Rapid analysis of organic farming insecticides in soil and produce using ultra-performance liquid chromatography/tandem mass spectrometry
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- Drożdżyński, D. & Kowalska, J. Anal Bioanal Chem (2009) 394: 2241. doi:10.1007/s00216-009-2931-5
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A new method for the analysis of three ecological insecticides, namely azadyrachtin, spinosad (sum of spinosyn A and spinosyn D) and rotenone, in produce and soil samples is presented. Investigated compounds are one of the most significant insecticides authorized for organic farming crop protection in many countries. Extraction of the pesticides from plant and soil matrices was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The method entailed a single extraction of the investigated compounds with acidified acetonitrile followed by a dispersive solid-phase extraction cleanup step prior to the final determination by reverse-phase ultra-performance liquid chromatography/tandem quadrupole mass spectrometry (UPLC-MS/MS). Validation studies were carried out on cabbage, tomato and soil samples. Recoveries of the spiked samples were in the range between 67% and 108%, depending on the matrix and the spiking level. Relative standard deviations for all matrix–compound combinations did not exceed 12%. The limits of quantification were ≤0.01 mg kg−1 in all cases, except for azadirachtin. The developed method was applied to the analysis of real samples originating from organic farming production.
In scientific literature, several methods for extraction of ecological pesticides have been proposed. The most important are simple homogenization and extraction with acetone, methanol, and acetonitrile–dichloromethane mixture, empore disk extraction, and microwave-assisted extraction with dichloromethane. The obtained extracts were cleaned-up by liquid–liquid extraction (LLE), silica gel solid-phase extraction (SPE) or dispersive SPE (dSPE). The final determination of selected pesticides was usually performed using liquid chromatography with mass spectrometric detection (HPLC-MS), liquid chromatography with diode array detection (HPLC-DAD), or by colorimetric methods [7–16, http://www.quechers.com].
In this work, a new method for the simultaneous analysis of three organic farming insecticides (azadirachtin, spinosad, and rotenone) in plant and soil samples by using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation technique followed by an UPLC/MS/MS determination is presented.
Acetonitrile (super gradient) was purchased from Labscan (Dublin, Ireland). Acetic acid (HPLC purity) was purchased from J. T. Baker (Deventer, Netherlands). Citrate buffer extraction tubes containing magnesium sulfate, sodium chloride, sodium citrate dibasic sesquihydrate, and sodium citrate tribasic dihydrate (4 g + 1 g + 0.5 g + 1 g) were purchased from Supelco (Bellefonte, USA). Primary secondary amine (PSA) cleanup tubes (900 mg MgSO4 + 150 mg PSA), PSA/octadecylsilyl (C-18) cleanup tubes (900 mg MgSO4 + 150 mg PSA + 150 mg C-18) and PSA/graphitized carbon black (GCB) cleanup tubes (900 mg MgSO4 + 150 mg PSA + 45 mg GCB) were also purchased from Supelco. Distilled water was obtained using a Millipore Elix 3 system and a Simplicity equipment (Billerica, MA, USA). For chromatographic analyses, methanol and water, both with addition of 0.1% ammonium acetate (Riedel-de Haën, Hanover, Germany, LC-MS purity) were applied. Nitrogen for organic extract evaporation (technical purity of 99.995%) and collision gas argon (purity 99.9998%) were supplied by Air Products Company (Siewierz, Poland). Syringe filters PTFE, 0.45 µm (Waters Inc., Milford, USA) were applied for samples processing.
Certified analytical standards of pesticides (Dr. Ehrenstorfer, Augsburg, Germany) were used. Purity of standards was as follows: rotenone—96.0%, spinosad—91.0%, and isoproturon-D6 (internal standard)—98.5%. Azadirachtin was obtained as 100 µg mL−1 stock solution in methanol. Rotenone and spinosad stock solutions were prepared at concentrations of about 1000 µg mL−1. Purity was included in the calculation of actual concentration of each standard solution. Of these stock solutions, a single composite mixture of the three pesticides was prepared, of which subsequent dilutions to obtain working standards were made. The single composite mixtures at appropriate concentrations were used to calibrate the UPLC/MS/MS system and spike samples in recovery experiments.
A Heidolph Multi-Reax laboratory shaker (Kelheim, Germany) for extraction and cleanup of samples and a centrifuge Rotina 420R (Hettich, Tuttlingen, Germany) for extracts centrifugation were used. Evaporation of organic extracts obtained after cleanup was performed under a stream of nitrogen using a Visidry Drying Attachment (Supelco, Bellefonte, USA). A Bandelin Sonorex Super RK 1034 ultrasonic bath (Berlin, Germany) was employed for dissolving the residues obtained after nitrogen drying in injection solvent.
Determinations were performed using a Waters ACQUITY UPLC ultra-performance liquid chromatograph system (Milford, USA) interfaced to a triple quadrupole mass spectrometer (Waters Inc., Micromass, Quattro Premier XE). The instrument was equipped with autosampler and column thermostats. A nitrogen generator NM30-LA (Peak Scientific, Renfrewshire, Scotland, UK) was used to deliver the nebulizer and desolvation gas to the mass spectrometer. The instrument was controlled using Waters MassLynx 4.0 software and data were evaluated using Waters TargetLynx software. Reverse-phase UPLC analysis was performed using a Waters ACQUITY UPLC column (BEH C18 2.1 × 100 mm, 1.7 µm). Temperature of the column and autosampler was thermostated at 30 °C. Sample extract volumes of 5 µL were injected into the system. The column was eluted with the mobile phase: water with 0.1% ammonium acetate (A) and methanol with 0.1% ammonium acetate (B) at the flow rate of 0.3 mL min−1 using gradient mode. Gradient was programmed to increase the amount of B from an initial content of 10–100% in 5 min and to return to the initial conditions (10% B) in 1 min (from 5 to 6 min). These conditions were maintained for 7 min.
UPLC-MS/MS conditions used for investigated organic farming pesticides
Retention time (min)
Dwell time (ms)
Cone voltage (V)
MRM transitions m/z, (collision energy (eV))
Ion ratio, %
743 > 725 (30)
743 > 665 (30)
Isoproturon D6 (I.S.)
213 > 78 (20)
395 > 213 (25)
395 > 192 (25)
732 > 142 (30)
732 > 98 (40)
746 > 142 (30)
746 > 98 (40)
Plant material of high water content and leafy vegetables
A 10-g comminuted sub-sample was placed in a polypropylene centrifuge tube (50 mL), then 50 µL of internal standard solution (isoproturon-D6 at 150 µg mL−1), 100 µL acetic acid and 10 mL acetonitrile were added, and the mixture was shaken vigorously on a laboratory shaker for 5 min. Further, 0.5 g disodium hydrogen citrate sesquihydrate, 1 g trisodium citrate dihydrate, 4 g anhydrous magnesium sulfate, and 1 g sodium chloride were added, and the mixture was immediately hand-shaken for 1 min, then centrifuged at 4,500 rpm for 2.5 min.
A 5-g soil sub-sample was placed in a polypropylene centrifuge tube (50 mL), then 5 mL water, 50 µL of internal standard solution (isoproturon-D6 at 150 µg mL−1), 100 µL acetic acid and 10 mL acetonitrile were added and the mixture was shaken vigorously on a laboratory shaker for 5 min. Further, 0.5 g disodium hydrogen citrate sesquihydrate, 1 g trisodium citrate dihydrate, 4 g anhydrous magnesium sulfate, and 1 g sodium chloride were added, and the mixture was immediately hand-shaken for 1 min, then centrifuged at 4,500 rpm for 2.5 min.
Crops of high water content
A 5-mL aliquot of the supernatant was transferred into a polypropylene centrifuge tube (15 mL) containing 900 mg MgSO4 and 150 mg PSA. The tube was vortexed for 1 min and centrifuged at 4,500 rpm for 2.5 min. A 2-mL aliquot of the supernatant was transferred into a glass test tube. The extract was evaporated to dryness under a stream of nitrogen and the residue was re-dissolved in 0.5 mL of 0.1% ammonium acetate in methanol using ultrasonic bath followed by an addition of 0.5 mL of 0.1% ammonium acetate in water and vortexed. The obtained mixture was filtered through a PTFE syringe filter (0.45 µm), if needed, prior to its injection into the UPLC-MS/MS system.
The procedure is similar to the above described but the supernatant was transferred to a polypropylene centrifuge tube (15 mL) containing 900 mg MgSO4, 150 mg PSA, and 45 mg GCB.
A 5-mL aliquot of the supernatant was transferred into a polypropylene centrifuge tube (15 mL) containing 900 mg MgSO4, 150 mg PSA, and 150 mg C-18. The tube was vortexed for 1 min and centrifuged at 4,500 rpm for 2.5 min. A 2-mL aliquot of the supernatant was transferred into a glass test tube. The extract was evaporated to dryness under a stream of nitrogen and the residue was re-dissolved in 0.25 mL of 0.1% ammonium acetate in methanol using ultrasonic bath followed by an addition of 0.25 mL of 0.1% ammonium acetate in water, then vortexed. The obtained mixture was filtered through the PTFE syringe filter (0.45 µm), if needed.
Validation of developed analytical protocols was performed by using spiked control samples of tomato, cabbage, and soil. The recovery was determined for six replicates at three spiking concentrations of 0.01, 0.05, and 0.1 mg kg−1. The intermediate precision was calculated as the relative standard deviation (RSD) for each concentration level. The linearity of the calibration curves was evaluated at a concentration range between 0.01 and 0.5 µg mL−1 using six calibration solutions prepared in blank control sample extracts. Calculations of recoveries were done by using the peak areas. The calculations were done with reference to isoproturon-D6 which was used as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were determined as three times and ten times the standard deviation of the absolute recovery at the lowest spiking level for which the validation was achieved . The method recovery and intermediate precision data were assessed according to the EU requirements described in the SANCO/3131/2007 document [18, 19].
Results and discussion
Choice of method and determinative technique
Method validation results
Recoveries (%), relative standard deviations, RSD (%), limits of detection (LOD) and limits of quantification (LOQ) obtained by UPLC–MS/MS analysis of tomatoes, cabbage and soil at three spiking levels of 0.01, 0.05, and 0.1 mg kg−1 (n = 6)
Average recovery (%) (RSD, %)
LOD (mg kg−1)
LOQ (mg kg−1)
0.01 mg kg−1
0.05 mg kg−1
0.1 mg kg−1
Application to real samples
Up to now, the developed method has been applied for analysis of 20 real samples originating from ecological farms. These were eight samples of cabbage, eight samples of potatoes, and four samples of soil. Internal quality control was implemented to assure the quality of the results. A spiking recovery was always determined together with the analyzed real samples to check the extraction efficiency of the method. In the analyzed organic samples, no residues of the target ecological insecticides were detected which on one hand provided an evidence of the proper use of the ecological insecticides by the growers from which the tested samples were collected but on the other hand, the method cannot be fully validated without any positive findings in real samples. Efforts to collect suitable samples from organic farming experimental plots are in progress.
A new method for the simultaneous analysis of three organic farming insecticides in plant and soil samples is presented in this paper. To the knowledge of these authors, this is the first analytical report in which application of UPLC/MS/MS to the analysis ecological pesticides is presented. The proposed analytical methodology allows for the single determination of selected organic farming insecticides residues at trace levels with excellent analytical performance. The LOQs values were ≤0.01 mg kg−1 for all target pesticides, except for azadirachtin. Satisfactory validation parameters such as trueness, precision, and linearity proved fitness for purpose of the developed method.
This work was financially supported by the Polish Ministry of Science and Higher Education, Grant No. NN 310 4358 33r. The authors thank Stanisław Walorczyk, Ph.D., for helpful discussions.