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Determination of carbamates in soils by liquid chromatography coupled with on-line postcolumn UV irradiation and chemiluminescence detection
⁎Corresponding author. joseantonio.murillo@uclm.es (José A. Murillo Pulgarín)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In this study, high performance liquid chromatography (HPLC) coupled with a simple and fast sample pre-treatment based on the use of the UV-irradiation in a simple continuous flow system for the chemiluminescent quantification of pesticide carbamates in soils was developed and validated. HPLC was used to separate thiodicarb, bendiocarb and carbaryl in soil extracts. The eluates emerging from the column tail were mixed with an alkaline solution of Co2+ in EDTA and irradiated with UV light to induce photolysis of the carbamates, in order to obtain free radicals and other reactive species capable of oxidizing luminol and producing photoinduced chemiluminescence (PICL) as a result. Measurements of CL intensity were plotted as a function of time to obtain chromatographic peaks. Under the optimum operating conditions for the combined HPLC-PICL system, CL peak areas were linearly related to analyte concentrations. The limit of detection were 0.05 mg L−1 for thiodicarb, 0.09 mg L−1 for bendiocarb and 0.17 mg L−1 for carbaryl. A simple extraction procedure using 98% methanol as solvent ensured complete dissolution of the analytes in spiked soils with recoveries from 87 to 120%. The proposed method is a simple, fast, accurate choice for quantifying the target pesticides in soils.
Keywords
Insecticides
Photodecomposition
Free radicals
UV detection
1 Introduction
The presence of pesticides in the environment is an issue of great concern owing to their well-known hazardous effects on living organisms. Intensive use of insecticides, herbicides and many other agrochemicals has led to their accumulation in soil; as a result, soil has become a natural reservoir of pollutants which are sooner or later leached to subsurface waters (Hossain et al., 2015) and contaminate water resources.
The insecticidal action of carbamates is based on their ability to inhibit the enzyme acetylcholinesterase, which degrades acetylcholine. This inhibitory effect facilitates build-up of acetylcholine at synapses and result in uncontrolled movement, paralysis, convulsions or even death (Moser et al., 2015). Carbamic insecticides can be absorbed by the skin and from the gastrointestinal tract. Once absorbed, they are immediately distributed into internal tissues (Krieger et al., 2002), where they can cause oxidative stress by producing extremely toxic free radicals (Ribera et al., 2001; Banerjee et al., 1999).
The chemiluminescence (CL) of luminol and similar substances has been used to develop new methods for the analysis of pesticides including carbamates in environmental samples. For instance, Huerta-Pérez et al. (2004) quantified carbaryl in water by its enhancing effect on the chemiluminescence of the luminol-KMnO4 system.
Tsogas et al. (2006) found carbaryl to produce CL emission upon oxidation by KMnO4 and used the effect to quantify the pesticide in water samples. Carbaryl in water was also quantified by Soto-Chinchilla et al. (2005), using another CL reaction involving the oxidation of bis(2,4,6-trichlorophenyl)oxalate by hydrogen peroxide with imidazole as catalyst. Hydrolysis and derivatization of carbaryl gave the fluorophore required for CL emission, with a detection limit of 31 ng mL−1. Huertas-Pérez and García-Campaña (2008), reported a reverse HPLC method for separating various carbamates extracted from water and vegetables based on the enhancement of the CL of the luminol-KMnO4 system as a post-column detection reaction.
Luminol becomes chemiluminescent upon oxidation by free radicals or oxygen reactive species (ROS) such as those resulting from the decomposition of H2O2 in the presence of metal cation such as Cu2+, Co2+, Fe3+, the cations acting as catalysts for the reaction. The reaction produces aminophthalate as an electronically excited intermediate that relaxes to the ground state with release of excess energy as a visible light (García-Campaña and Baeyens, 2001).
Therefore, any substance producing free radicals or ROS on decomposing may be able to promote the oxidation of luminol and give CL emission. Irradiation of many pesticides with UV light has been found to facilitate their decomposition by promotion to their excited singlet states and then to triplet states by intersystem crossing. The excited states can subsequently undergo homolysis, heterolysis or photoionization (Burrows et al., 2002).
According to Herweh and Hoyle (1980), the UV photodegradation of n-aryl carbamates involves an excited singlet state that undergoes homolytic cleavage of the C—N bond to give a pair of free radicals. Upon irradiation with UV light, thiocarbamate herbicides decompose via a mechanism involving C—S bond homolysis and giving formamide and disulphide —possibly forming by combination of two sulphur radicals— as the main products (Marco and Hayes, 1979).
Climent and Miranda (1996) found the photolysis of various carbamates by effect of UV irradiation to cleave C—S or C—N bonds and give free radicals. Similarly, the photodecomposition of carbofuran involves the cleavage of its C—O bond to give phenoxide anion and an acylium cation.
In this work, thiodicarb, carbaryl and bendiocarb were separated by HPLC with an acetonitrile/water mixture as solvent and then used to obtain the reactive species needed to alter the CL emission of luminol. The concentration of each individual carbamate was determined from the increase or decrease in luminol CL intensity it caused.
2 Materials and methods
2.1 Reagents
All reagents used were analytical-grade and solvents HPLC-grade. Ultra-pure water from a Milli-Q plus system (Millipore Corp, Bedford, MA, USA) was used throughout. Acetonitrile, methanol and luminol were supplied by Sigma-Aldrich (St Louis, MO, USA). Sodium hydroxide, EDTA disodium salt and CoCl2·6H2O were purchased from Panreac (Barcelona, Spain). The carbamates (carbaryl, thiodicarb, bendiocarb and oxamyl) were obtained from Riedel de Haën (Seelze, Germany).
A stock solution of each carbamate was prepared by dissolving an amount of 50 mg in methanol in a 50 mL volumetric flask. An aliquot of 500 µL from each stock solution was transferred to a 10 mL volumetric flask and made up with methanol to obtain a 50 µg mL−1 working solution. Calibration curves were constructed from standard solutions spanning the concentration range 0.1–3.0 mg L−1. Oxamyl at a 500 mg L−1 concentration was used as internal standard (ISTD).
2.2 Instrumentation and software
The HPLC system consisted on a PU-1585 quaternary pressure pump from Jasco (Easton, MD, USA) coupled to a UV-1575 multichannel UV/Vis detector (Jasco UV-1575) also from Jasco. Reverse-phase separations was carried out on a C18 Ultrabase AV-3053 column (150 mm × 4.6 mm, particle size 5 µm). The samples were manually injected into the chromatographic system via an injection valve with a 20 µL sample loop.
Chemiluminescence intensity was measured with a Camspec Chemiluminescence Detector CL-2 (Leeds, UK) equipped with a Hamamatsu 45773-20 photosensor module with spectral response from 300 to 900 nm and spiral-type flow cell of 120 μL (Sawston, Cambridge, UK). The detector was interfaced to a computer via an analogue-to-digital converter and data were acquired by using the software Clarity v. 2.4.1.77 from Data Apex (Prague, Czech Republic) to integrate chromatographic peaks in order to calculate the peak area under the CL response signal.
The post-column reagents for the CL reaction and the Co2+/EDTA/NaOH system were delivered via a Gilson minipuls 3 peristaltic pump. UV irradiation was done with a low-pressure 4.5 W Hg PSA 10.570 UV Cracker lamp from PS Analytical (Orpington, UK). A Teflon tube 30 cm long × 1 mm i.d. was spiral coiled around the lamp to irradiate the eluates.
2.3 General procedure
Fig. 1 shows the HPLC-PICL system used. Separation was carried out at room temperature (25 ± 1 °C) in the C18 column, using an isocratic binary mobile phase consisting of 40:60 acetonitrile/water pumped at a flow rate of 1 mL min−1. The peristaltic pump was used to simultaneously pump the luminol/NaOH solution to the detector cell and the Co2+/EDTA/NaOH solution to a T-piece for mixing with the eluate emerging from the column tail. This mixture was passed through the 50 cm Teflon tube coiled around the UV lamp for irradiation and the resulting stream then transferred to the detection cell for reaction between luminol and the irradiation products. The resulting CL emission was measured in the form of a chromatographic peak.
Scheme of the HPLC-PICL manifold.
Quantitation of the carbamates was based on the increase or decrease in CL intensity and expressed in terms of peak area. Calibration curves were constructed from mixtures of three carbamates, which were quantified with the aid of the fourth (oxamyl, the internal standard, ISTD), using the following equation:
2.4 Procedure for sample preparation
Two soil samples collected from the province of Ciudad Real, Spain, were transferred to the laboratory, cleared of stones and other residues by hand, air-dried, ground and sieved to a particle size of 2 mm or less. In parallel, a Certified Reference soil 020-050 (RTC, Salisbury, UK) was used as sample matrix for spiking with the carbamates. Soil extracts were obtained as follows: an amount of 5 g of soil was homogenized in a porcelain mortar and supplied with 20 mL of 98% methanol, the resulting suspension being magnetically stirred for 15 min and filtered through Whatman no. 2 paper, the filtrate being transferred to 10 mL centrifuge tubes and centrifuged at 3000 rpm for 10 min. Finally, the clear supernatants were stored in volumetric flasks in the dark.
Once the soil samples were checked to contain none of the target carbamates by chromatographing their extracts, they were spiked with aliquots of the thiodicarb, carbaryl and bendiocarb standards plus 30 µL of the ISTD to obtain concentrations spanning the 0.1–3.4 mg L−1 range. The chromatographic system was used to assess recovery of the analytes from each soil extract (matrix).
2.5 Validation of the method
The results of the proposed method were validated by comparison with those provided by the same HPLC system but used in combination with UV/Vis spectroscopy at 230 nm as post-column detection/quantitation technique.
3 Results and discussion
3.1 Influence of the chemical conditions for UV irradiation and luminol concentration
The first variable to be optimized was the chemical conditions for UV irradiation of the carbamates emerging from the C18 column. For this purpose, the tube coiled around the UV lamp was filled with various types of media by using a T-piece to mix the eluates from the column with an appropriate solution to facilitate photodecomposition of the carbamates.
Burrows et al. (2002) found the photodegradation rate of carbaryl and propoxur to increases with increasing pH. Mixing the eluate with a 0.02 mol L−1 NaOH solution produced luminol CL that was detected as a chromatographic peak. In addition, the presence of dissolved metal ions may boost photodegradation in pesticides by facilitating redox processes yielding free radicals in Photo-Fenton type reactions Litter (1999). For example, Co2+ ion dissolved in a 0.02 mol L−1 NaOH solution containing EDTA to form a metal chelate enhances luminol CL (Murillo Pulgarín et al., 2011a; Murillo Pulgarín et al., 2011b; Sakai et al., 1989).
As it is known, the presence of Co2+ affects to luminol CL emission but it was investigated its effect in the photodegradation process of pesticides. For this, the sequence of mixing of the reactants was modified, so that, the eluate emerging from the chromatographic column was directly driven to the UV lamp while luminol was mixed with the Co/EDTA/NaOH solution, without being irradiated, in a T-piece just before entering the CL detection cell. No CL emission was observed under these conditions, which indicates that the presence of divalent cobalt during UV irradiation of the carbamates facilitated their photodecomposition
Increasing the Co(II) and EDTA concentrations to 0.5 mmol L−1 led to smaller peaks indicating reduced CL emission. A similar effect of the concentration of metal ion (specifically, Cu2+ or Fe3+) was previously observed by Litter (1999) that required identifying the concentration maximizing pesticide photodegradation. In this work, we used 0.1 mmol L−1 as the optimum concentration.
As can be seen from Fig. 2, the presence of cobalt ion and EDTA in the alkaline medium led to increased CL peaks. Besides, irradiation and the presence of cobalt proved essential to obtain CL emission. This suggests that UV energy is the agent causing the formation of the intermediate substances effecting luminol oxidation. Based on the test results, a solution containing a 20 mmol L−1 concentration of NaOH, and one of 0.1 mmol L−1 of both Co(II) and EDTA, was used for irradiation.
Effect of the chemical conditions for irradiation of solutions containing 2.7 mg L−1 carbaryl and 3.3 mg L−1 thiodicarb on CL emission.
The effect of the UV irradiation time was also evaluated. For this purpose, four Teflon tubes of variable length were used to irradiate samples for 20, 30, 60 and 90 s. CL peaks increased with increasing irradiation time up to 60 s, corresponding to a tube length of 55 cm which was selected for further testing.
The concentration of luminol influenced the intensity of its CL emission, which reached maximum value and levelled off at 3.5 mmol L−1 —the value selected for further testing.
3.2 Influence of the mobile phase and reactant flow rates
The luminol CL intensity was strongly influenced by the rate of mixing of the reactants. This required optimizing the flow rates of the HPLC mobile phase and the peristaltic pump. As can be seen from Fig. 3, CL peak areas increased with increasing flow rate of the mobile phase and CL reactants, and peaked at 0.7 and 2.25 mL min−1, respectively. Although a mobile phase rate of 1.0 mL min−1 led to slightly decreased peak areas, it was selected in order to obtain retention times of 5–8 min and reduce analysis times as a result.
Influence of the HPLC and CL reactant flow rates on CL emission.
3.3 Chromatographic separation of carbamates
Table 1 summarizes the optimum operating conditions for the HPLC-PICL system. In the chromatogram (Fig. 4), the base line with an intensity value above zero indicates that the luminol is being oxidized by some reactive oxygen species formed by the UV irradiation of the mobile phase. It is possible that oxygen molecules dissolved in the mixture acetonitrile-water used as the mobile phase can absorb the UV energy reaching electronically excited states able to react with luminol to give a constant CL signal background.
Variable
Condition
Concentrations:
Co2+ and EDTA
0.1 mmol L−1 each in NaOH 0.02 mmol L−1
Luminol
3.5 mmol L−1 in NaOH 0.02 mmol L−1
Peristaltic pump flow-rate
2.25 mL min−1
Eluent
Acetonitrile-Water (40:60)
Mobile phase velocity
1.0 mL min−1
Sample loop
20 µL
Chromatograms for thiodicarb, bendiocarb and carbaryl as obtained with oxamyl as internal standard. (a) CL detection; (b) UV detection.
In the presence of oxamyl, thiodicarb or carbaryl, other reactive species or free radicals can be formed as consequence of homolytic cleavages of C-O or C-N bonds, and these free radicals can increase the rate of luminol oxidation resulting in the positive CL peaks showed in the chromatogram.
By contrast, UV irradiation of bendiocarb inhibited luminol CL, giving negative peaks. According to Climent and Miranda (1996), the UV irradiation promotes the photochemical cleavage of the carbonyl-oxygen bond in bendiocarb molecule to give an acyl-aryloxy radical, which after rise to 2.2-Dimethyl-1,3-benzodioxol-4-ol. In previous works, Murillo Pulgarín et al. (2017, 2018) found that phenol compounds such as 1-naphthol or flavonoids are able to inactivate reactive oxygen molecules or free radicals, forming more stable phenolic free radicals, with a reduction of excited species able to oxidize luminol, inhibiting consequently the emission of chemiluminescence.
3.4 Analytical performance
Calibration curves for the four carbamates were constructed by using standard solutions containing them at concentrations over the range 0.1–3.4 mg L−1. The standards were injected in triplicate and calibration curves constructed by plotting CL peak areas against analyte concentrations. According with results of the Table 2, the CL detection has several advantages over the UV detection. For instance, the CL showed to have more sensibility for the quantification of bendiocarb, with a slope of 55.00 mg L−1 from the regression equation, while for the UV calibration curve this vale was of only 21.12 mg L−1. In addition, the CL detection allows detecting insecticides concentrations of 0.12, 0.11 and 0.14 mg L−1 for thiodicarb, bendiocarb and carbaryl, respectively, which represents concentrations in soils between 0.22 and 0.24 mg Kg−1, while for UV detection these detection limits were 0.53, 0.80 and 0.92 mg L−1. Sa standard deviation of the intercept.
Carbamate
Dynamic range (mg L−1)
Calibration equation
R2
Sa
LDa (mg L−1)
LQb (mg L−1)
Using the Chemiluminescence detection
Thiodicarb
0.25–3.00
A = 49.38 C + 10.36
0.996
1.94
0.12
0.40
Bendiocarb
0.25–3.00
A = 55.00 C + 28.58
0.997
2.05
0.11
0.37
Carbaryl
0.25–3.00
A = 58.56 C − 2.21
0.995
2.76
0.14
0.47
Oxamyl (ISTD)
0.17–1.70
A = 128.0 C + 26.8
0.994
7.29
0.17
0.57
Using the UV detection
Thiodicarb
0.82–6.54
A = 72.10C + 7.65
0.994
12.64
0.53
1.78
Bendiocarb
1.21–9.64
A = 21.12C − 6.01
0.994
5.64
0.80
2.67
Carbaryl
1.34–10.68
A = 58.57C + 2.74
0.994
17.98
0.92
3.07
In other work, Huertas-Pérez and García-Campaña (2008) employed the HPLC chromatography and Luminol CL detections after KMnO4 oxidation to determine carbaryl in water and vegetable sample with a detection limit of 0.01 mg L−1. However, this procedure was not applied to soil samples. Qiu et al. (2013) proposed the direct determination of this carbamate in several samples, including soil, by flow injection analysis using CL produced in the carbaryl reaction with Ce(IV) in a nitric acid medium containing rhodamine 6G as sensitizer. In this case, a detection limit of 0.05 mg L−1 had been obtained but the procedure can be employed only for carbaryl.
Analysis of a soil sample spiked to contain a 1.0 mg kg−1 concentration of each carbamate four times on the same day resulted in relative standard deviations of 7.5–9.0% and the inter-day analysis of the same sample led to similar precision, concretely, relative standard deviations varied between 7.5 and 9.5%.
3.5 Analysis of samples and validation of the method
Spiked soil extracts were analysed by using the response factors shown in Table 3 in combination with the relative response factor representing the CL detector response for each carbamate relative to oxamyl (the internal standard). Various samples were analysed for each one of the three soils considered. As can be seen from Table 4, the recoveries obtained (87–120%) indicate that the soil matrix had no effect on the chromatographic separation of the analytes or their detection via luminol CL.
Carbamate
Response factor
Relative response factor
Thiodicarb
0.0285
3.407
Bendiocarb
0.0165
5.885
Carbaryl
0.0775
1.253
Oxamyl (ISTD)
0.0971
1.000
Soil
Sample spiked
Thiodicarb
Bendiodicarb
Carbaryl
Theoretical (mg Kg−1)
Found (mg Kg−1)
Recovery (%)
Theoretical (mg Kg−1)
Found (mg Kg−1)
Recovery (%)
Theoretical (mg Kg−1)
Found (mg Kg−1)
Recovery (%)
Soil I
1
1.20
1.31
109
1.80
1.88
104
1.0
0.90
90
2
1.50
1.67
111
0.00
0.0
0
3.1
3.30
106
3
2.20
2.28
104
1.20
1.36
113
1.7
1.81
106
Soil II
4
1.00
1.20
120
0.00
0.0
0
0.85
0.92
108
5
1.00
1.17
117
0.00
0.0
0.0
0.0
0.0
0.0
6
0.00
0.00
0
0.00
0.0
0.0
2.40
2.75
115
CRSa
7
0.50
0.53
106
0.00
0.0
0.0
0.78
0.74
95
8
1.50
1.31
87
1.90
2.02
106
1.50
1.57
105
9
0.35
0.38
109
0.85
0.90
106
0.57
0.58
102
10
0.35
0.38
109
0.85
0.90
106
0.57
0.58
102
The proposed method was validated by comparing the previous results with those obtained by chromatographic method using UV–Visible spectrophotometric detection. A total of 7 samples containing the analytes at levels within the application range of the proposed method were analysed. The results of the two methods were subjected to least-squares pair analysis (Massart et al., 1988). This statistical technique considers the effects of various types of error. The presence of random of errors in the test method causes points to scatter around the least-squares line, and the calculated slope and intercept to slightly depart from unity and zero, respectively, as a result. Random errors can be estimated from the standard deviation in the y-direction (also called “the standard deviation of the estimate of y on x”). A proportional systematic error causes a change in b, so the difference between b and unity provides an estimate of the error. A constant systematic error reflects in a non-zero value for the intercept. If both methods provided identical concentrations in the same samples then the least-squares analysis should give a zero intercept and a unit slope. As can be seen from Fig. 5, the results of the two detection systems were significantly correlated.
Determination of the carbamates with the proposed method and the UV–Vis detection method used for comparison.
4 Conclusions
A method based on chromatographic separation coupled to on-line post-column UV irradiation and luminol CL detection for the determination of carbamates in soil was developed. The method has good selectivity for the target species, is operationally simple and fast, and allows the accurate determination of carbaryl, thiodicarb and bendiocarb in soil. Also, it uses a straightforward, fast extraction procedure providing extracts that require no clean-up for processing. The proposed method thus has a high potential for the simultaneous monitoring of the target N-methylcarbamates in environmental samples.
Acknowledgments
The authors are grateful for the financial support from the “Consejería de Educación, Cultura y Deportes” and “Fondo Europeo de Desarrollo Regional (FEDER)” Project no. PEII11-0351-7802.
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