Analytical and Bioanalytical Chemistry

, Volume 394, Issue 8, pp 2241–2247

Rapid analysis of organic farming insecticides in soil and produce using ultra-performance liquid chromatography/tandem mass spectrometry

Authors

    • Institute of Plant Protection, National Research Institute
  • Jolanta Kowalska
    • Institute of Plant Protection, National Research Institute
Original Paper

DOI: 10.1007/s00216-009-2931-5

Cite this article as:
Drożdżyński, D. & Kowalska, J. Anal Bioanal Chem (2009) 394: 2241. doi:10.1007/s00216-009-2931-5

Abstract

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.

Keywords

Ecological insecticidesSoilFoodQuEChERSUPLC-MS/MS

Introduction

In this paper we describe a method for the sensitive and selective determination of three ecological insecticides (azadirachtin, spinosad and rotenone), which are widely used for pest control in organic farming crop production. Azadirachtin is a feeding deterrent for many insects and a growth disruptant for most insects and many other arthropods. This is a limonoid of the tetranortriterpenoid type extracted from the oil obtained from mature seeds of the neem tree (Azadirachta indica). Notably, it has a low toxicity against nontarget and beneficial organisms and causes less disruptance to ecosystems than conventional insecticides. The spinosyns, macrocyclic lactones, are active on a wide variety of insect pests, especially lepidopterans and dipterans. The spinosyns are derived from a naturally occurring Actinomycetes bacterium, Saccharopolyspora spinosa. The active substance spinosad consists of the two main components: spinosyn A and spinosyn D. Rotenone is a non-systemic botanical insecticide obtained from leguminous plants such as Derris elliptica, Lonchocarpus nicou, and Tephrosia vogelii. It is a non-selective insecticide, easily decomposing in the presence of light [16]. Figure 1 presents the structures of the determined ecological insecticides (http://www.alanwood.net/pesticides/).
https://static-content.springer.com/image/art%3A10.1007%2Fs00216-009-2931-5/MediaObjects/216_2009_2931_Fig1_HTML.gif
Fig. 1

Formulae of objected compounds: a azadirachtin, b rotenone, c spinosyn A, d spinosyn D

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 [716, 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.

Experimental

Chemicals

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.

Equipment

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.

Chromatography

UPLC conditions

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.

MS/MS conditions

The interface conditions were optimized for maximum intensity of the precursor ions and were as follows: nebulizer and desolvation (drying gas) N2 flows were set at 100 and 700 L h−1, respectively, source block and desolvation temperatures were 120 and 350 °C, respectively. Argon was used as a collision gas at the pressure of 6.9 × 10-3 mbar. Selection and tuning of multiple reaction monitoring (MRM) transitions was performed individually for each analyte on the instrument used in this work. All the compounds were analyzed using a positive electrospray ionization mode (ESI+). MS/MS scanning was performed only over 2 min, between 0.5 and 2.5 min. The conditions applied for the investigated pesticides are given in Table 1.
Table 1

UPLC-MS/MS conditions used for investigated organic farming pesticides

Pesticide

Retention time (min)

Dwell time (ms)

Cone voltage (V)

MRM transitions m/z, (collision energy (eV))

Ion ratio, %

Quantification

Identification

Azadirachtin

0.98

50

40

743 > 725 (30)

743 > 665 (30)

15

Isoproturon D6 (I.S.)

1.08

75

30

213 > 78 (20)

Rotenone

1.20

75

30

395 > 213 (25)

395 > 192 (25)

85

Spinosyn A

1.98

75

45

732 > 142 (30)

732 > 98 (40)

6

Spinosyn D

2.19

75

45

746 > 142 (30)

746 > 98 (40)

7

Sample preparation

Extraction

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.

Soil samples

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.

Cleanup

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.

Leafy vegetables

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.

Soil samples

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 experiments

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 [17]. 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

The QuEChERS sample preparation approach was chosen in this work, since it has become an increasingly popular and still developing extraction/cleanup technique in the area of pesticide residue analysis. In our work, enhanced recoveries of the target pesticides were achieved by using an acidic citrate-buffered extraction. The proposed method is characterized by a low consumption of chemicals and materials as well as a shortened time of extraction and cleanup steps, which took approximately 30 min for ten samples per technician to prepare sample extracts ready for the UPLC/MS/MS analysis. The use of commercially available tubes packed with buffer salts and dSPE adsorbents allowed to avoid tedious preparation of these mixtures. The application of ultra-performance liquid chromatography with tandem quadrupole mass spectrometry (UPLC/MS/MS) allowed for shortening of the final determination time down to 5 min, within which the data acquisition time took about 2 min only (from 0.5 to 2.5 min of the analytical run). The remaining time was necessary for the system conditioning. The major advantage of UPLC over classical HPLC was that UPLC produced narrower peaks giving increased signal-to-noise ratios and thus improved quantitative and qualitative determination of the targeted pesticides in plant and soil matrices by mass spectrometric detection (MS/MS). The MRM traces of the target pesticides obtained from the UPLC-MS/MS analysis of a cabbage sample spiked at 0.05 mg kg−1 are shown in Fig. 2.
https://static-content.springer.com/image/art%3A10.1007%2Fs00216-009-2931-5/MediaObjects/216_2009_2931_Fig2_HTML.gif
Fig. 2

Extracted MRM traces of the target pesticides obtained by UPLC-MS/MS analysis of a spiked cabbage sample at the level of 0.05 mg kg−1

Method validation results

Matrix effects were studied by multiple injections (n = 6) of the pesticides standards prepared in extracts of each matrix to be analyzed and calculating the relative responses with reference to those obtained with the standard prepared in extract of the reagent blank. As seen in Fig. 3, an enhancement effect of 11–16% was determined for cabbage and a diminishment effect of −1–7% and −3–26% was determined for soil and tomato, respectively. However, for any analyte–matrix combination, the average relative response was in the range between 70% and 120%.
https://static-content.springer.com/image/art%3A10.1007%2Fs00216-009-2931-5/MediaObjects/216_2009_2931_Fig3_HTML.gif
Fig. 3

Matrix-induced signal enhancement and suppression effects observed for the target compounds in the analyzed matrices. The signal in reagents blank extract served as a reference and was set to 100%

Linearity of the calibration curves for azadirachtin, rotenone, spinosyn A, and spinosyn D was studied by using six analytical standard concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 µg mL−1 prepared in blank sample extracts of tomato, cabbage and soil, separately. For all investigated pesticides in the selected concentration range, the regression equations yielded R2 ≥ 0.99 for each analyte and matrix except for azadirachtin in cabbage where the R2 was 0.98. For the determination of trueness (recovery) and intermediate precision (RSD) of the proposed method, blank samples spiked with the analytical standards were analyzed. The obtained average recoveries fell within the acceptance ranges recommended for pesticide residue analysis by the EU guidelines, i.e., the average recovery was between 70% and 120%, with RSD ≤ 20% with the exception of azadirachtin in cabbage spiked at 0.01 mg kg−1 where the average recovery was 67%. The trueness and precision parameters were obtained by using an internal standard method and matrix-matched-standards for more accurate quantification. The results for six replicates at each spiking level obtained for all tested pesticide–matrix configurations are given in Table 2. As can be seen, the proposed analytical methodology is characterized by excellent analytical performance parameters and allows for the single determination of selected organic farming insecticides residues at the trace levels. The LOQs values ranged between 0.006–0.01, 0.003–0.012, and 0.006–0.009 mg kg−1 for tomato, cabbage, and soil, respectively (Table 2) [18]. In addition, we performed an experiment in which we determined method LOD and LOQ using soil samples spiked with the target compounds at 0.02 mg kg−1 (n = 6) and allowed to interact with the soil for 3 h at room temperature (an average time from sample collection to arrival in the laboratory) before proceeding with the extraction and cleanup. The LODs were 0.0066, 0.0023, 0.0017, and 0.0021 mg kg−1 and the LOQs were 0.02, 0.008, 0.006, and 0.007 mg kg−1 for azadirachtin, rotenone, spinosyn A, and spinosyn D, respectively. The obtained values were in good agreement with those obtained for samples extracted immediately after spiking, except for azadirachtin (Table 2). This proved acceptable ruggedness of the developed method. Nevertheless, we noticed some instability and degradation of azadirachtin in matrix-containing extracts after just 1 day of storage. Hence, to obtain valid results, the samples should be processed as quickly as possible, and the combination of the QuEChERS-based methodology and rapid UPLC/MS/MS analysis is particularly suited for this purpose.
Table 2

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)

Pesticide

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

Tomatoes

Azadirachtin

83 (10)

91 (6)

90 (4)

0.0030

0.010

Rotenone

106 (8)

98 (5)

99 (3)

0.0024

0.008

Spinosyn A

101 (6)

104 (5)

98 (2)

0.0018

0.006

Spinosyn D

101 (6)

108 (4)

95 (3)

0.0018

0.006

Cabbage

Azadirachtin

67 (12)

86 (10)

94 (6)

0.0036

0.012

Rotenone

71 (3)

72 (3)

76 (3)

0.0009

0.003

Spinosyn A

83 (4)

93 (4)

81 (3)

0.0012

0.004

Spinosyn D

87 (7)

89 (4)

87 (4)

0.0021

0.007

Soil

Azadirachtin

90 (9)

93 (8)

101 (6)

0.0027

0.009

Rotenone

104 (7)

101 (6)

103 (6)

0.0021

0.007

Spinosyn A

92 (8)

93 (6)

88 (6)

0.0024

0.008

Spinosyn D

96 (6)

91 (5)

83 (5)

0.0018

0.006

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.

Conclusion

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.

Acknowledgments

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.

Copyright information

© Springer-Verlag 2009