You are on page 1of 11

Anal Bioanal Chem (2011) 400:30973107 DOI 10.

1007/s00216-011-4967-6

ORIGINAL PAPER

Miniaturized liquidliquid extraction coupled with ultra-performance liquid chromatography/tandem mass spectrometry for determination of topramezone in soil, corn, wheat, and water
Yuanbo Li & Fengshou Dong & Xingang Liu & Jun Xu & Jing Li & Caihong Lu & Yunhao Wang & Yongquan Zheng

Received: 7 January 2011 / Revised: 21 March 2011 / Accepted: 28 March 2011 / Published online: 21 April 2011 # Springer-Verlag 2011

Abstract A rapid and simple miniaturized liquidliquid extraction method has been developed for the determination of topramezone in soil, corn, wheat, and water samples using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-electrospray ionization (ESI)/ MS/MS). The established method for the extraction and purification procedure was based on liquidliquid partitioning into an aqueous solution at a low pH (pH2.5), followed by back-partitioning into water at pH>9. Two precursor, product ion transitions for topramezone were measured and evaluated to provide the maximum degree of confidence in the results. Under negative ESI conditions, quantitation was achieved by monitoring the fragment at m/z=334 and the qualitative fragment at m/z=318, whereas also collecting the corresponding parent ion at m/z=362. Chromatographic separation was achieved using gradient elution with a mobile phase consisting of methanol and a 0.01% aqueous ammonium hydroxide solution. Recovery studies for soil, corn, wheat, and water were conducted at four different topramezone concentrations (5 or 10, 50, 100, and 1,000 g kg1); the overall average recoveries ranged from 79.9% to 98.4% with intra-day relative standard deviations (RSD) of 3.1~8.7% and inter-day RSD of 4.3~7.5%. Quantitative results were determined from
Y. Li : F. Dong : X. Liu : J. Xu : J. Li : C. Lu : Y. Zheng (*) Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture, Beijing 100193, China e-mail: yongquan_zheng@yahoo.com.cn Y. Wang China Green Food Development Center, Ministry of Agriculture, Beijing 100181, China

calibration curves of topramezone standards containing 1500 g L1 with an R2 0.9994. Method sensitivities expressed as limits of quantitation were typically 6, 8, 9, and 1 g kg1 in soil, corn, wheat, and water, respectively. The results of the method validation confirmed that this proposed method was convenient and reliable for the determination of topramezone residues in soil, corn, wheat, and water. Keywords Topramezone . UPLC-MS/MS . Determination . LLE

Introduction Topramezone, [3-(4,5-dihydro-1,2-oxazol-3-yl)-4-mesyl-otolyl](5-hydroxy-1-methylpyrazol-4-yl)methanone (Fig. 1), is a new, highly selective herbicide compound that controls a wide spectrum of annual grass and broadleaf weeds in corn and wheat [1]. Topramezone is the first herbicide belonging to a new chemical class called pyrazolones or benzoyl pyrazoles [2] and was commercially introduced in 2006 [1, 3]. Its efficacy is the result of inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) in target plants, leading to chlorophyll loss and necrosis in the growing shoot tissues [1]. Topramezone inhibition of 4-HPPD in mammalian systems results in elevated serum tyrosine levels (http://toxnet.nlm.nih. gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+7500) [4]. As a consequence of these elevated tyrosine levels, topramezone causes adverse effects in the eye, liver, kidney, pancreas, and thyroid in rats (http://toxnet.nlm.nih.gov/cgibin/sis/search/a?dbs+hsdb:@term+@DOCNO+7500) [4].

3098

Y. Li et al.

O C O CH3 S O N O CH3

OH N CH3 N

Fig. 1 The chemical structure of topramezone

Furthermore, developmental toxicity studies in rats and rabbits showed that topramezone increased incidences of skeletal variation and alterations in skeletal ossification sites (http://www.impactherbicide.com/PDFs/IMPACTMSDS.pdf) [4]. In an oncogenicity study in rats, topramezone caused a significant increase in rates of thyroid follicular cell adenomas and combined adenomas/carcinomas in both sexes (http:// pmep.cce.cornell.edu/profiles/herb-growthreg/sethoxydimvernolate/topramezone/topram_impact_reg_0307.pdf) [4]. Topramezone can be persistent in aerobic soils (half-life> 125 days; http://sitem.herts.ac.uk/aeru/footprint/en/Reports/ 686.htm) [4]. Drift and/or runoff were identified as the primary routes leading to the presence of topramezone residues in aquatic ecosystems; topramezone residues may also be present in irrigation water and may be phytotoxic to irrigated non-target plants [4]. Consequently, topramezone poses a potential hazard to human health and the environment. Although topramezone has been in use for several years, we are unaware of any studies that have been reported in the literature on methods that can be used to detect the residue of this herbicide in food and environmental samples. Moreover, topramezone is currently registered for use on corn in China. Thus, development of a quick and reliable analytical method for topramezone is timely and highly desirable to help evaluate its impact on humans and the environment. As is well known, among the different techniques for chromatographic and mass analysis, LC-mass spectrometry (MS), especially LC-tandem mass spectrometry (MS/MS), has proven to be a powerful technique for the determination of drug and pesticide residues at the trace level because of its high selectivity, precision, and sensitivity [59]. Recently, ultra-performance liquid chromatography (UPLC) has been developed as an innovative and powerful separation technique that is based on the use of columns containing stationary phases with a particle size (<2 m) that is smaller than conventional LC [10]. This feature has led to a higher resolution and sensitivity and a shorter analysis time [11]. In MS/MS, the use of a multiple reaction monitoring (MRM) mode results in a significant decrease in detection

limits due to an increased signal-to-noise ratio. UPLC in combination with tandem MS has been shown to be a more robust analytical tool for pesticide residue analysis in different matrices [1215]. Despite the considerable advances in separation and quantification, traditional liquidliquid extraction (LLE) is still among the most popular techniques for routine sample preparation [16]. LLE is recognized as an attractive method for screening tests of unknown pesticides [17, 18] not only because of its simplicity, efficiency, and robustness, as well as the minimal operator training involved, and the wealth of available analytical data but also because of its wide acceptance throughout many standard methods. However, this classic technique is usually labor-intensive and timeconsuming; in addition, LLE frequently requires the use of large amounts of chemicals that could be harmful to the operator, expensive, and environmentally hazardous [19, 20]. In this work, the miniaturized LLE method that has been developed could overcome these shortcomings, exhibiting advantages such as reduced sample and reagent consumption, as well as the exclusion of an evaporation process, which minimizes waste generation. Thus, the developed method is more environmentally friendly, rapid, inexpensive, and easy to use. For sample preparation in soil and water matrices, an aliquot of the extracted aqueous solution was removed, acidified, partitioned with dichloromethane (DCM), and then back-partitioned into 0.05% aqueous ammonium hydroxide (NH4OH) solution (alkaline acceptor). Topramezone in corn and wheat samples was extracted into acidified DCM and back-partitioned into 0.05% aqueous NH4OH solution. The pH of all samples was checked prior to partitioning, and the instrument recovery was tested within each analytical series. The purpose of this study is to describe a novel and efficient analytical method for the determination of topramezone residues in corn, wheat, soil, and water. To our knowledge, this is the first report to establish an analytical method for topramezone, and the developed method was validated by application to the analysis of authentic samples. This method was also used to conduct the required studies needed to gain product registration in many parts of the world. Therefore, this method serves as the standard accepted method against which other topramezone residue methods might be assessed.

Experimental Chemicals and reagents Topramezone analytical standard (purity 99.8%) was obtained from BASF Co., Ltd. (Beijing, People's Republic of China (PRC)). LC grade methanol was purchased from

Miniaturized liquidliquid extraction coupled with UPLC-MS/MS

3099

Sigma-Aldrich (Steinheim, Germany); analytical grade dichloromethane (DCM), NaCl, hydrochloric acid (HCl, 38%), and ammonium hydroxide (NH4OH, 30%) for pesticide residue analysis were purchased from Beihua Fine-chemical Co. (Beijing, PRC). Ultra-pure water was obtained from a Milli-Q system (Bedford, MA, USA). The mobile phase solvents were distilled and passed through a 0.22-m pore size filter (Tengda, Tianjin, PRC) before use. Standard stock solutions of topramezone (100 mg L1) were prepared in methanol. The standard solutions required for construction of a calibration curve (1, 5, 10, 50, 100, and 500 g L1) were prepared from stock solutions by serial dilution with a 10:90 methanol:0.01% NH4OH aqueous solution. The matrix-matched standard solutions were similarly prepared (1, 5, 10, 50, 100, and 500 g L1) by adding the concentrated blank sample extract (soil, corn, wheat, and water) to each serially diluted standard solution. All solutions were protected against light with aluminum foil and stored in a refrigerator in the dark at 20 C, and the working standard solutions underwent no degradation for 3 months. Instrumentation and LC-MS/MS analytical conditions Chromatographic separation of topramezone was performed on a Waters Acquity UPLC system. The system included a Waters Acquity UPLC binary solvent manager, an Acquity UPLC manager, and an Acuity column heater equipped with a Waters Acquity UPLC BEH Shield RP18 column (1002.1 mm, 1.7 m particle size; Milford, MA, USA). This column was packed with a C18 reverse-phase bonded to an ethylene-bridged hybrid (BEH) substrate, which incorporated an embedded carbamate group into the bonded phase ligand. Its shield ligand demonstrates increased retention of phenolic compounds versus straight chain alkyls. Gradient UPLC elution was performed with methanol (LC grade) as mobile phase (A) and 0.01% (v/v) ammonium hydroxide in water as mobile phase (B). Separation of the analyte was performed at room temperature at a flow rate of 0.3 mL min1 with an elution gradient of 10% to 90% A in 3.0 min, followed by 10% A in 0.1 min and holding at 10% A for 2.0 min. The system was re-equilibrated at 10% A in 0.1 min for 2.0 min. The separation and stabilization were achieved in 5.1 min. The temperature in the autosampler was set at 5 C, and the injected sample volume was 10 L. A triple-quadrupole mass spectrometer (TQD, Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source was used for topramezone detection. MS/MS detection was performed in negative ionization mode, and the monitoring conditions were optimized for topramezone. The nebulizer gas was 99.95% nitrogen, and the collision gas was 99.999% argon with a pressure of

2103 mbar in the T-wave cell. The typical conditions were as follows: the capillary voltage was set at 3.0 kV, and the cone voltage was 50 kV; the source temperature and desolvation temperature were held at 120 C and 350 C, respectively; the cone and desolvation gas were set at flow rates of 50 and 500 L h1, respectively; 362 (m/z) was selected as the precursor ion, and its quantitative and qualitative product ions were 334 (m/z) and 318 (m/z), respectively, when the collision energies were both 23 V. Multi-reaction monitoring mode (MRM) was selected as the scan mode. For UPLC analysis, Masslynx NT v.4.1 (Waters) software was used to process quantitative data obtained from the calibration standards and samples. Under the described conditions, the retention time of topramezone was approximately 1.28 min. Sampling Water samples were obtained from the Institute of Plant Protection located in the Haidian region in Beijing. Soil (sandy loam), wheat, and corn samples were collected from wheat and corn trial plots at the Institute of Plant Protection in Beijing. Topramezone was not applied to these four matrices, and thus, the sample should not have been contaminated by the target analyte. Soil, wheat, and corn samples were placed into polyethylene bags, and water samples were placed into plastic bottles. Samples were transported to the laboratory and stored in the dark at less than 18 C until analysis. Soil samples were collected at depths ranging from 0 to 30 cm at 15 randomly selected points. After collection, the soil samples were air-dried at room temperature, homogenized, and passed through a 2-mm sieve; treated samples were kept in the dark until analysis, which was carried out within a few days. Corn and wheat samples Samples of wheat and corn were ground in a mechanical mortar for 5 min, passed through a 20-mesh screen, and then mixed. Amounts of 1.00.01 g of the ground samples were weighed into a 10-mL Teflon centrifuge tube. The fortified samples were spiked with the standard solutions of topramezone at different levels. The tubes containing spiked samples were vortexed for 30 s and allowed to stand for 2 h at room temperature to distribute the pesticide evenly and to give them time to interact with the sample matrix. Next, 2 mL of 1 N NaCl-saturated HCl and 5 mL of DCM were added to the samples. The tubes were capped and immediately vortexed vigorously for 3 min at maximum speed and then centrifuged for 5 min at a relative centrifugal force (RCF) of 2,077g. Subsequently, 2 mL of the DCM layer (bottom layer) was accurately removed using a volumetric pipette and transferred to a new 10-mL

3100

Y. Li et al. Fig. 2 UPLC-MS/MS chromatograms of a blank soil sample, b soil spiked at 10 g kg1 topramezone, c blank corn sample, d corn spiked at 10 g kg1 topramezone, e blank wheat sample, f wheat spiked at 10 g kg1 topramezone, g blank water sample, and h water spiked at 5 g kg1 topramezone

Teflon centrifuge tube. A volume of 2 mL of 0.05% aqueous NH4OH solution was added. Samples were vortexed vigorously for 1 min at maximum speed and then centrifuged for 3 min at 2,077g. The resulting aqueous (top) layers were filtered through 0.22-m nylon syringe filters to be injected into the UPLC-MS/MS system. Soil and water samples Dried, homogenized soil and water samples (100.1 g) were weighed into a 50-mL Teflon centrifuge tube and spiked at different levels with topramezone. The tubes containing spiked samples were vortexed for 30 s and allowed to stand for 2 h at room temperature to distribute the pesticide evenly and to give them time to interact with the sample matrix. Next, the soil samples were extracted with 10 mL of water, vortexed vigorously for 3 min at maximum speed, and centrifuged for 5 min at 2,077g (not required for water sample). A 2-mL aliquot of the aqueous sample solution was accurately transferred using a volumetric pipette into a 10-mL Teflon centrifuge tube, and 0.5 g of NaCl was added. A volume of 2 mL of 1 N NaCl-saturated HCl and 4 mL of DCM were then added. Samples were vortexed vigorously for 1 min and then centrifuged for 3 min at 2,077g. Next, 2 mL of the DCM layer (bottom layer) was removed using a volumetric pipette and transferred into a new 10-mL Teflon centrifuge tube; 2 mL of a 0.05% aqueous NH4OH solution was added. Samples were vortexed vigorously for 1 min and then centrifuged for 3 min at 2,077g. The resulting aqueous (top) layers were filtered through 0.22-m nylon syringe filters to be injected into the UPLC-MS/MS system. Application to real samples Real samples including corn and soil were collected from our residual study field, which trial is mainly for registration of topramezone commercial formulations on corn in China. The soil and corn samples were collected at harvest time. All samples were placed in polyethylene bags and transported to the laboratory, and the subsamples were then kept frozen at 20 C until analysis. Wheat samples were bought from the local market, and water samples were collected from the Jingmi Irrigation Canal in Beijing. Quantification and identification Experimental data were calibrated and quantified using QuanLynx in MassLynx 4.1 software. Standard solution calibration curves were prepared and utilized for quantifi-

cation of topramezone in all of the samples. Two transitions of the target compound precursor ion were monitored, and the most intense ion (m/z 334) was used for quantification. The retention time of topramezone was used to ensure correct peak identification. Confirmation of the presence of topramezone in a sample was based on the following criteria: (1) the signal-to-noise ratio (S/N) for each product ion was 3:1, (2) the retention time of the product ion in the sample matched the mean retention time of the product ions in the standards within 5%, and (3) the area ratio obtained from the product ions in the sample was within 10% of their mean in the standards [21]. Validation study To evaluate the performance of the developed method, the method was validated according to a conventional validation procedure that included the following parameters: specificity, linear range, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, and stability. To verify the absence of interfering species around the retention time of the analyte, 20 blank samples (soil, corn, wheat, and water) were analyzed. The linearity of the method was studied by analyzing the standard solutions and the different matrices in triplicate at six concentrations ranging from 1 to 500 g L1. Satisfactory linearity was obtained when the correlation coefficient (R2) was higher than 0.9994 based on measurement of the analyte peak areas. Blank analyses were performed to check interference from the matrix. To differentiate between extraction efficiency and matrix-induced signal suppression/enhancement, the slope ratios of the linear calibration functions were calculated, and the signal suppression/enhancement (SSE) due to matrix effects was determined. The LOD for topramezone was considered to be the concentration that produced a signal-to-noise (S/N) ratio of 3, and the LOQ was defined based on a S/N ratio of 10; the LOQ was estimated from the chromatogram corresponding to the lowest point used in the matrixmatched calibration. The recovery assays were carried out to investigate the method accuracy and precision. Five replicates of spiked samples (soil, corn, wheat, and water) at four levels (5 or 10, 50, 100, and 1,000 g kg1) were prepared on three different days. Topramezone was extracted and purified according to the above procedure. The precision in these conditions for repeatability, expressed as relative standard

Miniaturized liquidliquid extraction coupled with UPLC-MS/MS

3101

3102 Fig. 2 (continued)

Y. Li et al.

deviation (RSD), was determined by the intra-day and interday assays. The stability of topramezone was determined in the solvent and in the matrix. Stability of the stock solutions was tested monthly by injection of new prepared working solution, and soil, corn, wheat, and water matrix-matched standards of 10 g mL1 were analyzed monthly, and all the samples' related stability were stored at 20 C. The pH of all of the samples was checked prior to partitioning.

molecular ions [MH] in the full-scan mass spectra. The voltages of the electrospray capillary and cone were also optimized. Mass fragments of the precursor ions were produced by collision-induced dissociation (CID) using 2.98 mTorr argon. Precursor ions [MH] in negative mode were further fragmented at various collision energies (1040 V), and the SRM was optimized to achieve the highest sensitivity. Two intense fragment ions were observed in the product ion spectra; the major fragment ions at m/z 334 and 318 were chosen in MRM acquisition for topramezone analysis. Optimization of LC parameters Modification of the mobile phase was performed because the mobile phase composition has a significant effect on peak shapes and the retention behavior of the analyte in the LC column as well as on the MS response [22]. Methanol and acetonitrile with different additives, including formic acid and ammonium hydroxide at various concentrations, were tested as mobile phases. A solvent system consisting of methanol and 0.01% aqueous ammonium hydroxide solution, which provided the most efficient chromatographic condition, was ultimately selected as the mobile phase system using gradient elution. Basic ammonium hydroxide is known to promote the deprotonation of acidic species in topramezone

Results and discussion Optimization of MS/MS parameters Full-scan and MS/MS mass spectra were obtained from the infusion of 1 mg L1 standard solution of topramezone in methanol/0.01% aqueous ammonium hydroxide (10:90, v/v) at a flow rate of 10 L min1. ESI in both positive and negative ion modes was evaluated, and negative mode was found to offer higher precursor ion signal intensities and better fragmentation patterns than positive mode; consequently, negative ESI mode was selected for topramezone analysis. In the negative ESI mode, the analyte formed predominantly deprotonated

Miniaturized liquidliquid extraction coupled with UPLC-MS/MS

3103

(pKa =4.06) [MH] (http://sitem.herts.ac.uk/aeru/footprint/ en/Reports/686.htm), and as a result, an increase in the signal is observed in the negative ESI interface. Moreover, the addition of ammonium hydroxide improved the peak shape and sensitivity. Chromatographic conditions were optimized to achieve good resolution, to increase the topramezone signal and to minimize analysis times. Typical UPLC-MS/MS chromatograms of blank and fortified sample are shown in Fig. 2; there were no interference peaks in the pertinent region of the chromatogram, and the analysis time of the herbicide was about 1.28 min. The major issue in the optimization of the chromatographic method was the choice of standard dissolution media due to the great variability in response that was observed (Fig. 3). Methanol and acetonitrile were selected as the most common dissolution media based on their effective solubility. Interestingly, in this study, pure methanol as the dissolution agent caused the column to produce two separate peaks, and the target compound exhibited poor retention behavior. Similarly, pure acetonitrile as the dissolution agent resulted in bad peak shape and low sensitivity. Based on principles of chromatography, dissolution media with a composition similar to that of the mobile phase should be used, which in this study is approximately 90% of 0.01% aqueous ammonium hydroxide solution/10% methanol. This dissolution media yields compound separation with an appropriate peak shape and sensitivity (Fig. 3). Therefore, as a compromise, 10% methanol in 0.01% aqueous ammonium hydroxide solution was chosen as the dissolution media for the standard solutions. Miniaturized liquidliquid extraction In general, liquidliquid partitioning is the fundamental method utilized for the extraction of acid herbicides from

various matrices [23]. Easy, economical, and miniaturized LLE was optimized and used in our present work. To determine the solvent with optimal extraction efficiency, several solvents were tested. Commonly used solvents including cyclohexane, chloroform, carbon tetrachloride, dichloromethane, toluene, and ethyl acetate were selected because they provide a broad range of polarities. The results were confirmed by the respective solvent solubility in water, and the solvents were further evaluated by their extraction efficiency. Compared to other organic solvents, dichloromethane has a relatively high solubility and is suitable for LLE, and was eventually chosen as the solvent and obtained good recoveries. Similarly, the volume of the extraction solvent was also evaluated in this study. Two, 3, 5, and 10 mL of dichloromethane were introduced to the extraction procedure. Compared with the extracted results, 3 and 5 mL of dichloromethane could obtain the equivalent recoveries better than other two tested volume. According to the requirement of the experiment, 5 and 4 mL dichloromethane were selected for the extraction of A (corn and wheat) and B (water and soil), respectively. Topramezone is a relatively strong acid (pKa = 4.06) and is more stable at low pH. Therefore, the pH of the extraction is also important and should be suitably controlled. In the present study, the solubilities of topramezone in water were 0.51 and 100 mg mL1 at pH=3.1 and pH> 9.0 (http://dsp-psd.pwgsc.gc.ca/Collection/H113-7-2006-9E. pdf), respectively. The pH values of the sample solution ranging from 2 to 10 on extraction were investigated. After optimizing the pH values in this experiment, the best results were confirmed as follows: an aliquot of the extract was removed and purified using liquidliquid partitioning at low pH (pH2.5), followed by back-partitioning at a high pH into 0.05% aqueous ammonium hydroxide solution (pH 9.5). Using these conditions, good extraction and purification results were obtained.

Fig. 3 Chromatograms of different dissolution media for the standard mixture: a dissolved with 10% methanol in 0.01% aqueous ammonium hydroxide solution, b dissolved with pure methanol, and c dissolved with pure acetonitrile

3104 Table 1 Linear regression parameters of calibration curve of topramezone and calculation of signal suppression/enhancement (SSE) in solvent and in matrices (soil, corn, wheat, and water) SSE=(slope matrix-matched calibration/slope standard calibration in solvent)100% Matrix Calibration range (g kg1) Standard calibration curve Slope Solvent Soil Corn Wheat Water 1500 1500 1500 1500 1500 12.667 12.364 12.069 11.713 11.509 y-intercept 48.439 49.613 42.027 50.423 32.792 R2 0.9997 0.9994 0.9998 0.9995 0.9998 98 95 92 91 SEE (%)

Y. Li et al. P value

0.290 0.202 0.238 0.195

Method validation Matrix effects and linearity To evaluate matrix effects, the signal suppression enhancement (SSE) for each analyte in each matrix was calculated; the SSE was defined as the percentage of the matrixmatched calibration slope divided by the slope of the standard calibration in solvent. To achieve this objective, the proposed method was applied to 20 blank samples in different matrices to evaluate the specificity of the method, and the results show that no interference was detected at the expected retention time (Fig. 2). Linear regression results for the standard solution and matrix-matched calibration curves are shown in Table 1; no significant suppression or enhancement for topramezone was observed, as evidenced

by the slope ratios, which were within 10% of the slope ratio=100% (9198%), and the high linearity (R2 >0.9994) of the calibration curves. Furthermore, we also used twotailed paired t test with a probability of 95% to evaluate the SSE. The P values imply that the data between pure solvent and sample matrix (soil, corn, water, and wheat) are not significantly different (P>0.05). Consequently, quantification was performed with the external calibration using standards in pure solvent in this study. Precision and accuracy Evaluation of the recoveries and RSDs of topramezone studies was performed to validate the UPLC-MS/MS method by spiking samples (soil, corn, wheat, and water) at four levels (5 or 10, 50, 100, and 1,000 g kg1;

Table 2 Accuracy and precision of the proposed method in the four studied matrices Sample Spiked level (g kg1) Intra-day (n=5) Day 1 Average recoveries (%) Soils 10 50 100 1,000 10 50 100 1,000 10 50 100 1,000 5 10 50 100 81.4 82.6 85.1 79.9 86.1 93.4 93.7 98.4 94.3 90.3 91.2 88.1 93.6 93.9 89.7 90.6 RSD (%) 5.8 4.0 5.3 4.9 8.1 5.5 5.9 5.1 5.3 6.4 7.9 5.4 6.9 6.5 4.2 3.6 Day 2 Average recoveries (%) 82.5 85.6 86.3 83.7 91.0 94.6 95.1 97.0 91.3 89.1 90.0 93.3 94.4 92.5 91.7 89.8 RSD (%) 7.1 6.6 5.5 6.0 7.9 4.2 6.2 5.1 7.5 4.4 5.9 4.3 6.8 5.0 4.5 5.4 Day 3 Average recoveries (%) 84.1 83.5 83.7 82.1 94.2 91.6 94.1 96.2 89.7 92.9 93.7 90.5 92.5 93.1 90.5 92.8 RSD (%) 6.2 5.2 4.5 3.1 6.7 6.7 8.4 5.5 8.7 4.5 3.7 5.6 7.2 7.4 6.3 4.7 6.1 5.2 5.6 4.9 7.5 5.4 6.7 4.6 7.0 5.1 6.1 5.2 6.5 5.6 4.3 4.4 Inter-day (n=15) RSD (%)

Corn

Wheat

Water

Miniaturized liquidliquid extraction coupled with UPLC-MS/MS


120 100 80 60 40 20 0 soil wheat corn

3105
120 100 80 60 40 20 0 soil wheat corn

LLE ODS GCB PSA Florisil

LLE ODS GCB PSA Florisil

Fig. 4 The extraction effectiveness of the proposed method (LLE) compared with the optimized QuEChERS method: a recoveries (percent) of the LLE with acetonitrile as the extraction solvent with

different sorbents and b recoveries (percent) of the LLE with 5% (v/v) formic acid in acetonitrile as the extraction solvent with different sorbents (n=5)

Table 2). The recoveries were calculated using the sixpoint standard calibration curves. As Table 2 shows, the mean recovery values that were obtained were in the acceptable ranges of 79.986.3%, 86.198.4%, 88.1 94.3%, and 89.794.4% from soil, corn, wheat, and water, respectively. The intra-day precision was measured by comparing the standard deviation of the recovery percentages of a set of spiked samples during the same day. The inter-day precision was determined by analyzing spiked samples for three distinct days. In general, the intra-day RSDs (n=5) and inter-day RSDs (n=20) for the proposed method ranged from 3.18.7% to 4.37.5%, respectively.

Figure 2 shows chromatograms of the topramezone blank and the different spiked samples. The results of the recovery studies demonstrated that this method and UPLC-MS/MS analysis achieved satisfactory recovery, precision, and sensitivity for pesticide analysis in corn, wheat, soil, and water matrices. Limits of detection (LODs) and quantitation (LOQs) The LODs and LOQs were based on the minimum amount of target analyte that produced a chromatogram peak with a sign-to-noise ratio of 3 and a peak ten times the background

Real soil sample 55 1 .2 8 100

MRM of 2 Channels ES-

Real corn sample 53


100

MRM of 2 Channels ES-

362 > 334 (tomramezone) 4.35e4

1.28

362 > 334 (tomramezone) 3.22e3

1.40

1.00
Real soil sample 55
100
1.28

2.00

3.00

4.00
100

1.00
1.28

2.00

3.00

4.00

MRM of 2 Channels ES-

Real corn sample 53

MRM of 2 Channels ES-

362 > 318 (tomramezone) 1.92e4

362 > 318 (tomramezone) 1.56e3

2.02 2.15 1.40 2.40

3.60

Time

Time

1.00

2.00

3.00

4.00

1.00

2.00

3.00

4.00

Fig. 5 Sample chromatograms of a real soil sample and b real corn grain sample

3106

Y. Li et al.

chromatographic noise, respectively. Estimated LOD values for all of the matrices were 2, 3, 3, and 0.4 g kg1 topramezone based on five replicate extractions and analyses of spiked samples of soil, corn, wheat, and water, respectively. The LOQs corresponding to the lowest level for fortified topramezone were determined to be 6, 8, 9, and 1 g kg1 in soil, corn, wheat, and water matrices, respectively, based on five replicate measurements. These LODs and LOQs are below the MRL required by the US and the EU [24, 25], indicating that the proposed method is suitable for the quantification of topramezone in the studied matrices. In addition, the stability of topramezone in the solutions was studied, and there was no significant difference (P>0.05) observed under the two types of storage treatment as described in Experimental section. Comparison of the extraction method In order to show the advantages of the proposed method, an evaluation was performed to compare the extraction effectiveness of currently proposed method to QuEChERS (quick, easy, cheap, effective, rugged, and safe), which is a major development in sample preparation that involves a streamlined approach to pesticide residue analysis [26]. The comparison of the two methods was carried out by spiking five replicate samples (corn, wheat, and soil) at the same concentration level (100 g kg1). External matrix-matched standards were analyzed to acquire more realistic results. Figure 4 presents the results for the mean recoveries of the two methods in the three matrices (corn, wheat, and soil). During the process of sample preparation in the QuEChERS method, the extraction and purification procedure was also optimized to obtain a more satisfactory recovery. The influence of different dispersive sorbents on the purification and recovery of topramezone extracts, including primarysecondary amine (PSA), C18 (ODS), florisil, and graphitized carbon black (GCB), were investigated. Acidified acetonitrile (5% (v/v) formic acid in acetonitrile) was also evaluated in place of acetonitrile as the extraction solvent for optimal extraction efficiency; unfortunately, recovery using the QuEChERS method (less than 70%) was consistently unable to fulfill the strict requirement of a recovery range of 70 120%. In contrast to the QuEChERS method, it was concluded that the proposed method could obtain a satisfactory recovery, and importantly, this method is also simple, rapid, inexpensive, and easy to use, minimizes the use of both sample and organic solvents, and uses direct injection. Application to real samples To assess the applicability of the validated method, the effectiveness of this method in measuring trace levels of

topramezone was evaluated by analyzing soil and corn samples collected from our residual study trial field. Wheat samples and water samples were collected from the local market and Jingmi Irrigation Canal in Beijing, respectively. A total of 20 samples were analyzed; three positive corn samples and four positive soil samples were detected, and those samples contained topramezone in the range 0.01 0.13 mg kg1. The herbicide was not detected in real wheat or water samples with the proposed analytical method. Some typical chromatograms are presented in Fig. 5.

Conclusions A simple UPLC-MS/MS method for the quantification of topramezone in soil, corn, wheat, and water samples has been developed and validated. Careful optimization of UPLC-MS/ MS conditions revealed that ESI in negative mode was more suitable for the analysis of topramezone. To satisfy the requirement of the standard dissolution media, 10% methanol in 0.01% aqueous ammonium hydroxide solution was chosen. A miniaturized LLE method was optimized to ensure satisfactory recoveries and sample purification, and the pH was controlled during the partitioning procedure. The LODs for topramezone in soil, corn, wheat, and water were calculated to be at 2, 3, 3, and 0.4 g kg1, respectively, and the recovery percentages ranged from 79.9% to 98.4% in the four matrices. When compared with the conventional QuEChERS method, the proposed method produced relatively higher recoveries and was also less time-consuming and cheaper in terms of consumable and equipment requirements. In addition, the linearity, precision, and reproducibility were validated successfully, demonstrating the suitability of this method for herbicide analysis. In conclusion, this work demonstrates that our newly developed UPLC-MS/MS method is a reliable analytical method for the determination of topramezone in soil, corn, wheat, and water in routine analyses.
Acknowledgments This work was financially supported by the foundation established by the Agricultural Ministry of the PRC, National Basic Research Program of China (The 973 Program, Grant No. 2009CB119000), the National Natural Science Foundation of China (31071706 and 30900951), and the Public Service Sector Research and Development Project (200903054 and 200903033).

References
1. Grossmann K, Ehrhardt T (2007) Pest Manag Sci 63:429439 2. Siddall TL, Ouse DG, Benko ZL, Garvin GM, Jackson JL, McQuiston JM, Ricks MJ, Thibault TD, VanHeertum JC, Weimer MR, Turner JA (2002) Pest Manag Sci 58:11751186 3. Schonhammer A, Freitag J, Koch H (2006) J Plant Dis Prot 23:10231031

Miniaturized liquidliquid extraction coupled with UPLC-MS/MS 4. USEPA/Office of Pesticides Programs. Pesticide fact sheet: topramezone, p. 2. Available at http://www.epa.gov/opprd001/factsheets/ topramezone.pdf. Accessed 10 Aug 2005 5. Dong FS, Liu XG, Cheng L, Chen WY, Li J, Qin DM, Zheng YQ (2009) J Sep Sci 32:36923697 6. Liu XG, Dong FS, Li S, Zheng YQ (2008) J AOAC Int 91:1110 1115 7. Hayama T, Takada M (2008) Anal Bioana Chem 392:969 976 8. Radisic M, Grujic S, Vasiljevic T, Lausevic M (2009) Food Chem 113:712719 9. Shao B, Zhao R, Meng J, Xue Y, Wu GH, Hu JY, Tu XM (2005) Anal Chim Acta 548:4150 10. Marin JM, Gracia-Lor E, Sancho JV, Lopez FJ, Hernandez F (2009) J Chromatogr A 1216:14101420 11. Mellors JS, Jorgenson JW (2004) Anal Chem 76:54415450 12. Giordano A, Fernandez-Franzon M, Ruiz MJ, Font G, Pico Y (2009) Anal Bioana Chem 393:17331743 13. Leandro CC, Hancock P, Fussell RJ, Keely BJ (2007) J Chromatogr A 1144:161169 14. Mezcua M, Aguera A, Lliberia JL, Cortes MA, Bago B, Fernandez-Alba AR (2006) J Chromatogr A 1109:222227

3107 15. Montoro EP, Gonzalez RR, Frenich AG, Torres MEH, Vidal JLM (2007) Rapid Commun Mass Spectrom 21:35853592 16. Lambropoulou DA, Albanis TA, Biochem J (2007) Biophys Methods 70:195228 17. Fatoki OS, Awofolu RO (2003) J Chromatogr A 983:225236 18. Mahara BM, Borossay J, Torkos K (1998) Microchem J 58:3138 19. Silvestre CIC, Santos JLM, Lima JLFC, Zagatto EAG (2009) Anal Chim Acta 652:5465 20. Ahmed FE (2001) Trends Anal Chem 20:649661 21. Center for Veterinary Medicine, USFDA. Guidance for industry 118: mass spectrometry for confirmation of the identity of animal drug residue-final guidance. Center for Veterinary Medicine, USFDA. Available at http://www.fda.gov/cvm/Guidance/guide118.pdf 22. Rubert J, Soler C, Manes J (2010) Talanta 82:567574 23. Koesukwiwat U, Sanguankaew K, Leepipatpiboon N (2008) Anal Chim Acta 626:1020 24. USDA. Pesticide MRL database. Available at http://www. mrldatabase.com. Accessed Dec 2010 25. Pesticide MRL database. Available at http://ec.europa.eu/sanco_ pesticides/public/index.cfm. Accessed Dec 2010 26. Koesukwiwat U, Lehotay SJ, Miao S, Leepipatpiboon N (2010) J Chromatogr A 1217:66926703

You might also like