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Abstract [Objectives] This study was conducted to establish an analytical method for the determination of ethephon residue in walnuts by UPLCMS/MS.
[Methods] This study was conducted to establish an analytical method for the determination of ethephon residue in walnuts by UPLCMS/MS.
[Results] The limit of quantification was 0.1 mg/kg and the recoveries were 94%-105% with the relative standard deviations of 0.82%-2.64% at the spiked levels of 0.1-5.0 mg/kg.
[Conclusions] The accuracy and sensitivity of the method meet the requirements of pesticide residue analysis， and the experimental method is simple to operate.
Key words Walnut; Ethephon; Residue analysis; LCMS/MS
Ethephon is a plant growth regulator that promotes ripening. It is widely used in accelerating the ripening of fruits and vegetables such as passion fruit， bananas， apples， etc. It is also gradually used for accelerating the ripening of rice and other cereals and nuts such as walnuts[1-3]. In recent years， the use of ethephon has increased year by year. Longterm use of foods with excessive ethephon will accelerate the aging of the digestive tract， causing peptic ulcers and certain damage to the brain and kidneys[4].
There are a lot of literatures on the analysis of ethephon residue in fruits， vegetables and grains[5-8]， but the analysis method of the residue in nuts such as walnuts has not been reported yet. Therefore， it is necessary to develop a method for determining the residual amount in nuts. To this end， in this study， we established a method for the determination of ethephon residue in walnuts by LCMS/MS method， which also can be used for the rapid determination of ethephon in high oilcontaining matrix samples.
Materials and Methods
Materials and reagents
Chromatographic acetonitrile （TEDIA， USA）; chromatographic methanol （TEDIA， USA）; formic acid （chromatographic pure， Tianjin Guangfu Technology Development Co.， Ltd.）; PSA （particle size 40-63 μm， 60A， CNW）; C18SPE cartridge （500 mg/3 ml， Tianjin BonnaAgela Technologies）.
Standard
Ethephon standard： purity 96.0%， Dr. Ehrenstorfer GmbH.
Instruments and equipment
Ultrahigh performance liquid chromatography/tandem mass spectrometry （UPLCMS/MS） （ACQUTY Ultra Performance LCMS/MS， Waters， USA）; one hundred thousandth electronic balance （sartorius BT25S， Sartorius Scientific Instruments （Beijing）Co.， Ltd.）; electronic balance （B5002， Shanghai LiangpingInstrument Co.， Ltd.）; water bath thermostat oscillator （WHY2， Jintan Jincheng Guosheng Laboratory Instrument Factory）; ultrasonic cleaner （KQ600B， Kunshan Ultrasonic Instrument Co.， Ltd.）; low speed centrifuge （SC3610， Anhui USTC Zonkia Scientific Instruments Co.， Ltd.）; glass instrument airflow dryer （KQC， Gongyi Yuhua Instrument Co.， Ltd.）; volumetric flask， glass pipette， etc. Experiment methods
Standard solution preparation
A certain amount of ethephon standard was accurately weighed and dissolved and diluted to 10 mlwith chromatographic acetonitrile. The standard solution was transferred to a brown standard stock bottle， and then diluted to 100 mg/L for use. The 100 mg/L ethephon standard solution was diluted with walnut blank medium to 2 000， 1 000， 500， 200， 100， 50 and 20 μg/L standard solutions， which were determined under mass spectrometry conditions. A standard curve was finally drawn with the concentrations of ethephon standard working solutions plotted on the abscissa and the peak areas plotted on the ordinate.
Recovery test
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed， and added with the ethephon standard working solutions to the concentrations of 0.1， 0.5 and 5.0 mg/kg， respectively. Each of the three addition levels was done with five replicates in parallel. The prepared solutions were determined， as well as the concentration of the blank sample.
Sample extraction method
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed into a 50 ml centrifuge tube， and added with 20 ml of 0.1% formic acid solution. The mixture was ultrasonically extracted for 20 min， mechanically oscillated for 30 min and then centrifuged at 4 000 r/min for 5 min.Next， 5 ml of the supernatant was transferred to a 10 ml centrifuge tube and centrifuged at 12 000 r/min for 10 min.
The C18SPE cartridge was activated with 3 ml of methanol and 3 ml of pure water in sequence. After the liquid was drained， 4 ml of the above extract was pipetted into the C18SPE cartridge. The first 1 ml of the eluent was discarded， and then the eluate was collected in a 15 ml centrifuge tube and mixed well. The collected eluate was finally filtered with a 0.22 μm hydrophilic filter membrane for LCMS/MS determination.
Instrument determination conditions
Column： Waters ACQUITY UPLC R BEH C18， 100 mm × 2.1 mm × 1.7 μm; column temperature： 40 ℃; mobile phase： water/methanol （V/V=98/2） +0.1% formic acid∶ 0.1% formic acid methanol=95∶ 5; flow rate： 0.3 ml/min; injection amount： 10 μl.
Ionization mode： ESI-; thin tube voltage： 2.5 kv; ion source temperature： 150 ℃; cone gas flow： 50 L/Hr; desolvation gas temperature： 350 ℃; desolvation gas flow： 700 L/Hr; monitoring mode： MRM mode.
Results and Analysis Determination results of standard solutions
The 2 000， 1 000， 500， 200， 100， 50 and 20 μg/L standard working solutions were detected by UPLCMS/MS. The results showed that the peak area had a good linear relationship with the concentration in this concentration range. The ethephon standard curve equation was y=9.464 5x-133.24， R2=0.999 9. The results are shown in Table 1.
Results of recovery test
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed， and added with the ethephon standard working solutions to the concentrations of 0.1， 0.5 and 5.0 mg/kg， respectively. Each addition level was done with five replicates in parallel. Purification， extraction and determination were performed according to above methods. The recovery results are shown in Table 2.
The recoveries were 94%-105% with the relative standard deviations of 0.82%-2.64% at the spiked levels of 0.1-5.0 mg/kg. The minimum addition concentration in the recovery test was selected as the limit of quantitation， i.e.， 0.1 mg/kg.
Discussion and Conclusions
Selection of extraction solvent
Ethephon has a strong polarity and is easily soluble in polar solvents such as acetonitrile， methanol and ethyl acetate. There
fore， when treating walnut samples， a polar solvent was selected as the extraction solvent. First， when using acetonitrile， acetonitrilewater， methanol and methanolwater for extraction， since ethephon in the walnut matrix is extracted with the organic reagents， a large amount of impurities such as oils and the like which are easily dissolved in organic reagents are extracted together. And the extracts should be further purified with C18cartridge， but the purification effect is still not good. The extraction effect of ethephon is good under acidic conditions in reported literatures， and the polarity of ethephon is relatively large， so 0.1% formic acid aqueous solution was selected for the extraction of ethephon in walnuts.
Selection of mobile phase
The method used Waters ACQUITY UPLC R BEH C18column for analysis. With water/methanol （V/V=98/2）+0.1% formic acid： 0.1% formic acid methanol solution as the mobile phase， the ethephon peak had a better shape， and the separation effect and ionic strength were best.
Optimization of MS conditions
In this study， the electrospray ion source （ESI） was selected for condition optimization， and the ethephon standard solution （concentration： 1.0 mg/L） was used to scan the parent ion under ESI+ and ESI- conditions， respectively. The results showed that the ethephon had a higher response value under the ESI- condition， and the secondorder MS fragment analysis was carried out， obtaining two ion pairs： 142.8/78.7 and 142.8/106.8， of which 142.8/106.8 was used as the quantitative ion pair. The residual limit of ethephon in walnuts in China is 0.5 mg/kg. In the standard analytical method， the residual ethephon in fruits and vegetables needs to be derivatized and extracted， and the operation steps are complicated. The related literatures reported are all about the detection of ethephon residues in fruits and vegetables， and the methods are less practical for the matrixes with higher oil contents such as nuts. In this study， 0.1% formic acid aqueous solution was used for extraction， purification was performed with a C18SPE column evolution， and determination was carried out by UPLCMS/MS. Under the electrospray ionization negative voltage mode， the waters ACQUITY UPLC R BEH C18column was used to perform analysis， and a method for detecting the residual amount of ethephon was established. The method has the advantages of simple operation， less solvent extraction and fast detection speed， and can be applied to the detection of ethephon residues in nut crops such as walnuts.
Conclusions
The method of extraction with formic acid aqueous solution， purification by solidphase extraction and determination by UPLCMS/MS can quickly and effectively realize the residue analysis of ethephon in walnuts， and the accuracy and precision of the method better satisfy the requirements.
References
[1] LI J， LIAO JY， LU YX， et al. Effect of ethephon and ethanol treatment on ripening quality of passion fruit[J]. Journal of Shanxi Agricultural Sciences， 2019， 47（9）： 1509-1512， 1527. （in Chinese）
[2] LIU L， LIU XP， HUANG L， et al. Effect of postharvest ethylene degreening， treatment on the quality of “Eureka” lemon[J]. Food and Fermentation Industries， 2004（4）： 28 （in Chinese）
[3] LI WG， MING Y. Evaluation of the efficacy of 40% ethylene dilute agent for tomato growth regulation and tomato quality determination[J]. Xin Nongye， 2018（12）： 32-33. （in Chinese）
[4] YU WH， GAO YQ， ZHAO W， et al. Study on mutation of ethephon in mice[J]. Chinese Journal of Pesticide Science， 2006， 8（2）： 184-186. （in Chinese）
[5] CAO HE， XIA H， LU P， et al. Determination of ethephon residues in bean sprout samples by static headspace gas chromatography[J]. Agrochemicals， 2011， 50（4）： 286-288. （in Chinese）
[6] DONG QJ， ZHAO JH， LI Y. GC determination of residual amount of ethephon in cucumber after derivatization with diazomethane[J]. Physical Testing and Chemical Analysis （Part B： Chemical Analysis）， 2011， 47（9）： 1071-1073. （in Chinese）
[7] ZHANG Y， DING CR， LU Y. Detection of ethephons residues in fruits and vegetables by high performance liquid chromatography/tandem mass spectrometric method[J]. Xinjiang Agricultural Sciences， 2015， 52（1）： 157-161. （in Chinese）
[8] LI EH， WU BB， ZHANG W， et al. Determination of ethephon residue in barley by HPLCMS/MS[J]. Pesticide Science and Administration， 2017， 38（10）： 36-40. （in Chinese）
[Methods] This study was conducted to establish an analytical method for the determination of ethephon residue in walnuts by UPLCMS/MS.
[Results] The limit of quantification was 0.1 mg/kg and the recoveries were 94%-105% with the relative standard deviations of 0.82%-2.64% at the spiked levels of 0.1-5.0 mg/kg.
[Conclusions] The accuracy and sensitivity of the method meet the requirements of pesticide residue analysis， and the experimental method is simple to operate.
Key words Walnut; Ethephon; Residue analysis; LCMS/MS
Ethephon is a plant growth regulator that promotes ripening. It is widely used in accelerating the ripening of fruits and vegetables such as passion fruit， bananas， apples， etc. It is also gradually used for accelerating the ripening of rice and other cereals and nuts such as walnuts[1-3]. In recent years， the use of ethephon has increased year by year. Longterm use of foods with excessive ethephon will accelerate the aging of the digestive tract， causing peptic ulcers and certain damage to the brain and kidneys[4].
There are a lot of literatures on the analysis of ethephon residue in fruits， vegetables and grains[5-8]， but the analysis method of the residue in nuts such as walnuts has not been reported yet. Therefore， it is necessary to develop a method for determining the residual amount in nuts. To this end， in this study， we established a method for the determination of ethephon residue in walnuts by LCMS/MS method， which also can be used for the rapid determination of ethephon in high oilcontaining matrix samples.
Materials and Methods
Materials and reagents
Chromatographic acetonitrile （TEDIA， USA）; chromatographic methanol （TEDIA， USA）; formic acid （chromatographic pure， Tianjin Guangfu Technology Development Co.， Ltd.）; PSA （particle size 40-63 μm， 60A， CNW）; C18SPE cartridge （500 mg/3 ml， Tianjin BonnaAgela Technologies）.
Standard
Ethephon standard： purity 96.0%， Dr. Ehrenstorfer GmbH.
Instruments and equipment
Ultrahigh performance liquid chromatography/tandem mass spectrometry （UPLCMS/MS） （ACQUTY Ultra Performance LCMS/MS， Waters， USA）; one hundred thousandth electronic balance （sartorius BT25S， Sartorius Scientific Instruments （Beijing）Co.， Ltd.）; electronic balance （B5002， Shanghai LiangpingInstrument Co.， Ltd.）; water bath thermostat oscillator （WHY2， Jintan Jincheng Guosheng Laboratory Instrument Factory）; ultrasonic cleaner （KQ600B， Kunshan Ultrasonic Instrument Co.， Ltd.）; low speed centrifuge （SC3610， Anhui USTC Zonkia Scientific Instruments Co.， Ltd.）; glass instrument airflow dryer （KQC， Gongyi Yuhua Instrument Co.， Ltd.）; volumetric flask， glass pipette， etc. Experiment methods
Standard solution preparation
A certain amount of ethephon standard was accurately weighed and dissolved and diluted to 10 mlwith chromatographic acetonitrile. The standard solution was transferred to a brown standard stock bottle， and then diluted to 100 mg/L for use. The 100 mg/L ethephon standard solution was diluted with walnut blank medium to 2 000， 1 000， 500， 200， 100， 50 and 20 μg/L standard solutions， which were determined under mass spectrometry conditions. A standard curve was finally drawn with the concentrations of ethephon standard working solutions plotted on the abscissa and the peak areas plotted on the ordinate.
Recovery test
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed， and added with the ethephon standard working solutions to the concentrations of 0.1， 0.5 and 5.0 mg/kg， respectively. Each of the three addition levels was done with five replicates in parallel. The prepared solutions were determined， as well as the concentration of the blank sample.
Sample extraction method
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed into a 50 ml centrifuge tube， and added with 20 ml of 0.1% formic acid solution. The mixture was ultrasonically extracted for 20 min， mechanically oscillated for 30 min and then centrifuged at 4 000 r/min for 5 min.Next， 5 ml of the supernatant was transferred to a 10 ml centrifuge tube and centrifuged at 12 000 r/min for 10 min.
The C18SPE cartridge was activated with 3 ml of methanol and 3 ml of pure water in sequence. After the liquid was drained， 4 ml of the above extract was pipetted into the C18SPE cartridge. The first 1 ml of the eluent was discarded， and then the eluate was collected in a 15 ml centrifuge tube and mixed well. The collected eluate was finally filtered with a 0.22 μm hydrophilic filter membrane for LCMS/MS determination.
Instrument determination conditions
Column： Waters ACQUITY UPLC R BEH C18， 100 mm × 2.1 mm × 1.7 μm; column temperature： 40 ℃; mobile phase： water/methanol （V/V=98/2） +0.1% formic acid∶ 0.1% formic acid methanol=95∶ 5; flow rate： 0.3 ml/min; injection amount： 10 μl.
Ionization mode： ESI-; thin tube voltage： 2.5 kv; ion source temperature： 150 ℃; cone gas flow： 50 L/Hr; desolvation gas temperature： 350 ℃; desolvation gas flow： 700 L/Hr; monitoring mode： MRM mode.
Results and Analysis Determination results of standard solutions
The 2 000， 1 000， 500， 200， 100， 50 and 20 μg/L standard working solutions were detected by UPLCMS/MS. The results showed that the peak area had a good linear relationship with the concentration in this concentration range. The ethephon standard curve equation was y=9.464 5x-133.24， R2=0.999 9. The results are shown in Table 1.
Results of recovery test
A certain amount of treated walnut blank sample （5.00 g） was accurately weighed， and added with the ethephon standard working solutions to the concentrations of 0.1， 0.5 and 5.0 mg/kg， respectively. Each addition level was done with five replicates in parallel. Purification， extraction and determination were performed according to above methods. The recovery results are shown in Table 2.
The recoveries were 94%-105% with the relative standard deviations of 0.82%-2.64% at the spiked levels of 0.1-5.0 mg/kg. The minimum addition concentration in the recovery test was selected as the limit of quantitation， i.e.， 0.1 mg/kg.
Discussion and Conclusions
Selection of extraction solvent
Ethephon has a strong polarity and is easily soluble in polar solvents such as acetonitrile， methanol and ethyl acetate. There
fore， when treating walnut samples， a polar solvent was selected as the extraction solvent. First， when using acetonitrile， acetonitrilewater， methanol and methanolwater for extraction， since ethephon in the walnut matrix is extracted with the organic reagents， a large amount of impurities such as oils and the like which are easily dissolved in organic reagents are extracted together. And the extracts should be further purified with C18cartridge， but the purification effect is still not good. The extraction effect of ethephon is good under acidic conditions in reported literatures， and the polarity of ethephon is relatively large， so 0.1% formic acid aqueous solution was selected for the extraction of ethephon in walnuts.
Selection of mobile phase
The method used Waters ACQUITY UPLC R BEH C18column for analysis. With water/methanol （V/V=98/2）+0.1% formic acid： 0.1% formic acid methanol solution as the mobile phase， the ethephon peak had a better shape， and the separation effect and ionic strength were best.
Optimization of MS conditions
In this study， the electrospray ion source （ESI） was selected for condition optimization， and the ethephon standard solution （concentration： 1.0 mg/L） was used to scan the parent ion under ESI+ and ESI- conditions， respectively. The results showed that the ethephon had a higher response value under the ESI- condition， and the secondorder MS fragment analysis was carried out， obtaining two ion pairs： 142.8/78.7 and 142.8/106.8， of which 142.8/106.8 was used as the quantitative ion pair. The residual limit of ethephon in walnuts in China is 0.5 mg/kg. In the standard analytical method， the residual ethephon in fruits and vegetables needs to be derivatized and extracted， and the operation steps are complicated. The related literatures reported are all about the detection of ethephon residues in fruits and vegetables， and the methods are less practical for the matrixes with higher oil contents such as nuts. In this study， 0.1% formic acid aqueous solution was used for extraction， purification was performed with a C18SPE column evolution， and determination was carried out by UPLCMS/MS. Under the electrospray ionization negative voltage mode， the waters ACQUITY UPLC R BEH C18column was used to perform analysis， and a method for detecting the residual amount of ethephon was established. The method has the advantages of simple operation， less solvent extraction and fast detection speed， and can be applied to the detection of ethephon residues in nut crops such as walnuts.
Conclusions
The method of extraction with formic acid aqueous solution， purification by solidphase extraction and determination by UPLCMS/MS can quickly and effectively realize the residue analysis of ethephon in walnuts， and the accuracy and precision of the method better satisfy the requirements.
References
[1] LI J， LIAO JY， LU YX， et al. Effect of ethephon and ethanol treatment on ripening quality of passion fruit[J]. Journal of Shanxi Agricultural Sciences， 2019， 47（9）： 1509-1512， 1527. （in Chinese）
[2] LIU L， LIU XP， HUANG L， et al. Effect of postharvest ethylene degreening， treatment on the quality of “Eureka” lemon[J]. Food and Fermentation Industries， 2004（4）： 28 （in Chinese）
[3] LI WG， MING Y. Evaluation of the efficacy of 40% ethylene dilute agent for tomato growth regulation and tomato quality determination[J]. Xin Nongye， 2018（12）： 32-33. （in Chinese）
[4] YU WH， GAO YQ， ZHAO W， et al. Study on mutation of ethephon in mice[J]. Chinese Journal of Pesticide Science， 2006， 8（2）： 184-186. （in Chinese）
[5] CAO HE， XIA H， LU P， et al. Determination of ethephon residues in bean sprout samples by static headspace gas chromatography[J]. Agrochemicals， 2011， 50（4）： 286-288. （in Chinese）
[6] DONG QJ， ZHAO JH， LI Y. GC determination of residual amount of ethephon in cucumber after derivatization with diazomethane[J]. Physical Testing and Chemical Analysis （Part B： Chemical Analysis）， 2011， 47（9）： 1071-1073. （in Chinese）
[7] ZHANG Y， DING CR， LU Y. Detection of ethephons residues in fruits and vegetables by high performance liquid chromatography/tandem mass spectrometric method[J]. Xinjiang Agricultural Sciences， 2015， 52（1）： 157-161. （in Chinese）
[8] LI EH， WU BB， ZHANG W， et al. Determination of ethephon residue in barley by HPLCMS/MS[J]. Pesticide Science and Administration， 2017， 38（10）： 36-40. （in Chinese）