Simultaneous determination of 15 pesticide residues in Chinese cabbage and cucumber by liquid chromatography-tandem mass spectrometry utilizing online turbulent flow chromatography

Sufang Fan,Junmei Ma,Meirong Cao,Juan Wang,Leilei Zhang,Yan Zhang,?,Qiang Li,?,Jia Chen

aHebei Key Laboratory of Forensic Medicine,College of Forensic Medicine,Hebei Medical University,Shijiazhuang 050017,China

bHebei Food Safety Key Laboratory,Hebei Food Inspection and Research Institute,050227,Shijiazhuang,China

cCollege of Food Science and Technology,Hebei Agricultural University,071001,Baoding,China

Keywords:

Pesticide residues

Online turbulent flow chromatography

Liquid chromatography-tandem mass

spectrometry

ABSTRACT

In this experiment,a liquid chromatography tandem mass spectrometry method was built to determine 15 pesticide residues in Chinese cabbage and cucumber samples based on online turbulent flow chromatography purification.After modified quick,easy,cheap,effective,rugged,and safe(QuEChERS)extraction,extracts were directly injected to the TLX(TurboFlow Liquid Xcalibur)system and brought to TurboFlowTMcolumns for on-line purification and then transferred to analytical column for further separation and analysis.TurboFlowTMcolumns types,transfer flow rate,and transfer time were optimized.Limits of detection and limits of quantification of the method obtained for 15 pesticide residues were ranged between 0.2–1.0 μg/kg and 0.5–2.0 μg/kg in Chinese cabbage and cucumber samples.Recoveries of pesticide residues were in range of 75.3%–103.7%.Matrix effects for 15 pesticides were in range of 5.6%–106.6%.The developed method has been successfully used for the determination of 15 pesticide residues in real samples.

1.Introduction

As one kind of leaf vegetable,Chinese cabbage is widely cultivated in China.Cucumber is very popular in China for its usage both as vegetable and fruit.While Chinese cabbage and cucumber are often attacked by various insects and/or pests[1],in order to prevent or reduce losses[2],pesticides are widely used in the cultivation of Chinese cabbage and cucumber.The functions of pesticides are not only to provide reliable supplies of agricultural produce at affordable prices to consumers,but also to ensure high profits to farmers[3–5].Pesticides play a key role in the control of pests.On the other hand,it also brings unexpected negative effects to human and animal health[6].The overuse of pesticides has caused many environmental problems,such as the destruction of the natural balance, widespread resistance of pests,environmental pollution,and hazards to no-target organisms and humans[7].Due to the potential hazards,the pesticides in Chinese cabbage and cucumber are harmful to our health[1].The standard of maximum residue limits(MRLs)in many countries are becoming more strict,with the purpose of protecting consumer’s health[8].

Because the concentrations of pesticide residues in Chinese cabbage and cucumber are low,it is necessary to build a sensitive multi-residue method to detect the pesticide residues[9].Gas chromatography or liquid chromatography tandem mass spectrometry have been widely used in the detection of pesticide residues[10].High resolution mass spectrometry,such as time-of- flight(TOF)and quadrupole/orbitrap mass spectrometry,have also used in the pesticide analysis[11–13].In order to determine pesticide residues,a proper sample preparation technique is required to isolate and concentrate the target compounds[14].Traditional sample preparation and purification methods include solid-phase extraction(SPE),solid-phase microextraction(SPME),gel permeation chromatography(GPC),microwave-assisted extraction(MAE),and supercritical fluid extraction(SFE)[15].The quick,easy,cheap,effective,rugged,and safe(QuEChERS)method has been widely used in the pesticide residues analysis,which was introduced by Anastassuades et al.[16,17].The technique involves micro-scale extraction using acetonitrile and purifies by dispersive solid-phase extraction(d-SPE)[18].Since the first report,QuEChERS has been gaining significant popularity.Primary secondary amine(PSA),ethylenediamine-N-propyl silanized silica gel,is usually used in QuEChERS as one kind of bulk sorbent to remove various polar organic acids,polar pigments,some sugars,and fatty acids[19].In the current study,modified QuEChERS extraction is usually used in the sample pretreatment[17,20–22].However,many results showed that the influence of co-elutes was significant for some kinds of pesticides after the purification of QuEChERS.So,it is necessary to eliminate the influence the co-elutes when QuEChERS is used.

In our study,online purification based on turbulent flow chromatography(TFC)was used to simultaneously detect 15 pesticide residues(imidacloprid,3-hydroxycarbofuran,acetamiprid,pirimicarb,aldicarb,propoxur,carbofuran,carbaryl,isoprocarb,metalaxyl,carbendazim,isazofos,diflubenzuron,chlorbenzuron,and phoxim,which are often detected or inspected in the routine monitor) in Chinese cabbage and cucumber samples.The online TFC is one kind of online solid phase extraction technology developed by Thermo Scientific.This online automated system uses turbulent columns to separate analytes of interest from their complex matrices,depending on diffusion,chemistry,and size exclusion[23,24].The TurboFlowTMcolumns have large particles,which lead to more interstitial space.TurboFlowTMtechnology uses two mechanisms:it exploits the difference between large and small molecules and it incorporates column chemistry[25].After online purification,the target analytes were transferred to the analytical column for further separation.The online TFC column is very effective in the complex substrate purification compared to the traditional of fline solid phase extraction[26].The online TFC purification has been used in the direct determination of free state low molecular weight compounds in serum[27],organophosphate,pyrethroid metabolites in human urine samples[28],17-hydroxyprogesterone,and rostenedione,testosterone,cortisol in human serum[29],10 steroid metabolites in serum[30],irinotecan,and SN38 in human plasma[31].The object of this experiment was to develop a liquid chromatography-tandem mass spectrometry method based on TFC to determine 15 pesticide residues in Chinese cabbage and cucumber.After modified QuEChERS extraction,samples were purified by online TFC and finally determined by mass spectrometry,and the matrix effect of the method was reduced.

2.Materials and methods

2.1.Reagents and materials

Fifteen pesticide standards were supplied by the Agro-Environmental Protection Institute,Ministry of Agriculture,Tianjin,China.A total of 10 mg/L stock solutions of 15 pesticides were prepared in acetonitrile and stored at 4?C.The working solutions were prepared before use.Acetonitrile with HPLC-grade was purchased from Fisher Scientific and the formic acid with HPLC-grade was supplied by Fluka Analytical.Anhydrous sodium chloride(NaCl)with analytical reagent grade was obtained from the Sinopharm Chemical Reagent(Beijing,China).High purity water used in the experiment was obtained from a MilliQ purification system(Millipore,Bedford,MA,USA).Chinese cabbage and cucumber samples were bought from a local market[24].

Fig.1.Schematic diagram of the TFC,(a)valve configuration during sample loading and clean-up,(b)valve configuration during sample transfer,(c)valve configuration during sample elution and loop fill.

2.2.Instruments

A transcend and AriaTMsystem was used for sample purification,which contained a TLX system and a LX system.On the TLX system,compounds of interest are retained on the TurboFlowTMcolumn while the sample matrix molecules flow to waste.The TLX autosampler injects sample directly into the TurboFlowTMcolumn,a large-volume loop(100 μL)was equipped for injection.

A multiple column module(MCM)was equipped in the system and six types of TurboFlowTMcolumns and two analytical columns were configurated in our system.The six types of TurboFlowTMcolumns were as follows:XL C18(0.5×50 mm,silica type),XL C18-P(0.5×50 mm,silica type),Cyclone(0.5×50 mm,polymer type),Cyclone-P(0.5×50 mm,polymer type),Cyclone-MAX(0.5×50 mm,polymer type),and Cyclone-MCX(0.5×50 mm,polymer type)[24].Thermo C18(1.7 μm,50 × 2.1 mm)and Phenomenex C18(1.7 μm,100 × 2.1 mm)were used as analytical columns.The TurboFlowTMcolumns were supplied by Thermo Fisher Scientific.

As presented in Fig.1,the connection of TurboFlowTMcolumns and analytical columns was carried out by four 6-port valves.The loading pump was equipped with TurboFlowTMcolumns,which was a response for the online purification,and an eluting pumpwas configured for the analytical column,which was a response for further separation.When the TLX system was used,the samples were injected to the TurboFlowTMcolumn for online purification and compounds of interest were retained on the TurboFlowTMcolumn while the sample matrix molecules flowed to the waste.When the online purification process finished,the target analytes were brought to analytical column for further separation.The online purification and analytical analyses were automatically controlled by AriaTMoperating software 1.6.3(Thermo Scientific,Waltham,USA).

Table 1The TFC-LC conditions.

A triple quadrupole mass spectrometer(TSQ Vantage,Thermo Scientific,Waltham,USA)was used as the detector,and electron spray ionization(ESI)was equipped.Xcalibur was used for data acquisition.

2.3.Samples preparation

A total of 10 g Chinese cabbage and cucumber samples were weighed and placed in 50 mL centrifuge tubes,10 mL acetonitrile was added and samples were mixed thoroughly for 1 min with a vortex mixer(IKA,Staufen,German).After the addition of 1 g anhydrous NaCl and 4 g anhydrous magnesium sulfate,the samples were shaken vigorously for 1 min and centrifuged by a 3K15 centrifuge(Sigma,Osterode,German)for 4 min at 9500 r/min[17].1 mL supernatant was filtered using a 0.22 μm organic membrane filter.The filtered fluid was then transferred into a sample vial for further cleanup and analysis[24].In the experiment,the extracts were purified by on-line TFC rather thand-SPE.

2.4.Online TFC conditions

The online purification process was controlled by the AriaTMTLX system.As shown in Fig.1,there were four steps in the whole process,including sample loading,sample transfer,sample elution,and column equilibration[23].The purpose of the sample loading step was to keep the target analytes on the Turbo flowTMcolumn and remove other compounds.The aim of sample transfer step was to carry the target analytes from the purification column to the analytical column.The sample elution was to separate and analyze the target analytes and the column equilibration was to flush the column and prepare for the next analysis.The whole process was finished by column switching.A XL C18(0.5×50 mm,silica type)was employed as the Turbo flowTMcolumn.Samples were injected into the TLX injection port and were brought to the XL C18column for online solid phase extraction.In order to avoid overload,samples were diluted before analysis or reduce injection volume[25].

The TFC-LC conditions were shown in Table 1.The injection volume was 20 μL.The sample was directly injected into the XL C18 TurboFlowTMcolumn with a flow rate of 2.0 mL/min.Formic acid in water(0.1%,V/V)was used as the mobile phase,the analytes were retained,and other sample matrix was eluted to the waste.The next step was transfer;0.1% formic acid solution was used to transfer the target compounds from the TurboFlowTMcolumn to the analytical column and the transfer time was 120 s.After the transfer,the XLC18column was re-washed by methanol for 60 s and then washed with methanol for 60 s.The flow rate was 1.0 mL/min.The mobile phase(1.0 mL/min,acetonitrile/isopropanol/acetone 1:1:1,V/V)was used to wash the TurboFlowTMcolumn.The flow rate was 1.0 mL/min and the wash time was 180 s.The loop was filled by methanol for 180 s with a flow rate of 1.0 mL/min.Finally,the TurboFlowTMcolumn was equilibrated using water containing 0.1% formic acid for 330 s at a flow rate of 1.0 mL/min[25].

2.5.HPLC analysis

After online purification,target analytes were brought into the analytical column for further separation and analysis,and this step was finished by valve switching.The analytical column was in backflush mode for 1 min,then the valve was switched back and the separation analysis was started.The Phenomenex C18column was used as the analytical column.Methanol and 0.1% formic acid solution were used as the mobile phase, and the elution conditions were presented in Table 1.

3.Results and discussion

3.1.Optimization of mass spectrometry

In order to optimize the parameters of mass spectrometry,standard solutions of 15 pesticides were directly injected into mass spectrometry using a syringe pump.The operating parameters of mass spectrometry were as follows:the spray voltage was 3500 V,the flow rate of the sheath gas(N2)was 4.6 L/min,the flow rate of the auxiliary gas(N2)was 8.8 L/min,the capillary temperature was 350?C,and the vaporizer temperature was 320?C.

A positive scan mode was used in our experiments,and[M+H]+ions were selected as precursor ions.Selected reaction monitoring(SRM)mode was used to monitor the precursor-to-production transition.The collision energy of the analyte was confirmed through compound optimization[24].All parameters of mass spectrometry were presented in Table 2.The parameter of the S-lens was a voltage added on the tube lens which could accelerate ion transmission.

Table 2Parameters of mass spectrometry.

3.2.Optimization of the TFC liquid chromatography conditions

3.2.1.Optimization of LC conditions

Several related conditions of the online TFC procedure,such as liquid chromatography(LC)conditions,TurboFlowTMcolumn type,transfer flow rate,and transfer time,should be optimized.

In order to achieve the optimal separation effect,it is necessary to select the most suitable column and the best elution conditions.Two analytical columns were compared,which were the Thermo C18column(1.7μm,50×2.1mm)and the Phenomenex C18column(1.7μm,100×2.1mm).Peak shapes of the compounds were better when the Phenomenex column was used,so the Phenomenex C18column was chosen as the analytical column in this experiment.The chromatographic performance of methanol and acetonitrile as the organic phase was compared. The response of compounds were higher when methanol was used.When0.1% formic acid was added to the aqueous phase, the response of analytes were relative higher.As a result,methonal and 0.1% formic acid solution were chosen as the mobile phase.

Different elution conditions were tested in order to get the best separation effect,and the optimized elution conditions were presented in Table 1.The samples were injected into the system when the organic phase content was low.The retained analytes were eluted successfully with the increase of organic solvent.A total of 15 pesticides were successfully separated within 11 min under the optimum separation conditions.The analytical column was reequilibrated with the initial mobile phase for 2.5 min after each analysis.

Fig.2.Response of 15 pesticides with six types of TurboFlowTMcolumns.

3.2.2.Optimization of online TurboFlowTMcolumns

Six different types of TurboFlowTMcolumns were investigated in order to find the most appropriate purification column.A suitable column must be able to retain the analyte to the maximum content and elute the analyte satisfactorily and avoid carry over between samples[24].

A mixed standard solution of 15 pesticides with contents of 100 ng/mL was injected into the six TurboFlowTMcolumns under the optimal HPLC conditions.The peak shapes and peak areas of analytes were compared.As shown in Fig.2,the response of 10 pesticides in XL C18was the highest,the response of four pesticides in Cyclone was the highest,and the response of one pesticide in Cyclone-P was the highest,so the XL C18column was chosen as the purification column for further experiments.

3.2.3.Optimization of transfer flow rate

The online purification was conducted in the TurboFlowTMcolumn.The analytes retained on the TurboFlowTMcolumn should be transferred to the analytical column for further separation.The transfer efficiency both depended on the transfer flow rate and the transfer time.A low transfer flow rate will lead to a slow transfer,which also lead to peak extension and longer retention.High transfer flow rate could improve the transfer efficiency but may lead to higher column pressure.In order to find the best transfer efficiency,transfer flow rate of 0.06,0.08,0.1,and 0.12 mL/min was investigated,and the transfer time was set at 120 s.When the transfer flow rate was 0.06 mL/min,the transfer was incomplete,and peak shapes of pesticides were not very well,and the peak areas were very small.As the transfer flow rate increased,the peak shapes of analytes got better,and peak areas increased.When the transfer flow rate reached 0.12 mL/min,the column pressure nearly reached the maximum limit.Intensities of 15 pesticides with different transfer flow rate were shown in Fig.3.Finally,the transfer flow rate of 0.10 mL/min was chosen in the following experiments.

3.2.4.Optimization of transfer time

Transfer efficiency depended on the transfer time and the transfer flow rate.With the increase of the transfer time,the transfer efficiency increased when the transfer flow rate was constant.In order to find the best transfer efficiency,the transfer times of 30,60,90,120,and 150 s were tested with transfer flow rate of 0.10 mL/min.When the transfer time was 30,60,or 90 s,no peak was detected,and when the transfer time was 120 s,responses of 15 pesticides were detected and the peak shapes were perfect.When the transfer time increased to 150 s,the peak areas of four pesticides were increased in small degrees,but the longer transfer time resulted in a longer analysis time.After comprehensive consideration,the transfer time was set as 120 s.

A chromatogram of 15 pesticides in Chinese cabbage with a spike level of 20 μg/kg under the optimal conditions was presented in Fig.4.

3.3.Method validation

Spiked samples were used to detect the method recovery,limits of detection(LODs),and limits of quantification(LOQs).Signal-tonoise of analytes with different spike levels were investigated,and the spiked levels of analytes with the signal-to-noise ratio of 3 and 10 were defined as LODs and LOQs[24].

Fig.3.Intensities of 15 pesticides with different transfer flow rate.

3.3.1.Method accuracy and precision

A stock standard solution was added to samples after weighting.Celery cabbage and cucumber samples spiked with 10 and 100 μg/kg were used to determine the method recovery.Recoveries of pesticides were in range of 79.2%–103.4% in Chinese cabbage samples and were in range of 75.3%–103.7% in cucumber samples.

Five consecutive extractions(n=5)of spiked samples were determined to study the precision of the method.The precision of the method was expressed by relative standard deviation(RSD).The RSDs of 15 pesticides in two kinds of samples were no higher than 10%.The recovery and precision of the method were listed in Table 3.

3.3.2.Matrix effect

The matrix effect is defined as a change in the analytical signal caused by anything else in the sample other than analyte[31].As first reported by Kebarle and Tang[32],the influence of the matrix effect is that it interferes with the ionization process,resulting in ionization enhancement or suppression.Due to the matrix effect,the quality of quantitative data may be adversely affected[33–36].

In our experiments, the ratio of the slope of the standard solution of matrix-matched and solvent-based standards was determined to calculate the matrix effect.When the percentage was larger than 100%,it indicated signal enhancement.When the percentage was lower than 100%,it indicated signal suppression[37].In this study,signal suppression was found in Chinese cabbage for all analytes.The matrix effect of pirimicarb was the most serious;almost 94% of the signal was suppressed.The signal suppression of imidacloprid, 3-hydroxycarbofuran, acetamiprid, and aldicarb in Chinese cabbage were also serious.Meanwhile the signal enhancement of imidacloprid was found in cucumber.Signal suppression was detected for the other 14 pesticides,but the degree of signal suppression was less than that in Chinese cabbage for most pesticides,which indicated that the purification effect of cucumber was better than Chinese cabbage.The data of matrix effect of 15 pesticides were given in Table 4.

3.3.3.Linearity,LODs and LOQs

Good linearity for all analytes were found with a concentration range 0.5–100 μg/kg.The correlation coefficients(R2)were better than 0.998 in two matrices.LODs of the method were in range of 0.1–1.0 μg/kg for all compounds,and LOQs were in range of 0.2–2.0 μg/kg.LODs,LOQs,and linearity of the method are presented in Table 4.

Matrix-matched calibration should be used in quantitative analysis and in real samples analysis in order to overcome matrix effect,and the LODs and LOQs were also matrix dependent.

3.4.Real sample analysis

The built method was used to determine the target ingredients in 10 celery cabbage samples and seven cucumber samples,which were bought from local markets.Imidacloprid was detected in one celery cabbage sample but not quantified(the concentration was lower than LOQ),carbendazim was detected in one cucumber sample and the concentration was 2.56 μg/kg.

4.Conclusions

Fig.4.Chromatogram of 15 pesticides in Chinese cabbage with spike level of 10 μg/kg under the optimal conditions.1.carbendazim;2.isoprocarb;3.carbaryl;4.propoxur;5.aldicarb;6.carbofuran;7.acetamiprid;8.3-hydroxycarbofuran;9.pirimicarb;10.imidacloprid;11.metalaxyl;12.phoxim;13.chlorbenzuron;14.di flubenzuron;15.isazofos.

Table 3Recoveries and precision of the method(n=5).

Table 4Parameters of the methods.

A liquid chromatography-tandem mass spectrometry method was built to determine 15 pesticide residues in Chinese cabbage and cucumber samples.Celery cabbage and cucumber samples were extracted according to QuEChERS method,while the extracts were purified by on-line TFC rather thand-SPE.The purification system could automatically perform online purification of 15 pesticides without the necessity of laborious clean up steps when using offline SPE.Online purification and separation of 15 pesticides were finished within in 16.5 min and the work efficiency was improved greatly.Compared to the traditional of fline SPE method,the online purification methodology had higher work efficiency and better precision.The developed method was successfully used in rapid screening of 15 pesticide residues in real samples[25].Because of the existence of matrix effect, it is necessary to use matrix-matched calibration for quantitative analysis in real sample determination.The built method has been successfully used for the analysis of 15 pesticide residues in real samples.

Declaration of Competing Interest

The authors have declared no conflict of interest.

Acknowledgements

This work was supported by National Key Research and Development Program of China(Project No.2018YFC1603400),Science and Technology Program of Hebei Province(Project No.19225503D)and Technical Support Project of State Administration for Market Regulation(Project No.2019YJ009).