Fast Determination of 22 Pesticides in Rice Wine by Dispersive Solid-Phase Extraction in Combination with GC-MS

A rapid method was developed for the determination of 22 pesticides in rice wine. The procedure involved an extraction with acetonitrile and a cleanup step using dispersive solid-phase extraction (d-SPE), and primary-secondary amine (PSA) and octadecylsilane (ODS) were used as sorbents. D-SPE had some advantages over some traditional preparations, especially in time and cost. Both the extraction and cleanup only cost about 15 min per a sample. Then the GC-MS was used for quantitative and qualitative analysis. Matrix-matched standards solution and analyte protectant were compared to decrease the matrix effect, and the former showed a better efficacy. Acceptable linearity was achieved in the range of 0.05 0.30 mg/L. After matrix-matched standards calibration, recoveries were between 60% and 140%. For the most of pesticides, precision and repeatability were less than 10% and 16%, respectively. The result indicated that the developed method was suitable for the determination of the multi-pesticides in rice wine.


Introduction
Rice wine, beer and wine are considered as the three ancient wines [1].Rice wine is an alcoholic beverage brewed from rice, and pesticides may be used during the period of rice growth.Rice is directly used for fermentation without any processing, and that causes the presence of pesticide residues in rice wine.
Many reports have been published for the determination of pesticides in alcoholic beverage.Major pretreatments of pesticides in food are liquid-liquid extraction (LLE) [2], solid-phase extraction (SPE) [3][4][5][6], and solidphase micro-extraction (SPME) [7,8].In 2003, dispersive solid-phase extraction (d-SPE) method was introduced for the determination of pesticides by Anastassiades et al. [9], and d-SPE was a simple and rapid technique with high recovery for the determination of pesticides in fruit and vegetables [10].In previous studies, the combination of several sorbents showed a better cleanup result than using primary-secondary amine (PSA) alone for some complex samples [11][12][13].However, few reports were introduced for the determination of the pesticides in rice wine.
In this study, a rapid method was introduced for the determination of multi-pesticides in rice wine by GC-MS/SIM.Pesticides in rice wine samples were extracted with acetonitrile and cleaned by d-SPE by using the combination of PSA and octadecylsilane (ODS).

Reagents and Standards
Acetonitrile (LC grade) was purchased from Merck (Darmstadt, Germany).PSA and ODS were provided by Bonna-Agela Technologies (Tianjin, China).All the standards of pesticide were obtained from Agricultural Environmental Protection Institution (Tianjin, China), and listed in Table 1.Polyethylene glycol (PEG300) and triphenyl phosphate (TPP) were purchased from SCRC (Beijing, China).Other chemicals were from Beijing Chemical Works (Beijing, China).All of the rice wines and olive oil were purchased from local supermarkets, and the wine samples were kept in a freezer (2˚C -4˚C).

Instrumentation
Separations of pesticides were performed on a gas chromatography equipped with a mass detector and an autosampler (GC-MS 2010 plus, Shimadzu, Japan).The ex- tractant of pesticides were injected with a splitless mode.DB-1701 capillary column (30 m × 0.25 mm × 0.25 μm) was provided by Agilent (Palo Alto, CA, USA).Helium was used as carrier gas, and the flow rate was kept at 1.2 mL/min.The column temperature was maintained at 40˚C for 1 min, and then ramped at 30˚C/min up to 130˚C, then at 5˚C/min up to 250˚C, and finally at 20˚C/min up to 280˚C.The injector temperature was 280˚C, the volume of injection was 1 μL.The MS ionization energy was 70 eV, the ion-source temperature was 230˚C, and the interface temperature was set at 280˚C.The MS detection was performed in Selection ion mode (SIM), and the characteristic of pesticides are listed in Table 1.

Sample Preparation
TPP (10.00 mg/L, dissolved by acetonitrile) was used as the internal standard (IS) solution, and 100 μL IS solution and 2.4 mL acetonitrile were added into 5.0 mL rice wine sample, and the mixture was vigorously shaken for 2 min.then NaCl (0.50 g) and anhydrous MgSO 4 (2.00 g) were added and then vortexed slightly for 1 min.The extract was then centrifuged for 5 min at 5000 rpm.An aliquot of 1.0 mL of the upper layer was transferred to a 2.0 mL micro-centrifuge tube containing 50 mg PSA, 50 mg ODS and 150 mg MgSO 4 , shaken for 2 min, and then centrifuged for 4 min at 8000 rpm.Finally, 500 μL of extract added 50 μL analyte protectant (prepared as it described by Xu et al. [14]) was placed into an autosampler vial for GC/ MS analysis.

Optimization of Sorbents
PSA, ODS, graphitized carbon black (GCB), and Florisilsilica were used as sorbents in d-SPE procedure [15,16].Among these sorbents, PSA can retain fatty acids and other organic acids, but only slightly reduces the color and vitamin.ODS has a remarkable cleanup ability for non-polar compound, such as some non-polar pigments and vitamins [17].GCB is a powerful sorbent for pigments, but some pesticides containing benzene ring can be adsorbed by GCB [12].As shown in Figure 1, peak areas of some pesticides reduced when GCB was used as the sorbent of d-SPE.Due to the light color of rice wine, GCB was not necessary.It is important for the cleanup to select an appropriate material as the sorbent.In previous works, some sorbents such as PSA, ODS, anhydrous MgSO 4 , and GCB, or their combination was selected for the cleanup of pigments in extracts of wine and grape [18][19][20].In this study, the combination of 50 mg PSA, 50 mg ODS, and 150 mg anhydrous MgSO 4 was selected in d-SPE for rice wine extracts cleanup.The SIM chromatogram of pesticide standards and the internal standard was shown in Figure 2.

Optimization of Temperature and pH
The low temperature minimizes the degradation of some heat-sensitive pesticides [11].Rice wine and other reagents were kept in freezer (4˚C) overnight before sample preparation.Keeping the samples in low temperature during the d-SPE procedure was important.
The pH of samples will be increased when PSA was added and the stability of some alkali-labile pesticides was affected by pH [18].Two ways were usually taken to resolve the problem, one was acidifying the extracts quickly to pH∼5, and another was the usage of citrate buffer [21].In this study, rice wine samples were acidity, and the pH of samples was not obviously increased with the addition of 50 mg PSA.

Matrix Effects
In GC analysis, analyte protectants and matrix-matched calibration solutions were proved to be effective approaches to minimizing matrix effect [22].The effects of analyte protectants were compared by Xu et al. [14], and the results showed that PEG300 and olive had a better effect.In our study, the combination of PEG300 and olive was used to be the analyte protectant, and the peak areas of pesticides were compared under the three situations (in pure solvent, matrix-matched calibration solutions and analyte protectant).As shown in Figure 3, both analyte protectant and matrix-matched calibration solution can reduce the matrix effect.The former had a better effect on diazinon, fenvalerate and cypermethrin, but for phorate, pyrimethanil, heptachlor, and chlorpyrifosmethyl, matrix-matched solution was better.In our study the method of matrix-matched calibration solution was selected for the minimizing of matrix effect.

Comparison with Other Preparation
Modern sample preparation techniques should be simple, reliable, cheap, and be similar to common analytical techniques, in order to minimize errors [23].Information of the major methods used for determining pesticides in alcoholic beverages was presented in Table 2.
d-SPE had advantages in time and cost, but SPE and some other methods had a better cleanup.

Methodology Evaluation
Linearity was investigated in the range 0.05 -0.30 mg/L with four calibration points, and the correlation coefficients for pesticides were listed in Table 3. Precision was less than 10%, and the recoveries were from 60% to 140% at three concentration levels (0.10, 0.20 and 0.30 mg/L).A SIM chromatography for blank rice wine sample and spiked rice wine was shown in Figure 4. LOQ, LOD, and other information were also listed in Table 3.

Conclusion
In this work, a rapid method was developed for the determination of multi-pesticides in rice wine by using d-SPE-GC-MS.The effect of different adsorbents was investigated, and the combination of different adsorbents was used for the d-SPE procedure with a high results.And then the approaches of minimizing the matrix effect were compared.And the developed method can be used for the determination of pesticides in rice wine samples.Names of pesticides are on Table 1.

Figure 1 .
Figure 1.GCB adsorbed some pesticides containing benzene ring.Names of pesticides are listed Table1.

Figure 2 .
Figure 2. GC-MS/SIM chromatogram of a pesticide standard of 1.0 mg/L for each pesticide and the internal standard.Names of pesticides are listedTable 1.

Figure 3 .
Figure 3.Comparison of pesticide peak areas in pure solvent, matrix-matched standards and adding analyte protectant of mixed PEG and olive oil in optimal concentrations.Names of pesticides are listed Table1.

Figure 4 .
Figure 4. SIM chromatography for (a) blank rice wine sample; (b) blank rice wine spiked with 0.3 mg/L of the pesticides.Names of pesticides are on Table1.