DETERMINATION OF CHLORPYRIFOS PESTICIDE BY EFFERVESCENCE LIQUID PHASE MICROEXTRACTION HPLC UV-VIS

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1 Journal Journal of Chemical of Chemical Technology Technology and Metallurgy, and Metallurgy, 52, 6, 52, 2017, 6, DETERMINATION OF CHLORPYRIFOS PESTICIDE BY EFFERVESCENCE LIQUID PHASE MICROEXTRACTION HPLC UV-VIS Usreg Sri Handajani, Yanuardi Raharjo, Bagas Wantoro Department of Chemistry, Faculty of Science and Technology Universitas Airlangga, Surabaya, Indonesia Received 05 January 2017 Accepted 20 July 2017 ABSTRACT Effervescence-LPME is a new method that combines the use of effervescence and LPME techniques which are considered green chemistry methods. It has been used to determine the levels of chlorpyrifos compounds in cabbage, continued by analysis with high performance liquid chromatography (HPLC) with UV-Vis detector. The objectives of this research were to determine and optimize parameters of the effervescence-lpme method, namely type of organic solvent, sample volume and salt concentration, together with the determination of linearity, limit of detection, recovery, and enrichment factor of the effervescence-lpme method. The application of the effervescence-lpme as a sample preparation method in the analysis of chlorpyrifos in cabbage with HPLC was also studied. The results for the optimized analytical parameters of the effervescence-lpme method were identified as: n-hexane as the organic solvent, sample volume of 10 ml, without salt addition. The results of the validation of the method indicate that a linier calibration curve for chlorpyrifos standard solution g/l has a correlation coefficient , limit of detection (LOD) g/l, recovery %, the coefficients of variation were within % and the enrichment factor This method can be applied to determine chlorpyrifos in cabbage. It was established that with these optimal conditions chlorpyrifos levels in cabbage could not be obtained, because its concentration is beyond the limit of detection. Keywords: effervescent-liquid Phase Microextraction, chlorpyrifos, High Performance Liquid Chromatography, cabbage. INTRODUCTION Chemical analysis consists of several stages of sampling, preservation, preparation, analysis, calculation and evaluation of analytical results. Widely employed sample preparation methods are destruction, extraction, and dispersion [1]. Extraction is the preparation technique most often used. Extraction techniques is divided as, namely liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The liquid-phase microextraction (LPME) and the solid-phase microextraction (SPME) are advanced extraction techniques. Microextraction technique is faster, easier and uses very little hazardous organic solvents. Several new methods for extraction have been proposed. The effervescenceassisted dispersive micro-solid phase extraction is one of these [2], but it still has many drawbacks, including relatively significant time consuming and the requirement for an elution process. Effervescence is a simple reaction between an acid and a carbonate salt that produces carbon dioxide and can assist phase separation of the organic solvent in the water phase of the solution [3]. Microextraction can be applied for sample preparation of for compounds in with low levels of concentration in nature, such as the pesticides. Organophosphate pesticides (OPPs) have been widely used in the world to reduce pest (OPT). They are 1056

2 Usreg Sri Handajani, Yanuardi Raharjo, Bagas Wantoro often used also in Indonesia, with names like diazinon, dimetoat, chlorpyrifos, and profenofos. Organophosphate pesticide compounds are suspected to inhibit the acetylcholinesterase enzyme (AChE), that controls the level of acetylcholine (ACh). AChE inhibition results in accumulation of Ach, inhibits neurotransmitions, and can cause muscle paralysis, seizures, bronchial constriction, and shortness of breath [4]. Because of the danger posed by organophosphate compounds, the government has set a safe limit for the concentration of organophosphates, like chlorpyrifos, of 0.5 mg/kg of the human body. The analysis of chlorpyrifos, contained in a plant can use chromatographic methods. Instruments for the analysis of pesticide chlorpyrifos levels should have a high accuracy for very low levels of the compound. They include High Performance Liquid Chromatography (HPLC), which can be used for the separation of a number of organic compounds; inorganic, and biological analysis of impurities; the analysis of volatile compounds, isolation and purification of compounds, separation of compounds that are structurally similar, separation of compounds of the trace elements. In modern high-speed liquid chromatography the UV-Vis detector is most widely used, because it can detect these compounds in a wide range [5]. The analysis with microextraction, including effervescence-assisted dispersive micro-solid phase extraction [2], has been developed for nitroaromatic compounds in river water and has produced a recovery of %, with a coefficient of variation of %. The effervescence assisted liquid phase microextraction [6] for triazine in water has shown a recovery of %, with a coefficient of variation of %. It needs an excess time to reach all areas in the sample, but the use of organic solvents is small, and without using additional tools. The effervescence-lpme method and HPLC UV- Vis instruments have been used to analyze the concentration of chlorpyrifos in cabbage. The studied parameters include organic solvent type, sample solution volume, and the effect of adding NaCl for ionic strength. The aim of this research was to determine and optimize parameters in effervescence-lpme method, namely the type of organic solvent, the sample volume, and the salt concentration together with the determination of linearity, limit of detection, recovery, and enrichment factor of the effervescence-lpme method. EXPERIMENTAL Material and Method The materials used in this study include the organic solvents methanol, toluene, chloroform and n-hexane. Chlorpyrifos, citric acid, sodium bicarbonate, and sodium chloride were also used. The sample was taken from cabbage plantations in Malang and Banyuwangi. The instruments used were High Performance Liquid Chromatograph (HPLC), Shimadzu LC-5A with an UV/ Vis detector, microsyringes, micropipettes, mortar, stir bars, an analytical balance, and a Buchner funnel. Optimization of the organic solvent Toluene, chloroform, and n-hexane were studied as suitable organic solvent. Hexane has been shown to be the best and so it was chosen for further analyses. Optimization of the sample solution volume The sample solution volume was optimized, using a variation of volume 5, 10, 15, and 20 ml. The other variables were kept constant: mass 1 g effervescent powder, organic solvent volume of 100 ml, concentration of 30 g/l standard solution, and NaCl concentration of 0 g/l. The resulting solution was analyzed by HPLC, then a graph between the volume of the sample solution to the chromatogram area was drawn. Optimization of salt addition The optimization of the NaCl addition was done using variation of 0.1, 0.2, and 0.3 g/l, while the rest of the variables were kept constant, namely, 1 g effervescent powder, organic solvent volume of 100 ml, concentration of the standard solution of 30 g/l. The resulting solution was analyzed by HPLC, then a graph between the volume of the sample solution to the chromatogram area was drawn. Sample preparation The cabbage was cut into small pieces and weighed to as much as 10 g. Then it was crushed with a mortar, put in a 50 ml beaker glass, stirred with 25 ml of 1057

3 Journal of Chemical Technology and Metallurgy, 52, 6, 2017 Fig. 1. Methanol and chlorpyrifos chromatogram. methanol and allowed to stand for 3 hours, covered with an aluminum foil. The mixture was then put in a 100 ml volumetric flask, diluted with water up to the mark, and homogenized. The solution was filtered with a Buchner funnel to separate solids and liquids (filtrate). The clear sample solution was poured in a brown glass bottle, wrapped in an aluminum foil and stored in a dark place at room temperature. This is done because the volatile chlorpyrifos can be easily degraded into compounds that are toxic. The samples were stored for a maximum of 7 days. Sample Analysis The extracts were analyzed using the HPLC instrument. The concentration of chlorpyrifos can be calculated using the linear regression equation, obtained from a calibration curve of a standard solution after extraction, using the effervescence-lpme techniques with the parameters that have been optimized. RESULTS AND DISCUSSION 1058 High Performance Liquid Chromatography (HPLC) The HPLC separation method is based on the difference in the migration between the two phases. The stationary phase used was C-18. A polar mobile phase and an nonpolar stationary phase were thus used. Based on the data contained in the packaging of Sigma-Aldrich, in the chlorpyrifos analysis as the eluent acetonitrile:water at a ratio of 80 : 20 (v/v) was used. The wavelength was set at 225 nm, the flow rate of the eluent used was 1.4 ml/min at a pressure of 25 x 10 kg/cm 2. In the chromatogram obtained, the methanol peak appeared at 1.4 min, while the peak of chlorpyrifos appeared at 5.4 min. The obtained chromatograms of methanol and chlorpyrifos can be seen in Fig. 1. Optimization of the organic solvent Optimization of the organic solvent for the analysis of chlorpyrifos with effervescence-lpme extraction was done with three organic solvents, namely toluene, chloroform, and n-hexane. For optimization of solvents, the rest of the variables were kept constant: mass of 1 g effervescent powder, organic solvent volume of 100 ml, concentration of 30 g/l standard solution, 10 ml volume of the sample solution, and no addition of salt. The extraction results for the organic solvents studied can be seen in Table 1. From the diagram optimization of organic solvent it can be concluded that the most appropriate solvent for the extraction of chlorpyrifos, using effervescence- LPME, is n-hexane which is able to extract more chlorpyrifos, more than any other solvents. The solubility of the component depends on the degree of polarity, which can be determined from the value of the dielectric constant of the solvent. The order of polarity of the studied organic solvent, based on the value of the dielectric Table 1. Data of organic solvents to chromatogram area. Organic solvent Chromatogram area (unit) toluene chloroform n-hexane

4 Usreg Sri Handajani, Yanuardi Raharjo, Bagas Wantoro Table 2. Data of sample solution volume to chromatogram area. Sample solution volume (ml) Chromatogram area (unit) constant is: n-hexane 2.0, chloroform 2.4, and toluene 4.8. The solvent n-hexane has a dielectric constant of the smallest and most nonpolar character. Chlorpyrifos is also nonpolar, so more chlorpyrifos extracted in n-hexane. Optimization of the volume of the sample solution In the chlorpyrifos analysis by effervescence-lpme the volume of the sample solution was varied at 5, 10, 15, and 20 ml. The values of the rest of the variables were kept constant, namely, mass of 1 g effervescent powder, organic solvent volume of 100 ml, concentration of the standard solution of 30 g/l, without the addition of salt. The organic solvent, in accordance with the results of the previous optimization, was n-hexane. The chlorpyrifos 30 g/l standard solution was prepared from a working solution of 100 g/l with water. The optimization results for the sample solution, in terms of the HPLC chromatogram area are given in Table 2. From Table 2 the optimum volume is of 10 ml sample solution, which has the greatest chromatogram area. For sample solution volumes below 10 ml the results are lower because the total volume of the sample solution is too small, so that the amount of analyte extracted is less. For volumes of the sample solution above 10 ml, the chromatogram shows smaller areas than for 10 ml, so the amount of analyte extracted becomes less. Optimization of the salt addition Optimization of the NaCl addition was tested using a variation of 0.1, 0.2, and 0.3 g/l. The other variables were kept constant. The organic solvent was n-hexane and the sample solution volume - 10 ml. The data for the extraction for the optimization of the NaCl addition can be seen in Table 3. Table 3. Data of NaCl concentration to chromatogram area NaCl concentration (g/l) Chromatogram area (unit) In Table 3, the chromatogram area is largest without the addition of NaCl, and after the addition of NaCl becomes smaller. The salt is very influential in the extraction process because it can improve recovery. With the addition of salt, extracted analytes will increase due to the effect of salting out, in which water molecules form a circle around the hydrated salt molecules. However, the addition of NaCl on the optimization in this study showed negative results. The compound chlorpyrifos has a value of log K o /w 4.96, which means that chlorpyrifos is nonpolar, therefore the addition of salt will lower the extraction recovery. Limit of detection (LOD) The calibration curve of the chlorpyrifos standard solution without extraction of effervescence-lpme has shown a limit of detection of 2.45 g/l. The calibration curve of the chlorpyrifos standard solution for extraction with effervescence-lpme has shown a LOD value of 1.39 g/l and LOQ value of 4.64 g/l. By comparing the value of the limits of detection we can conclude that the effervescence-lpme method is able to increase the sensitivity of the HPLC UV-Vis analyte response. Recovery (%) Based on data obtained from the standard curve of chlorpyrifos without effervescence-lpme extraction the recovery ranged from %, while the percent recovery obtained from the chlorpyrifos standard curve with the effervescence-lpme extraction ranged %. This means that the recovery of chlorpyrifos as analyte using effervescence-lpme techniques is approaching the actual concentrations of chlorpyrifos. The data in Table 4 show recovery of chlorpyrifos with effervescence-lpme techniques, car- 1059

5 Journal of Chemical Technology and Metallurgy, 52, 6, 2017 Table 4. Recovery of the chlorpyrifos standard solution. Concentration (g/l) Recovery (%) ried out in this study, of %, so it may be concluded that the application of effervescence-lpme in the determination of chlorpyrifos concentration has good accuracy. Coefficient of variation (precision) Precision is expressed by the value of standard deviation and the coefficient of variation. The coefficient of variation is used to view the reproducibility of an instrument. Based on Table 5, the precision value of the calibration curve, using a chlorpyrifos standard solution of effervescence-lpme, ranged from %, Table 5. The precision of chlorpyrifos. Concentration (g/l) Precision (%) which is lower than the calibration curve of chlorpyrifos standard solution without effervescence-lpme. This shows that precision of the HPLC instrument, when using effervescence-lpme is better when compared to the data without the effervescence-lpme method. Enrichment Factor The enrichment factor is the concentration factor of the magnitude that occurred during the extraction by effervescence-lpme. The chlorpyrifos concentration curve can be seen in comparison with the standard solution of chlorpyrifos, before and after extraction using effervescence-lpme shown in Fig. 2. It can be seen on Fig. 2, that there is 100 times enrichment of preparation using Effervescent-LPME. It can be concluded that the extraction process of effervescence-lpme has shown good results. Sample preparation The samples, used in this study, are cabbage samples taken at two places, namely from a plantation in Malang and from a plantation in Banyuwangi. Before the analysis, the sample was cut into small pieces and weighed to as much as 10 g. It was then crushed with a mortar, then put in a 50 ml beaker glass, stirred with 25 ml of methanol and allowed to stand for 3 hours, covered with an aluminum foil. The mixture was then put in a 100 ml volumetric flask, diluted with water up to the mark, and homogenized. The solution was filtered with a Buchner funnel to separate solids and liquids (filtrate). The clear Fig. 2. Chlorpyrifos concentration curve using effervescent-lpme extraction. 1060

6 Usreg Sri Handajani, Yanuardi Raharjo, Bagas Wantoro Table 6. Chromatogram area from sample analysis. Sample Chromatogram area Concentration (g/l) Malang plantation n.a. Banyuwangi plantation n.a. filtrate was poured into a brown bottle, which was then sealed with an aluminum foil and stored in a dark place at room temperature up to a maximum of 7 days. The storage of the samples prevented the compounds contained in the sample from damage or degradation. Sample Analysis The sample solution was analyzed using the same procedures as in the preparation of the manufacture of calibration curve with the chlorpyrifos standard solution, using effervescence-lpme. In the analysis of sample, the parameters used were: n-hexane as organic solvent with volume of 100 ml; sample solution volume of 10 ml, without addition of NaCl, and mass of 1 g effervescent powder. The equation of calibration curve using a chlorpyrifos standard solution of effervescence (LPME) is y = x After the calculation, the chlorpyrifos concentration obtained for each sample may be seen in Table 6. The data in Table 6 shows that chlorpyrifos in cabbage was not detected by the instrument because it is below the limit of detection. Thus, the levels of pesticide residues in the cabbage are under the maximum limit of pesticide residues on in agricultural products, namely 1 mg/kg. The concentration of chlorpyrifos in cabbage from Malang and Banyuwangi is below the threshold of chlorpyrifos in environment, but cannot be stated to be safe for consumption, because there is still the possibility of the presence of the compound or other pesticide residues. This study concerned only one type of pesticide, in which the active ingredient is chlorpyrifos. CONCLUSIONS The optimized analytical parameters for the effervescence-lpme method are: n-hexane as organic solvent, sample volume 10 ml, without salt addition. The result of validation shows that a linear calibration curve for the chlorpyrifos standard solution in the g/l has a correlation coefficient , the limit of detection (LOD) is 1.39 g/l, the recovery is %, the coefficients variations are % and the enrichment factor is This method can be applied to determine chlorpyrifos in cabbage. Acknowledgements The authors express gratitude to the Ministry of Research and Higher Education, Chemistry Department, Faculty of Science and Technology, Universitas Airlangga, for research grant and facility. REFERENCES 1. S. Mitra, Sampel Preparation Techniques in Analytical Chemistry, John Wiley & Sons. Inc. Canada, G. Lasarte-Aragones, R. Lucena, S. Cardenas, M. Valcarcel, Effervescence-assisted dispersive microsolid phase extraction, Journal of Chromatography A, 1218, 2011, M.J. Gonzalez-Rodriguez, A.G. Liebanas, J.L.M. Frenich, F.J.S. Vidal, Determination of pesticides and some metabolites in different kinds of milk by solidphase microextraction and low-pressure gas chromatography-tandem mass spectrometry, Analytical Bioanalytical Chemistry, 382, 2005, X. Liu, Z. Shen, P. Wang, C. Liu, Z. Zhou, D. Liu, Efferrvescence assisted on-site liquid phase microextraction for the determination of five triazine herbicides in water, Journal of Chromatography, 1371, 2014, J.L. Tadeo, S.B. Consuelo, G. Lorena, Analysis of Pesticide in Food and Environmental Samples, CRC Press, Boca Raton, D.J. Ecobichon, Casarett and Doull s Toxicology, McGraw-Hill, New York,