2.1 Apparatus, Chemicals and Materials
A Shimadzu GC/MS-QP2010 Ultra (Shimadzu,Japan), a J2 automated solid-phase extraction system (J2 Scientific,USA), a rotary evaporator (Switzerland, Germany),an electronic balance (Switzerland,Germany),a vortex mixer (Beijing, China),a universal pulverizer (Tianjin,China),an ultrasound cleaner (Shanghai,China) and the homogenate approach (Jiangsu,China) were used.
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 (Fairfield,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).
Panax notoginseng and Aucklandia lappa decne samples were purchased from local markets (Yunnan, China). Solid-phase extraction cartridges (SPE cartridges) containing C18, NH4 and Florisil were purchased from Agela Technologies (Tianjin, China).
2.2 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 flow rate of 1.20 mL.min-1 in a constant flow 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).
2.3 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 fluid 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.
2.4 Sample preparation
The samples were pulverized into fine 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 filtration, the extracts were transferred into a flat-bottomed flask and condensed on a rotary evaporator at 40°C under vacuum until a volume of 5 mL was reached. The concentrated solutions were purified 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 filtered through a 0.45 µm PTFE filter for GC-MS analysis.
2.5 Investigation of extraction and purification profiles
2.5.1 Investigation of ultrasonic extraction profiles
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(34) (Table 1) orthogonal test was applied.
Table 1 The factors and levels of the ultrasonic extraction orthogonal experiment
Factors
Levels
|
1
|
2
|
3
|
A (Solvent)
|
Methanol
|
Acetonitrile
|
Dichloromethane
|
B (Volume)/mL
|
20
|
40
|
60
|
C (Repetitions)
|
1
|
2
|
3
|
D (Duration)/min
|
15
|
30
|
45
|
2.5.2 Investigation of homogenate extraction profiles
The homogenate extraction approach of 17 OPPs from Panax notoginseng was under study, and the effects of factors such as time, revolution speed of homogenate extraction, extraction solvent and solvent volume on the extraction rate of 17 OPPs were examined through orthogonal experiments. The factors were screened, and an L9(34) (Table 2) orthogonal test was applied.
Table 2 The factor and level of the homogenate extraction orthogonal experiment
Factors
Levels
|
1
|
2
|
3
|
A (Solvent)
|
Methanol
|
Acetonitrile
|
Dichloromethane
|
B (Volume)/ mL
|
30
|
40
|
60
|
C (Revolution speed)/ r/min
|
10000
|
15000
|
20000
|
D (Time)/min
|
2
|
3
|
5
|
2.5.3 Investigation of purification profiles
The most effective purifying condition for 17 OPPs from Panax notoginseng was obtained experimentally by the orthogonal test design method. The influence of factors such as eluate, elution volume and times, and different solid-phase cartridges on the purification profiles were investigated. The factors were screened, and an L9(34) (Table 3) orthogonal test was applied.
Table 3 The factors and levels of the purification orthogonal experiment
Factors
Levels
|
1
|
2
|
3
|
A (Eluate)
(volume batching)
|
Acetone and N-hexane
(1:1,V/V)
|
Methylbenzene and Acetonitrile
(1:3,V/V)
|
Cyclohexane and Ethyl acetate
(1:1,V/V)
|
B (Volume)/mL
|
5
|
10
|
20
|
C (Solid-phase cartridges)
|
C18
|
NH4
|
Florisil
|
D(times)
|
1
|
2
|
3
|