Study of Analytical Methods for Determining 16 Organophosphorus Pesticide Residues in Panax Notoginseng and Aucklandia Lappa Decne by Automated Solid-Phase Extraction System and GC/MS

A fast and sensitive multi-residue analytical method was developed for the determination of organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne by comparing an ultrasonic extraction and homogenate extraction approach coupled with automated solid-phase extraction system and gas chromatography-mass spectrometry (GC/MS). Various factors affecting the homogenate extraction and purication eciency were investigated in detail. The optimum conditions for homogenate extraction of organophosphorus pesticide residues from Panax notoginseng and Aucklandia lappa decne were 60 mL dichloromethane, 10000 r/min of extracting revolution speed and 2 min of extracting time. The extracts were cleaned up by automated solid-phase extraction system and determined by GC/MS. Under the optimum conditions, the results of the homogenate extraction approach were compared with those of the ultrasonic extraction, and the homogenate extraction approach was used to extract the samples. The average recoveries of the method by a homogenate extraction approach coupled with automated solid-phase extraction system and GC/MS ranged from 72.56% to 96.70%, and RSDs were 0.05~0.14%, which allowed the determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne. The results indicated that the proposed method can apply for the determination of organophosphate pesticides residues in Panax notoginseng and Aucklandia lappa decne with high accuracy and precision. extraction, supercritical uid extraction, pressurized solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction, gel permeation chromatography, solid phase extraction, solid phase microextraction, molecularly imprinted polymers, matrix solid-phase dispersion, QuEChERS, cloud-point extraction, liquid phase microextraction, etc 18-22 . Automated solid-phase extraction system achieves articial intelligence in the sample pre-treatment techniques. Aucklandia has the of pain, regulating Qi and soothing the nerves. The application of OPPs in processes of rticially cultivated Panax notoginseng and Aucklandia lappa decne were unavoidable, but there are few standard of detective pesticide residue in Panax notoginseng and Aucklandia lappa decne. For these reasons above, a rapid and simple analytical method with high accuracy and precision for determining 17 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne by automated solid-phase extraction system and GC/MS was developed in this paper. The rapid and simple method for the simultaneous determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne was developed by comparing an ultrasonic extraction and homogenate extraction and optimizing purication conditions of automated solid-phase extraction system, which can meet the requirement of determining pesticide residues from Panax notoginseng and Aucklandia lappa decne. residues in Panax notoginseng and Aucklandia lappa decne by a homogenate extraction approach coupled with automated solid-phase extraction system and GC/MS. Under the optimum conditions, the results of the homogenate approach were compared with the ultrasonic extraction method, and the homogenate approach was used to extract the samples. The average recoveries of the method ranged from 72.56% to 96.70%, and RSDs were 0.05~0.14%, which meets the requirements for the determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne.


Introduction
Pesticide residues refer to pesticides, their toxic metabolites and degradation products and any impurities that may live on or in an biosome, farm products, or the environment after pesticides are used [1][2][3] .
Sometimes pesticide residues are inevitable; however, once the residue reaches its limitation, it can make a series of danger to humans and animals or other organism in the ecological system by the food cycle 4 . The organophosphate pesticides (OPPs) were widely used and leaded to the repeated incidents of poisoning, and therefore, self-poisoning with organophosphate pesticides is a signi cant global health problem [5][6][7] . Hence, a ban was imposed on the use of toxic pesticides, including OPPs, such as methamidophos, parathion, monocrotophos, methyl parathion, etc [8][9][10] .
The rules linking pesticides on plants and in agricultural products have be set up so far by a lot of international organizations and countries. With the remarkable enhancements in testing methods and analytical techniques of pesticide residues and the extensive attention of consumer safety in recent years, the developed countries have become strictly monitoring both the terms of pesticide class or amount and maximum residue levels (MRLs) in foodpesticide residues [11][12][13][14] . Multiresidue analytical systems, which have been developed worldwide, can simultaneously determine over three hundred pesticides 15 . The MRLs of 9 organochlorines (OCPs) were prescriptive in the Chinese pharmacopoeia, which involved benzene hexachloride (BHC), dichlorodiphenyltrichloroethane (DDT) and pentachloronitrobenzene (PCNB). Radix et Rhizoma Glycyrrhizae and Radix Astragali in the Pharmacopoeia of the People's Republic of China (Ch.P) were involved 16 . The rst draft of the quality standards from china in July 2001, which had something to do with green professional standards for import and export of o cinal plants and products, was issued and enforced in the ministry of foreign trade and economic cooperation. The 9 OCPs and aldrin were included according to the standards. The analytical methods of 3 pyrethroids and 12 OPPs were supplied separately in the Ch.P, but MRLs were not prescriptive. In order to improve the international regulations and standards of pesticide residues from Chinese traditional medicine, further research have been needed 17 . The main food safety issues focused on pesticide residues. Especially the MRLs of pesticide residues from many countries and health organizations had published. People have paid a more attention to analytical methods of remnant pesticides, so the analytical methods possessing sureness, simpleness, speediness, relatively steady, highly sensitive and repeatability is required. The sample pre-treatment and detected techniques have a crucial effect on the accuracy and precision of the analytical methods.
Aucklandia lappa decne belonging to asteraceae perennial herb which mainly produced in Yunnan places like Lijiang Naxi Autonomous County and Ludian County, Zhaotong City. The origin of arti cially cultivated Aucklandia lappa decne are distributed mainly over Sichuan,Yunnan, Guangxi and Guizhou provinces. Aucklandia lappa decne has the functions of stomach,tocolysis,relieving pain, regulating Qi and soothing the nerves. The application of OPPs in processes of rti cially cultivated Panax notoginseng and Aucklandia lappa decne were unavoidable, but there are few standard of detective pesticide residue in Panax notoginseng and Aucklandia lappa decne. For these reasons above, a rapid and simple analytical method with high accuracy and precision for determining 17 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne by automated solid-phase extraction system and GC/MS was developed in this paper. The rapid and simple method for the simultaneous determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne was developed by comparing an ultrasonic extraction and homogenate extraction and optimizing puri cation conditions of automated solid-phase extraction system, which can meet the requirement of determining pesticide residues from Panax notoginseng and Aucklandia lappa decne.
Acetone, dichloromethane,and n-hexane, all of analytical reagent grade, were from Tianjin University and were purchased from Tianjin Chemical Experiment Plant (Tianjin,China). Methylbenzene,acetonitrile,and methyl alcohol were all of HPLC grade and were purchased from Tedia (Fair eld,OH,USA). Anhydrous sodium sulfate and sodium chloride (Shanghai,China) were baked at 450°C for 4 h and stored in a desiccator before use. The standards of 17 OPPs (methamidophos, dichlorvos, acephate, omethoate, monocrotophos, phorate, phosphamidon, methyl parathion, fenitrothion, malathion, fenthion, chlorpyrifos, parathion, profenofos, triazophos, imidan, phosalone) with a solution concentration of 1000 mg/L were purchased from Quality Testing and Inspection Centre of the Ministry of Agriculture for Agricultural Products (Beijing, China).

GC/MS conditions
The separation was performed on an Rtx-5MS quartz capillary column (30 m × 0.25 mm, I.D. 0.25 µm) with a helium ow rate of 1.20 mL.min -1 in a constant ow mode using the following oven temperature program: 70 °C (1 min) to 250 °C (2 min) at a rate of 8°C.min -1 and 250 °C (2 min) to 300°C (10 min) at a rate of 5°C.min -1 . The injection inlet temperature was set at 250 °C, and a 1.0 µL volume was injected. Helium was the carrier gas. Using the splitless injection, the total analysis time was 45.5 min. The mass spectrometer was operated in electron ionization mode with an ionizing energy of 70 eV, an ion source temperature of 230°C,and an MS quad temperature of 280 °C. The scan ranged from m/z 45 to 500,and the solvent delay was 3.5 min. Under the above GC-MS conditions,the total ion chromatogram (TIC) of 17 OPPs was obtained ( Fig. 1).

Standard solution preparation
The 0.90 mg (accurate to 0.01 mg) individual pesticide standards of 17 OPPs were weighed and redissolved in methylbenzene to make 10mL,which was to obtain the mixed standard reserve uid of 17 OPPs. The standard stock mixtures were then diluted to 4.5 µg.mL -1 with acetone.
The series of mixed standard working solutions for 2.00, 1.00, 0.50, 0.25, 0.10, and 0.05 µg.mL -1 were made up by the dilution method step by step with acetone. The standard solutions were stored in the dark at -4°C before use.

Sample preparation
The samples were pulverized into ne powders, sieved through 80 mesh and dried at 60 °C for 4 h. One gram of powder was then weighed accurately, and 60 mL of dichloromethane was added. The mixture was extracted for 2 min via homogenate extraction at 10000 r/min, and then 1.0 g sodium sulfate anhydrous and 1.0 g sodium chloride were added. After ltration, the extracts were transferred into a at-bottomed ask and condensed on a rotary evaporator at 40°C under vacuum until a volume of 5 mL was reached. The concentrated solutions were puri ed with Florisil solid-phase extraction cartridges by an automated solid-phase extraction system. The solid-phase extraction cartridges with the adsorbed extracts were eluted with 5 mL of methylbenzene-acetonitrile (1:3, v/v) three times. The eluate solution was collected and evaporated until near dryness by a rotary evaporator with a water bath at 38°C. Finally, the residues were resuspended in 1.00 mL of acetone and ltered through a 0.45 µm PTFE lter for GC-MS analysis.
2.5 Investigation of extraction and puri cation pro les 2.5.1 Investigation of ultrasonic extraction pro les The extraction technology implemented for the analysis of 17 OPPs from Panax notoginseng was under study, and the effects of factors such as extraction repetition and duration, extraction solvent and solvent volume on the extraction rate of 17 OPPs were examined through orthogonal experiments. The extraction repetition and duration, extraction solvent and solvent volume were screened, and an L9(3 4 ) (

Investigation of puri cation pro les
The most effective purifying condition for 17 OPPs from Panax notoginseng was obtained experimentally by the orthogonal test design method. The in uence of factors such as eluate, elution volume and times, and different solid-phase cartridges on the puri cation pro les were investigated.
The factors were screened, and an L9(3 4 ) ( Table 3) orthogonal test was applied. Table 3 The factors and levels of the puri cation orthogonal experiment Single standard solutions of 17 OPPs was monitored with the full-scan mode (SCAN) by GC/MS.One quantitative ion and two or three qualitative ions of each compound were con rmed.The detection method for 17 OPPs from 4.5 µg/m was established with the selected ion monitoring mode (SIM). The results are shown in Table 4. Based on the orthogonal test and multiple standard addition method, the data in Table 5 were obtained using the ultrasonic and homogenate extraction methods. Both of intuitionistic analysis and square-difference analysis is applied to analyze the data from Table 5, and the results are shown in Table 6, from which indicated that the dominant in uence on the investigation factors for 17 OPPs from Panax notoginseng were the type of solvent used for the ultrasonic and homogenate extraction methods. For other factors with the ultrasonic and homogenate extraction, the effects on extraction e ciency were ranked as follows: C>B>D. The results showed the optimal conditions (A 3 B 1 C 1 D 2 ) for the extraction of 17 OPPs from Panax notoginseng by ultrasonic extraction were as follows: Panax notoginseng was extracted with approximately 20 mL of dichloromethane, and the extraction replication and duration times were one and 30 min, respectively. The results showed that the optimal conditions (A 3 B 3 C 1 D 1 ) for the extraction of 17 OPPs from Panax notoginseng by the homogenate extraction method were as follows: Panax notoginseng was extracted with approximately 60 mL of dichloromethane, and the extraction time was 2 min at 10000 r/min.

Selection of puri cation conditions
Chinese medicinal materials contain a variety of matrices; thus, a clean-up procedure is necessary. [19] Based on the orthogonal test and multiple standard addition method, the data in Table 7 are the puri cation pro les of the orthogonal L9(3 4 ) method implemented. The recovery results of the puri cation pro les are shown in Table 7. The results of the intuitionistic analysis and square-difference analysis are shown in Table 8. The results of the experiments and the intuitionistic analysis and square-difference analysis of experimental (Table 7 and Table 8) results showed that the dominant in uence on the puri cation recoveries of 17 OPPs was the type of eluting solvent. For other factors, the effects of the puri cation pro les were ranked as follows: B>D>C. The optimal puri cation conditions were a combination of A 3 B 3 C 1 D 1 that 3 extractions with approximately 5 mL of methylbenzene-acetonitrile (1:3, V/V) each time from Florisil solid-phase cartridges met the requirement for the puri cation of the 17 OPPs.

.1 Calibration curves, determination coe cients and LODs
To con rm that the optimized method was suitable for application, precision, recovery, linear range and detection limits (LODs) were evaluated for the analytical approach developed using samples of Panax notoginseng. Quantitative analysis was achieved using an external standard. The calibration curve was obtained by analyzing blank Panax notoginseng samples spiked with pesticides at the same levels (Table 9). For detection of the 17 OPPs by GC/MS, the LODs were 0.12~0.52×10 -3 μg. The acceptable linearity was indicated by the determination coe cients, which ranged from 0.9928 to 0.9999.

Results of methodology validation
Under the optimized extraction, puri cation and detection conditions for 17 OPPs in Panax notoginseng determined by orthogonal design, two analytical methods were developed. In the rst method, Panax notoginseng was extracted by ultrasonic extraction. The extracts were cleaned up by an automated solid-phase extraction system and determined by GC/MS. The second method used homogenate extraction. Experiments in which standards were added to the samples to determine the recovery rate were performed. The standard recovery test results for the two methods and calculation of the recovery rate are shown in Table 10. The average recoveries of the rst method ranged from 43.37% to 85.63%, with the relative standard deviations (RSDs) of 0.07~0.17% (n=3). The average recoveries of the second method ranged from 67.88% to 96.70%, with RSDs of 0.05~0.14% (n=3). The results showed that the average recoveries of the rst method were often relatively lower the second method. Therefore, the analytical method was developed by homogenate extraction and automated solid-phase extraction system(the speci c condition shown in 3.2 and 3.3). The average recovery (67.88%) of methyl parathion was just below 70%. The average recoveries of other OPPs ranged from 70% to120%, with RSDs of less than 10% . So the established analytical method can be applied to determining 16 organophosphorus pesticide residues in Panax notoginseng. Similarly,to further verify the viability of the method for Aucklandia lappa decne,recovery experiments were carried out,the results were satisfactory. The rapid and simple method for the simultaneous determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne was developed,which can meet the requirement of determining pesticide residues from Panax notoginseng and Aucklandia lappa decne.

Conclusion
Two sample pretreatment methods for the extraction of 16 OPPs from the traditional Chinese medicinal herb Panax notoginseng and Aucklandia lappa decne were developed. A comparison of the two methods of analyzing 16 OPPs in Panax notoginseng was performed. We can see that the homogenate extraction method is effective, with a high extraction rate and shortened extraction time. Therefore, a fast and sensitive multi-residue analysis method was developed for the determination of organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne by a homogenate extraction approach coupled with automated solid-phase extraction system and GC/MS. Under the optimum conditions, the results of the homogenate approach were compared with the ultrasonic extraction method, and the homogenate approach was used to extract the samples. The average recoveries of the method ranged from 72.56% to 96.70%, and RSDs were 0.05~0.14%, which meets the requirements for the determination of 16 organophosphorus pesticide residues in Panax notoginseng and Aucklandia lappa decne. Figure 1 The TIC of 17 OPPs