2Nagarjuna Agrichem Limited (R&D Center), Telangana, India
Keywords: Solid-Phase Extraction; Quechers; Imidacloprid; Method Validation; UFLC
A number of methods have been employed to measure imidacloprid residues: Photochemical fluorimetric method [1], electrochemical method, enzyme-linked immune sorbent assays [2], capillary electrophoresis, gas chromatographymass spectrometry (GC-MS) and High performance liquid chromatography HPLC [3]. Among these methods GC and LC are the most suitable methods. However, GC cannot be used directly to determine imidacloprid due to the poor volatility and polarity. Imidacloprid residues can be analyzed by derivatization and gas chromatography. In contrast to GC, HPLC is more effective and appropriate for the residual analysis of imidacloprid, and it has been successfully employed for assaying imidacloprid in the soils, water as well as in the vegetables [4]. High performance liquid chromatography (HPLC) appears to be a suitable alternative because of the thermolability and polarity of imidacloprid. The LC method gave good results for imidacloprid in groundwater, soils, fruits and vegetables but the limit of detection (LOD) was still too high for the present purpose [5]. Furthermore, the required method must satisfy strict quality criteria to be classified as a trace levels determination and low levels extraction procedure. Prakash Samnani, et al. [6] has been reported a simple and sensitive method for imidacloprid using HPLC and extraction procedure for imidacloprid which is a traditional method. The ultra fast liquid chromatography (UFLC) is quite popular for enhancing the laboratory's productivity. The combination of Prominence UFLC and new reversed-phase columns achieves ten times higher speed compared to a conventional HPLC system. Furthermore, a high-speed sample injection mechanism significantly reduces total analytical cycle time. With this combination, UFLC offers three times higher resolution than conventional HPLC so that the possibility of analysis is increased for samples that are not sufficiently separated by conventional HPLC. The UFLC yields good resolution, low sensitivity and fast analysis with area repeatability better than conventional HPLC.
The improvement in analysis is largely due to use of smaller particles used in the column (3.5 μm columns) and associated hardware to achieve higher pumping pressures, supported by faster injections and detection.
QuEChERS (Quick–Easy–Cheap–Effective–Rugged–Safe) is a sample preparation approach developed by Anastassiades, et al. [7] as a simple, rapid, effective, yet inexpensive, way to extract pesticide residues from fruits and vegetables, followed by a dispersive solid phase extraction (dSPE) cleanup of the extract. It is well established that QuEChERS can result in good recovery values not only for a large number of pesticides but also for a wide variety of other compounds. Kapoor U, et al. [8] have reported QuEChERS (quick, easy, cheap, effective, rugged, and safe) method of extraction procedure for imidacloprid in fruits, fruit juices, and baby foods followed by high-performance liquid chromatographic analysis, and imidacloprid residues were qualitatively confirmed by liquid chromatography-mass spectrometry. We developed a new extraction scheme with high recovery rates, coupled to a UFLC methodology in order to extract and quantify imidacloprid in soils, vegetables (cabbage and spinach). The present study will concern extensively to trace level determination of imidacloprid in vegetables (cabbage and spinach) and soil by validating and using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) and solid phase extraction method and ultrasound method followed by Ultra Fast Liquid Chromatography (UFLC). We modify the (QuEChERS) method for determination of imidacloprid. Comparison between (QuEChERS) extraction method, Solid phase extraction and ultrasound for its efficiency and sensitivity were carried out. More generally, such a method can be easily adapted for the analysis of fruits and vegetables.
Calibration mixture solution: Calibration mixtures of concentration levels 0.005, 0.01, 0.05, 0.1, 0.5 and 1.0 μg/mL were prepared in acetonitrile by diluting suitable aliquots of stock solution in acetonitrile and were stored at -20 ± 2°C.
The Figures 1, 2 and 3 depict a typical chromatogram of the separation of imidacloprid reference standard and recovery in vegetable and soil spike samples. The vegetable samples were analyzed by LC-MS Ion Trap for imidacloprid peak confirmation and other matrix effects.
Ratio approach: This method can only be applied to analytical procedures which exhibit baseline noise. It is determined by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing minimum concentration at which the analyte can be reliably detected. A S/N ratio of 3:1 is considered acceptable for estimating LOD (with Relative Standard Deviation (RSD) ≤ 10%) whereas for LOQ S/N ratio of 10:1 is considered appropriate (with Relative Standard Deviation (RSD) ≤ 3%).
Standard deviation of the response and slope: The LOD and LOQ may be expressed as LOD = 3.3 × σ/S and LOQ = 10 × σ/S where σ = the standard deviation of the response, S = the slope of the calibration curve of analyte. The slope S may be estimated from the calibration curve of the analyte. The value of σ may be taken from as standard deviation of analytical background responses of an appropriate Three of blank samples. The linear dynamic range of imidacloprid is shown in Figure 5.
The precision (% RSD) of the analytical method was determined by five replications in the duplicate injection of fortified substrate soil and cabbage and spinach sample extracts at LOQ, 10 × LOQ and 50 × LOQ levels. The accuracy (% recovery) of the method was determined by five replications (in duplicate) injection of fortified substrate soil, cabbage and spinach sample extracts at LOQ, 10 times and 50 times LOQ levels. Precision (% RSD) should not exceed 20% at lower levels.
QuEChERS method: A quantity of 10g soil and vegetables samples (cabbage and spinach) was weighed and transferred in Polyethylene (PFTE) 50 mL tube, then 20 mL acetonitrile was added and shaken vigorously for one minute, 2.0 gm anhydrous MgSO4 and 0.5 gm sodium chloride were then added and mixture shaken immediately for 1.0 minute. Centrifugation was carried out at 4000 rpm for 5 minutes and cleanup was done with 25 mg PSA (Primary secondary amine). Primary Secondary Amine (PSA) has been found as the most effective sorbent for removal of various matrices and significantly reducing the matrixenhancement effect. On the other hand, graphitized carbon black (GCB) is very useful for the removal of coloring substances (i.e. pigments) and sterols. Therefore, PSA itself and/or combination of GCB/PSA dual layer should be most effective for sample
Extraction procedure for imidacloprid with Ultrasound technique from vegetable samples: 10 g of vegetables sample (cabbage and spinach) was homogenized and was spiked at two levels 0.1 and 0.5 μg/g, with imidacloprid. The fortified sample was mixed with 25mL acetonitrile. This was placed on an ultrasound bath for 20 minutes and centrifuged at 3000 rpm for 30 minutes. This was found to improve the extraction efficiency of the imidacloprid. This procedure was repeated twice and the extracts were pooled. A volume of 5.0 mL extract was taken for cleanup by solid phase extraction.
Solid Phase Extraction method (SPE): 10 g of soil sample was weighed in reagent bottles of 50 mL capacity. 20 mL acetonitrile was added and the bottle was shaken on an orbital shaker for 10 minutes. This process was repeated twice. The extract was filtered through Whatman filter paper No.1 into 100 mL graduated tubes. From this, 20 mL aliquot of the extract was concentrated to less than 2.0 mL on the N-EVAPTM. The concentrate was suspended in 2 to 3 mL of methanol. A Bond- Elute cartridge was conditioned by rinsing with 5.0 mL of methanol on a vacuum and the extract suspension was loaded on the cartridge and the eluant was collected in a test tube. The test tubes were rinsed 2 times with 3 mL methanol. Each of the cartridge rinses was collected in the manifold. The eluate was concentrated to about 2.0 mL and final sample was made with methanol in 5 mL. This sample was directly injected to reverse phase Ultra Fast Liquid Chromatography (UFLC).
Similar results were found by us in present method validation for soil and vegetable samples. The repeatability of the method in the present study was determined for each fortification level by running a set of five recoveries each at different fortification levels for selected matrices. The % RSD for the resulting mean recovery rates ranged from 94.00 to 95.62% in soil with relative standard deviations between 0.25% to 1.61%. The % RSD for spinach and cabbage ranged varied from 0.87% to 2.15% and 0.89% to 1.00% in solid phase extraction method. In QuEChERS method % RSD range varied from 1.02% to 1.94% and 0.14% to 1.13% Imidacloprid recovery for Ultrasound technique in vegetable samples of spinach recovery range varied from 92.36 to 96.84 with % RSD 1.12 and 1.65%. Similarly, cabbage sample recovery range varied from 94.945 to 95.32% with %RSD was 0.98% to 1.40%. In ultrasound extraction, procedure recovery is good but at lower fortification levels 0.01 μg/g recovery is low. The vegetable samples were analyzed by LC-MS Ion Trap for imidacloprid peak confirmation and another matrix effect (Figure 4). We found the imidacloprid parent ion m/z 256 in positive mode and very sharp peak and no any matrix effect.
Parameters |
Substrate |
||||||
Cabbage |
Spinach |
Soil |
Cabbage |
Spinach |
|||
SPE |
QuEChERS Method |
SPE |
QuEChERS Method |
SPE |
Ultrasound technique |
||
Specificity |
No interference |
||||||
Fortification Levels (µg/g)
|
Precision (%RSD) |
||||||
0.01 |
1.71 |
1.02 |
1.00 |
0.14 |
0.25 |
- |
|
0.10
|
2.15 |
1.12 |
0.89 |
2.13 |
1.02 |
1.4 |
1.65 |
0.50
|
0.87 |
1.94 |
0.96 |
1.13 |
1.61 |
0.98 |
1.12 |
Accuracy (% Recovery) |
|||||||
0.01
|
96.82 |
95.00 |
90.34 |
89.39
|
95.00 |
- |
|
0.10
|
96.39 |
94.00 |
93.13 |
94.41 |
94.00 |
95.32 |
96.84 |
0.50
|
99.52
|
95.62
|
97.02 |
90.4
|
95.62
|
94.94 |
92.36 |
- Vilchez JL, El-Khattabi R, Fernandez J, Gonzalez-Casado, Navalon A. Determination of imidacloprid in water and soil sample by gas-chromatography-mass Spectrometry. J. Chrom. A. 1996;746(2):289-294. doi:10.1016/0021-9673(96)00402-5.
- Watanabe E, Eun H, Baba K, Arao T, Ishii Y, Endo S, Ueji M. Rapid and simple screening analysis for residual imidacloprid in agricultural products with commercially available ELISA. Analytica Chimica Acta. 2004;521(1):45-51. doi:10.1016/j.aca.2004.05.056.
- Zhou Q, Ding Y, Xiao J. Sensitive determination of thiamethoxam, imidacloprid and acetamiprid in environmental water samples with solid-phase extraction packed with multiwalled carbon nanotubes prior to high-performance liquid chromatography. Anal Bioanal Chem. 2006;385(8):1520-5.
- Baig SA, Akhter NA, Ashfaq M, Asi MR, Ashfaq U. Imidacloprid residues in vegetables, soil and water in the southern Punjab, Pakistan Journal of Agricultural Technology. 2012;8(3):903-916.
- Srivastava Ashutosh K, Srivastava MK, Patel DK, Mudiam MKR, Srivastava LP. Gas-Chromatographic Determination of Imidacloprid in Water. J. Environ. Res. and Development. 2012;7(2):643-651.
- Samnani P, Vishwakarma K, Pandey SY. Simple and sensitive method for determination of imidacloprid residue in soil and water by HPLC. Bull Environ Contam Toxicol. 2011;86(5):554-8. doi: 10.1007/s00128-011-0245-8.
- Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "Dispersive solid-phase extraction" For the determination of pesticide residues in produce. J AOAC Int. 2003;86(2):412-31.
- Kapoor U, Srivastava MK, Srivastava AK, Patel DK, Garg V, Srivastava LP. Analysis of imidacloprid residues in fruits, vegetables, cereals, fruit juices, and baby foods, and daily intake estimation in and around Lucknow, India. Environ Toxicol Chem. 2013;32(3):723-7. doi: 10.1002/etc.2104.
- Choning R, Schmuck R. Analytical determination of imidacloprid and relevant metabolite residues by LC MS/MS. Bulletin of Insectology. 2003;56(1):41-50.
- Method validation and quality control procedures for pesticide residues analysis in food and feed, Document NO. SANCO/10684/2009. SANCO;2009.