sulfate were purchased from Shanghai Sinopharm Chemical Co., Ltd.; Carbon-NH2 composite column ( Specification 1g) purchased from Shanghai Anpu Technology Co., Ltd.; ethyl acetate (chromatographically pure), acetone (chromatographically pure) supplied by ThermoFisher; water is deionized water; tobacco shredded tobacco is purchased from the market (smashed into smoke before use).
2.2 Preparation of extraction solvent and organic phosphorus standard
Extraction solvent: 450 ml of ethyl acetate was mixed with 50 ml of acetone to prepare a mixed solvent of ethyl acetate and acetone in a volume ratio of 9:1.
Blank tobacco extract: Take 2.0g of fumed organic phosphorus without soaking, add 10ml deionized water for 5min, add 4.0g NaCl, 20ml ethyl acetate and acetone mixed solvent, vortex for 2min, and purify by Carbon-NH2 composite column. To be used with the standard sample.
14 kinds of organic phosphorus pesticide residues with a concentration of 1.0ppm: Take 100ul 100ppm of each organophosphorus pesticide residue standard stock solution, add it to a 10ml volumetric flask, and dilute to volume with a blank tobacco extract to obtain a concentration of 1.0ppm. 14 kinds of organic phosphorus pesticide residues.
Preparation of a series of concentrations of organophosphorus pesticide residues: 5 ml of 1.0 ppm of 14 kinds of organophosphorus pesticide residues were added to a 10 ml solvent bottle and placed in the 4th position of the standard cleaning position of the TriplusRSH 3-in-1 automatic sample stage. Add 10ml of blank tobacco extract to the 10ml solvent bottle and place it in the 1st position of the standard cleaning position of the Triplus RSH 3 in 1 automatic sample stage for use as a dilution solvent; two solvent bottles on the Triplus RSH large volume cleaning position A suitable amount of ethyl acetate and acetone mixed solvent was separately added for washing the needle. Place an empty vial on the sample tray (position as shown below, Figure 2, 2, 3, 4, 5, 6), directly call the built-in automatic dilution file, you can automatically prepare a series of standard solutions (concentrations are 20ppb, 50ppb respectively , 100ppb, 200ppb and 500ppb).
2.3 Sample preparation
Accurately weigh 2.0g of smoke sample in a 50mL centrifuge tube, add 10.0ml deionized water, soak for 5min; add 4.0g NaCl and 20.0ml ethyl acetate acetone mixed solvent, vortex for 3min; take 10ml ethyl acetate acetone mixed Solvent-activated Carbon-NH2 composite column (add 2.0 g of anhydrous sodium sulfate at the upper end), dry it, take 10 ml of the extract, load the column (flow rate is about 1 drop/s); load the chromatographic bottle for GC-FPD analysis.
2.4 Sample spike
Accurately weigh 2.0g of cigarette sample in 50mL glass bottle, add 10.0ml deionized water, soak for 5min; add 1.0ml and 2.0ml 1.0ppm of 14 kinds of organic phosphorus pesticide residue; add 4.0g NaCl and 19.0ml And 18.0 ml of ethyl acetate and acetone mixed solvent, vortex for 3 min; 10 ml of ethyl acetate and acetone mixed solvent to activate the Carbon-NH2 composite column (add 2.0 g of anhydrous sodium sulfate at the upper end), and after drying, take 10 ml of the extract. Sample, pass through the column (flow rate approx. 1 drop/s); load into the chromatographic bottle for GC-FPD analysis.
2.5 Chromatographic conditions
Chromatographic conditions: column temperature: 75 ° C (7 min), 20 ° C / min to 200 ° C, 10 ° C / min to 280 ° C (6 min); splitless injection, no split time 7 min; inlet temperature: 280 ° C; Carrier gas: High purity nitrogen (99.999%), constant current mode, 1.2 mL/min. Liquid injection mode, injection volume: 30.0 μL, injection speed 100ul/s; FID detector: base temperature 300°C, detection cell temperature 150°C, air 115 mL/min, hydrogen 90mL/min.
3. Results and discussion
3.1 Standard chromatogram
3.1.1 Comparison of large volume splitless injection and standard splitless injection
Figure 2 shows the chromatograms of 14 organophosphorus pesticide residues at a concentration of 100 ppb using a large volume splitless injection (injection volume 30 ul, black) and a standard splitless injection (injection volume 1 ul, red). As can be seen from the figure, the use of large volume splitless injection can significantly improve the sensitivity of each organophosphorus pesticide residue. After calculation, using a large volume splitless injection of 30ul, the peak area of ​​most organophosphorus pesticide residues was 1ul higher than the standard splitless injection, with an average increase of nearly 25 times (see Table 1).
3.1.2 Effect of blank matrix extract on peak shape and sensitivity of organic phosphorus
Figure 3 is a chromatogram of 14 organophosphorus pesticide residues prepared in two different solvents at a concentration of 100 ppb, using a large volume injection with an injection volume of 30 ul. The standard chromatogram prepared with the blank tobacco extract and the standard chromatogram prepared from the mixed solvent of ethyl acetate and acetone were marked with red and blue, respectively. From the chromatograms of 14 organophosphorus pesticide residues prepared from these two different solvents, it can be seen that the standard prepared from the tobacco substrate extract is compared to the standard prepared from the mixed solvent of ethyl acetate and acetone. The peak shape and sensitivity have been significantly improved, especially methamidophos, dimethoate and bromide. The above phenomenon indicates that the matrix standard can significantly improve the peak shape and sensitivity of the target, especially organic phosphorus. In view of this, blank tobacco substrate extracts were used to prepare a series of organophosphorus pesticide residues to eliminate the effects of matrix effects.
3.2 Linearity and method detection limits
For the series of organophosphorus pesticide residues, the Triplus RSH automatic calibration function was used to prepare the concentrations: 20ppb, 50ppb, 100ppb, 200ppb and 500ppb. The above method was used to separate the samples, and the linearity in the concentration range of 20ppb-500ppb was investigated. The linear equations and correlation coefficients of 14 organophosphorus pesticide residues were calculated. The detection limit was calculated by 3 times the signal-to-noise ratio. Table 2. The experimental results show that the response of 14 organophosphorus pesticide residues in this concentration range has a good linear relationship with the concentration, and the correlation coefficient is greater than 0.998 (Fig. 4), and the detection limits are lower than the experimental results. CORESTA Guided Residue Limits (GRLs) [8]. This also demonstrates the high accuracy of the Triplus RSH and the reliability of large volume splitless injections.
3.3 Recovery and repeatability
According to the above method, 14 organophosphorus pesticide residues were added to the tobacco leaf samples containing no organophosphorus pesticide residues to be tested, and pre-treatment and GC-FPD analysis were performed separately, and according to the spike amount and The recovery rate was calculated from the actual measured amount, and the results are shown in Table 3. The experimental results show that under the above two spiked levels, the recovery rates of 14 organophosphorus pesticides except for dibromophosphorus are more than 120%, others are between 75% and 110%, and the relative standard deviations of six determinations are parallel. Below 4%, it is well suited to the need for multi-residue testing of pesticides.
to sum up
In this paper, the Thermo Scientific GC-FPD was combined with the large volume splitless component to improve the QuEChERS method (deionized water soaking, ethyl acetate acetone mixed solvent to extract the organic phosphorus pesticide residues in tobacco), and purified by CarbonNH2 composite column. Direct injection analysis by concentration. When the injection volume is 30 ul, the detection of each organophosphorus pesticide residue is improved by nearly 25 times compared with the conventional splitless injection of 1 ul. The method has simple and stable operation steps, and does not require a cumbersome and time-consuming solvent removal step, thereby avoiding loss of volatile pesticide residues; the detection limit for each organophosphorus pesticide residue is lower than the CORESTA guide residue limit requirement. At the same time, the tobacco samples were tested for spiked recovery of 0.5mg/Kg and 1.0mg/Kg. The recovery of 14 organophosphorus pesticide residues except for dibromophosphorus was more than 120%, and the others were between 75% and 110%. It can well meet the daily testing needs of organophosphorus pesticides.
references
[1] Zou Ximei, Lin Zhuguang. GC-EI/MS analysis method and application of multiple pesticide residues in tobacco, vegetable and fruit, Master's thesis of Xiamen University, 2009,1-101.
[2] Zhang Hongfei, Hu Qingyuan, Tang Gangling, Bian Zhaoyang, Wang Fang. Analysis of 29 organophosphorus pesticide residues in tobacco by gas chromatography-mass spectrometry, Chinese Journal of Tobacco, 2008, 14, 9-13.
[3] Shi Jie, Yang Jing, Liu Huimin, Hu Bin, Tao Pengli, Yan Huihui. Rapid GC/MS analysis of organophosphorus pesticide residues in tobacco, Tobacco Science and Technology, 2010, 9, 43-46.
[4] XS Chen, ZY Bian, HW Hou, FYang, SS Liu, GL Tang and QY Hu. Development and Validation of a Method for the Determination of 159 Pesticide Residues in Tobacco by Gas Chromatography Tandem Mass Spectrometry, J. Agric.Food Chem .2013,61,5746-5757.
[5] Lou Xiaohua, Gao Chuanchuan, Zhu Wenjing, Zhang Hongfei, Tang Gangling, Bian Zhaoyang, Ba Jinsha. Simultaneous determination of 202 pesticide residues in tobacco by PTV-GC-MS/MS, Tobacco Science and Technology, 2013, 8, 45-57 .
[6] M. Biedermann, A. Fiscalini, K. Grob. Large volume splitless injection with concurrent solvent recondensation: Keeping the sample in place in the hot vaporizing chamber, J. Sep. Sci. 2004, 27, 1157–1165
[7] P. Magni, T. Porzano, Concurrent solvent recondensation large sample volume splitless injection, J. Sep. Science (2003) 26, 1491-1498.
[8] Mueller L, KrzemienM R. The concep t and implementation of agrochemical guidance residue levels [ S ] / / CORESTA Agrochemical Residual Group. CORESTA Guide No. 1, 2003.
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