Determination of Fosthiazate Residue in Ginger by Gas Chromatography

Kailing PENG Yating XIE Chenzhong JIN Xiaoming OU Han XI Xiu LIU Qiangyong SONG Guiping LIANG Zhifu YANG Shunli XIAO Xuye ZHANG

Abstract [Objectives]An analytical method was established for determining fosthiazate residue in ginger by gas chromatography with flame photometric detector （GCNPD） to evaluate the safety of fosthiazate in ginger.[Methods]The fosthiazate residue in ginger was extracted with dichloromethane， and subjected to sodium chloride saltout and liquidliquid extraction with hexane and dichloromethane （the plant and soil samples had no need for the later two steps）. The analysis was performed on gas chromatograph equipped with a flame photometric detector （GCFPD）， and the fosthiazate residue was quantitatively determined by calculating the peak area.[Results]The results of degradation dynamics showed that the halflife of fosthiazate was 7.2-8.7 d in ginger plants， and was 9.9-19.8 d in the soil. The results of the final residue test showed that after applying the fosthiazate granules according to the recommended high dose （active component 58.2 g a.i/m2，WK） and low dose （38.8 g a.i/m2，WK） once， the final residual amounts of fosthiazate in the ginger and soil were estimated to be <0.02 mg/kg and <0.02-0.103 mg/kg， respectively， while no fosthiazate residue was detected in all control samples （<0.02 mg/kg）. The maximum residue limit （MRL） of fosthiazate in ginger was not established in China， and was 0.2 mg/kg in Japan.[Conclusions]The method was proved to meet the basic requirements of pesticide residue analysis on sensitivity， accuracy and precision and have good linearity. It is recommended that when using fosthiazate granules to control rootknot nematode and root rot in ginger， it is applied at the highest dose of 38.8 g ai/m2，WK once 30 d before ginger cultivation to treat the soil， and ginger is harvested in the ginger harvest period.

Key words Fosthiazate; Ginger; Residue; Gas chromatography

Fosthiazate is an organophosphate nematicide produced by Japan Ishihara Sangyo Kaisha Ltd.[1]. It has contact toxicity to nematodes and systemic action on plants. The molecular formula is C9H18NO3PS2， and the chemical structure is shown in Fig. 1. It has a boiling point at 198， a vapor pressure of 5.6≠10-4 Pa （25） and water solubility of 9.85 g/L （20）. Field experiments have shown that the control effect of fosthiazate increases with the increase of its concentration[2]， thereby increasing the risk of residual pollution to crops， foods and the environment. In addition， a high concentration of fosthiazate residue can be toxic to humans， including neurotoxicity， reproductive and developmental toxicity， carcinogenicity and acute toxicity[3]. Therefore， the analysis and determination of fosthiazate residue is very important to the assessment of food safety and the risk that might cause to human health.

Studies have evaluated the efficacy of fosthiazate in the control of nematicides in potato[4]， cucumber[5]， tomato[2]， banana[6]and tobacco[7]， while no studies have been reported on ginger. At present， the residual analysis of fosthiazate is mainly gas chromatography （GC）[8]， gas chromatographymass spectrometry （GCMS）[9-10]， ultra performance liquid chromatography （UPLC）[11]and ultra performance liquid chromatographytandem mass spectrometry （UPLCMS/MS）[12]. In order to evaluate the safety of fosthiazate in ginger， this study applied gas chromatography to study the residue of fosthiazate in ginger and soil in Hunan， Jiangsu， Yunnan and Shandong by field plot trial， and established the detection method of fosthiazate residue in ginger.

Materials and Methods

Experimental instruments

Gas chromatograph： GC2010 plus， with flame photometric detector （Shimadzu Corporation， Japan）; AL204 analytical balance （Mettler Toledo Instrument Co.， Ltd.， Switzerland）; BL310 Sartorius type 1% balance （Sartorius， Germany）; HeiVap rotary evaporator （Heidolph， Germany）; ZD85 gas bath thermostatic oscillator （Jiangsu Jintan Jincheng Guosheng Experimental Instrument Factory）; SHBB95T circulating water vacuum pump （Zhengzhou Greatwall Scientific Industrial and Trade Co.， Ltd.） Company）; SC3614 low speed centrifuge （Anhui USTC Zonkia Scientific Instruments Co.， Ltd.）.

Reagents

Sodium chloride， acetone， nhexane， dichloromethane： analytically pure; acetonitrile： chromatographically pure， Grace Chemical Company， USA; PSA： ANPEL Laboratory Technologies （Shanghai） Inc.; fosthiazate standard： 98.5%， Hunan Tian Ye Biotechnology Co.，Ltd.

Field trial design

Test plots were set according to the requirements in "Guideline on Pesticide Residue Trials" （NY/T 7882004）[13]. Each plot had an area of 20 m2，WK， with three replicates. Guard zones were set between the plots， and a control plot was also set.

Degradation dynamic test

Ginger plants

In the dynamic monitoring test of fosthiazate residue in ginger plants， the nematicide was applied at a dose of 58.2 g a.i/m2，WK 30 d before ginger cultivation. According to the field trail design， from each plot， 4-12 plants （whole plants with roots removed） with normal growth above the soil surface were collected according to the random sampling method， and the sampling amount was not less than 2 kg. The ginger plants were cut into small pieces smaller than 1 cm which were mixed thoroughly and divided by quartering method， and 500 g was filled into a sealed container which was labeled inside and outside and stored in a refrigerator at -20 for testing.

Soil

For the dynamic treatment of fosthiazate residue in the soil， one plot was set and applied with the nematicide separately. The plot had an area of 50 m2，WK， and applied with the nematicide at a rate of 58.2 g a.i/m2，WK （the preparation amount of 90 g/m2，WK）. Soil samples were collected at 2 h， 1， 2， 3， 5， 7， 10， 14， 21 and 30 d after application. A blank control was also provided， and guard intervals were set between the treatments.

Collection of soil dynamic samples： Soil was collected from the plot randomly at more than eight sampling points with a sampling depth of 0-10 cm. The sampling amount per plot was not less than 2 kg each time， and impurities such as gravel， weeds and plant roots were removed in a stainless steel container. The collected samples were mixed evenly， and 500 g was filled into a sealed container which was labeled inside and outside and stored in a refrigerator at -20 for testing.

Final residue test

The low dose was 38.8 g a.i/m2，WK， and the high dose was 58.2 g a.i/m2，WK. The application was performed according to the designed time and application method once， and ginger and soil samples were randomly collected during the harvest period. Each treatment was repeated 3 times， and a clear water blank was set. Guard intervals were set between the treatments.

Collection of ginger samples： According to the field trial scheme， the lower tuber was evacuated out with a spade at a distance of about 5 cm from the selected ginger plant by the chessboard type sampling method in each plot. The tuber was picked off， and the soil on the tuber was shaken off. In each plot， sampling was performed at nine points to collect no less than 2 kg of tubers. Soil and residues on the tubers were gently brushed off with a soft brush， and the tubers were chopped with a stainless steel knife， mixed well and divided by quartering method， so as to take about 300 g of the sample which was filled into a sealed container which was labeled inside and outside and stored in a refrigerator at -20 for testing.

Final residue samples of soil： In each test plot， sampling was performed randomly at 6-12 sampling points with a sampling depth of 0-15 cm， and 2 kg of soil sample was sampled from each plot. Impurities were removed from the collected soil sample. The soil was then crushed， sieved with a 1 mm sampling sieve （the soil with high water content cannot be sieved）， and mixed well， to take about 500 g of soil by quartering method， which was filled into a sealed container which was labeled inside and outside and stored in a refrigerator at -20 for testing.

Experimental methods

Chromatographic detection conditions

A GC2010 plus gas chromatograph with FPD detector （P light filter） and AOC20i autosampler was used for detection. The GC used a 30 m ≠ 0.53 mm ≠ 1 mm capillary column， which is coated with SE30 chromatography stationary liquid internally. During detection， the detection chamber had a temperature at 80; the temperature of the column oven was started at 140， which was kept for 1 min and raised at 10/min to 210， which was kept for 3 min and then raised at 100/min to 280， which was kept for 5 min; the carrier gas was nitrogen; the flow rate of the makeup gas was 30.0 ml/min; and the injection volume was 2 l.

Preparation of standard solutions

A certain amount of the fosthiazate standard （accurate to 0.000 1 g） was accurately weighed， and dissolved with acetonitrile into 500 mg/L mother liquor， which was then prepared to 0.02， 0.05， 0.1， 0.5， 1.0， 2.5， 5.0 and 10.0 mg/L standard solutions by dilution method. The series of standard solutions were stored in a refrigerator at 4 for later use. The above series of standard solutions were respectively determined under the above chromatographic conditions， and a standard curve was drawn with the peak area （y） as the ordinate and the injected mass concentration （X， mg/L） as the abscissa.

Extraction and purification of samples

Soil and plants： A certain amount of the soil or plant sample （10 g） was weighed into a 250 ml conical flask， which was added with 40 ml of dichloromethane and shaken on a shaker at 150 r/min for 30 min. The sample mixture was filtered with cotton to 250 ml roundbottom flask， and the residue was further extracted in 40 ml of dichloromethane with shaking for 10 min. The extracts were combined， and extraction was performed once more. The extract was evaporated to near dryness in a water bath at 45. After cooling to room temperature， 2.00 ml of acetone and 0.05 g of PSA were added at room temperature， followed by vortexmixing for 1 min and standing for 2 min. The supernatant was filtered with a 0.45 mm filter into a sample vial for testing.

Ginger： The ginger sample was extracted by the same extraction method as above. The concentrated neardryness extract obtained in a flask was washed with 20 ml of acetonitrile and poured into a separating funnel containing 20 ml of nhexane， followed by shaking for 1 min. The upper hexane phase was discarded， and the extraction was repeated once with nhexane. Then， the acetonitrile phase was added with 30 ml of 5% sodium chloride aqueous solution and extracted with 30 ml≠2 dichloromethane， and the dichloromethane phase was collected， evaporated to dryness under reduced pressure in a water bath at 45 and then cooled. Then， 2.00 ml of acetone and 0.05 g of PSA were added at room temperature， followed by vortexmixing for 1 min and standing for 2 min. The supernatant was filtered with a 0.22 mm filter into a sample vial for testing.

Recovery test

Into the blank ginger and soil samples， three different mass fractions of fosthiazate standard solutions （0.02， 0.2 and 10 mg/kg） were added， respectively， and 0.02， 0.2， 10 and 200 mg/kg fosthiazate standard solutions were added into the blank plant sample， respectively. Each mass fraction was repeated 5 times.

The recovery was calculated according to formula （1）：

X=ba≠100%（1）

Wherein a is the theoretical addition amount of fosthiazate （mg/kg）; and b is the actually measured amount of fosthiazate in the addition test （mg/kg）.

Results and Analysis

Standard curve

Under the above chromatographic conditions， the series of standard solutions of 0.02， 0.05， 0.1， 0.5， 1.0， 2.5， 5.0， 10.0 mg/L were determined， and the standard working curve of fosthiazate was obtained. The standard solutions of fosthiazate showed a linear relationship in the range of 0.02-10.0 mg/kg between the peak area （Y） and the injected mass fraction （x）， and the regression equation was Y=19 160 243.69x-1 422 610.75， R2=1.00. The results showed that the mass concentration of the fosthiazate standard solution and their corresponding peak area had a good linear relationship within the selected mass concentration range.

Accuracy and precision

The accuracy and precision of fosthiazate were expressed by recovery and relative standard deviation （RSD） （Table 1）. The results showed that when adding 0.02， 0.2 and 10 mg/kg fosthiazate into the ginger tuber and soil and 0.02， 0.2， 10 and 200 mg/kg fosthiazate into the plant， the recovery of fosthiazate was between 79.9% and 92.4% on the ginger tuber， with the relative standard deviation （RSD） of 1.2%-1.9%; the recovery of fosthiazate ranged from 84.0% to 98.9% on the soil， with the relative standard deviation （RSD） of 1.3% to 4.1%; and the recovery of fosthiazate was between 79.0% and 110.5% on the plant， with the relative standard deviation （RSD） of 0.8%-5.4%. The requirements for the determination of residues were satisfied.

Digestion dynamic test

The test results of 2017 showed that the residual amount of fosthiazate in plants and soil decreased with time， and the interval for application was exponentially related to the residual amount in ginger plants and soil. The digestion dynamic curve accorded with the firstorder kinetic equation Ct = C0e-kt. The firstorder kinetic equation and halflife of fosthiazate in ginger plants and soil are shown in Table 2.

It could be seen from Table 2 that during the test period at the four test sites， the original accumulation of fosthiazate at the Jiangsu test site was 136 mg/kg， and the original accumulations in the plant at the Shandong and Yunnan test sites were lower than the lowest detection concentration; the original accumulations in the soil were equivalent， in the range of 0.155-1.99 mg/kg; and the halflife in the plant was 7.2-8.7 d， and the halflife in the soil was 9.9-19.8 d， indicating that the digestion rates of fosthiazate in ginger plants and soil were both higher.

Final residue test

The final residual amounts in ginger and soil were determined by applying the agent at the high dose of 58.2 g a.i/m2，WK and the low dose of 38.8 g a.i/m2，WK once， respectively. The results of fosthiazate residue are shown in Table 3.

From the results of the final residue test， the residual amount of fosthiazate in ginger was <0.02 mg/kg， and the residual amount in the soil ranged from 0.02 to 0.103 mg/kg. Due to the complexity of the medication environment， there is no strict correlation between the application dose and the residual amount.

Conclusions and Discussion

Although there have been reports on the analysis of pesticide residues in ginger， no references were searched on the detection method of fosthiazate residue in ginger samples. In this paper， with ginger and soil as the research objects， the determination method of fosthiazate was established by gas chromatography with FPD detector. The standard curve obtained was Y=19 160 243.69x-1 422 610.75， R2=1.00. The method has the precision， accuracy and sensitivity in line with the requirements of pesticide residue standards， and is suitable for the determination of fosthiazate residue in ginger tubers， ginger plants and soil. It is recommended that when using fosthiazate granules to control rootknot nematode and root rot in ginger， it is applied at the highest dose of 38.8 g ai/m2，WK once 30 d before ginger cultivation to treat the soil， and ginger is harvested in the ginger harvest period.

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