Simultaneous Determination of Toxic Organophosphorus Insecticide

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Rao et al., 1:6 http://dx.doi.org/10.4172/scientificreports.327 Open Access Open Access Scientific Reports Scientific Reports Research article OpenOpen Access Access Simultaneous Determination of Toxic Organophosphorus Insecticide Residues in Honey Followed by Matrix Solid-Phase Dispersion Coupled to High-Performance Liquid Chromatography with Ultraviolet Detection Tentu Nageswara Rao*, Atmakuru Ramesh and Tentu Parvathamma Department of Analytical Chemistry, International Inisitituate of Biotechnology and Toxicology (IIBAT), Padappai, Kancheepuram 601301, Tamil Nadu, India

Abstract Matrix solid-phase dispersion has been developed for the extraction and preconcentration of organophosphorus insecticide residues (Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos) in honey. After extraction residues were determined by high performance liquid chromatographic method. The evaluated parameters included the type and amount of sorbent (silica gel, C18 and Activated Florisil) and the nature of eluent (n-hexane and dichloromethane). The best results were obtained using 2.0 g of honey sample, 2.0 g of C18 as sorbent and 20 mL of n-hexane -dichloromethane (1:1, (v/v)).The method was validated using honey sample spiked with insecticides at different concentration levels (0.01 and 0.1 µg/mL). Average recoveries (using each concentration six replicates) ranged 87-96%, with relative standard deviations less than 3%, calibration solutions concentration in the range 0.01- 2.0 mg/L and limit of detection (LOD) and limit of quantification (LOQ) were 0.003 mg/L and 0.01 mg/L respectively.

Keywords: Matrix solid-phase dispersion, Organophosphorus Organophosphorus compounds are mainly highly and moderately insecticides, HPLC-UV toxic to humans and warm-blooded animals. Upon entering into the body they affect the cholinergic synapses of the central nervous system Introduction and the peripheral nerve muscle connections [1]. A molecule of an organ phosphorous substance contains two kinds Many compounds of this group have an acute and moderate chronic of groups, the electronegative group ‘x’ forming a relatively unstable toxicity that manifests itself with the frequent introduction of toxic and anhydride bond with phosphorous and an alkoxy group having a sublethal doses. The high activity of phosphatases, carboxyesterases strong bond with phosphorous. The hydrolysis of organophosphorus and amidases in the organisms of warm blooded animals result in the compounds by phosphatases is the main metabolic way leading to a rapid decomposition of organophosphorus compounds into non toxic complete loss of toxicity in the organisms of humans, warm blooded water soluble products which is excreted through urine. The dermal animals, insects and plants. toxicity of organophosphorus insecticide is not high but toxicants like The toxic action of organophosphorus compounds on insects is due methyl-parathion are highly toxic when they get into the skin. to inhibition of the activity of acetylcholinesterase in the cholinergic Several methods have been used for the determination of synapses of the nervous system. (The space between nerve cells, organophosphorus insecticide residues using solid-phase extraction called a synapase). When organophosphorus compounds enter the (SPE), solid-phase micro extraction (SPME), supercritical fluid organism of an insect, the activity of the acetylcholinesterase sharply extraction (SFE), Single drop micro extraction (SDME) and matrix drops. Indications of poisoning appear very rapidly in hyperactivation, solid-phase dispersion (MSPD). However, none of the published next paralysis and then leading to death. Most organophosphorus researches to date have reported the simultaneous analysis of chemical insecticides have a high initial toxicity, and pests perish during the first classes such as Monocrotophos, Triazophos, Phosalone, Profenofos few hours after treatment. and Chlorpyrifos in honey. Substances of this group are good for controlling larvae and The matrix solid-phase dispersion (MSPD) technique was adult individual of pests but their ovicidal action is weak, which is developed by Barker in 1989 [2]. It has advantages over conventional associated with low permeability of egg shells. Oil solutions of some techniques because it employs small amounts of sample and solvent, organophosphorus compounds penetrate quite well into the eggs of and the extraction procedure consists of only a few experimental insects and mites, causing their death. steps. MSPD evolved from the solid-phase extraction (SPE) technique, The activity of an embryon’s cells and its growth are not violated modified for application to solid and semi-solid matrices. The MSPD but acetylcholine accumulates in the embryon. The larvae die owing to the inability of moving when it leaves the egg. This is why plantings should be treated with oil formulations of such a kind not long before *Corresponding author: Tentu Nageswara Rao, Department of Analytical Chemistry, International Institute of Biotechnology and Toxicology (IIBAT), the larvae hatch from the eggs. Kancheepuram, Tamil Nadu, India, E-mail: raoau@ rediffmail.com Upon perennial use of organophosphorus insecticides, specific Received August 18, 2012; Published September 16, 2012 resistance appears in insects or mites. Specific resistance to Citation: Rao TN, Ramesh A, Parvathamma T (2012) Residues in Honey Followed by organophosphorus compounds appears more rapidly in the species of Matrix Solid-Phase Dispersion Coupled to High-Performance Liquid Chromatography insects that produce several generations in a single season. But the most with Ultraviolet Detection. 1:327. doi:10.4172/scientificreports.327 dangerous is the development of resistance in phytophagous mites. Copyright: © 2012 Rao TN, et al. This is an open-access article distributed under The economical harm form this phenomenon is very great especially in the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and cotton-growing regions. source are credited.

Volume 1 • Issue 6 • 2012 Citation: Rao TN, Ramesh A, Parvathamma T (2012) Residues in Honey Followed by Matrix Solid-Phase Dispersion Coupled to High-Performance Liquid Chromatography with Ultraviolet Detection. 1:327. doi:10.4172/scientificreports.327

Page 2 of 4 procedure is based on the use of a sorbent, which acts as an abrasive in mortar for 30 min, to assess the homogeneity of the sample. The sample order to produce a modified “opening” of the solid matrix, facilitating was allowed to stand at room temperature for one hour, until analysis. the extraction process when using a suitable solvent for eluting the analytes. The use of MSPD for organophosphorus insecticides recovery Extraction procedure depends on the solubility of the organophosphorus insecticides in 2.0 g of honey sample was weighed out and homogenized with the eluting solvent, as well as the interactions between the matrix 2.0 g of C18 –bonded silica for 5 min. The homogenized sample components, sorbent and eluent [3]. Due to the lack of literature was transferred to an MSPD column consisting of a 20 mL capacity reports concerning the use of MSPD as an extraction technique for polyethylene syringe containing 2.0 g florisil and 2.0 g of anhydrous organophosphorus insecticides belonging to different chemical classes sodium sulfate. The elution was performed under vacuum with 20 mL from plants, soil water and food products, this paper presents an MSPD of n-hexane-dichloromethane (1:1). The eluent was collected into a 50 method for determination of residue of organophosphorus insecticides mL glass tube and then evaporated under gentle stream of nitrogen, in honey [4-7]. So, the present research considered five different with the water bath temperature set at 40-45ºC. Residue was dissolved chemical classes, namely Monocrotophos, Triazophos, Phosalone, with 5 mL of acetonitrile. Profenofos and Chlorpyrifos which analysis by high-performance liquid chromatography with ultraviolet detector (HPLC-UV). Chromatographic separation parameters Experimental The HPLC-UV system used, consisted Shimadzu high performance liquid chromatography with LC-20AT pump and SPD- Standards, reagents and samples 20A interfaced with LC solution software, equipped with a reversed Phase C18 analytical column of 250 mm×4.6 mm and particle size Certificated analytical standards of Monocrotophos (99.4%), 5.0 µm (Phenomenex) Column temperature was maintained at 30°C. Triazophos (98.2%), Phosalone (99.2%) Profenofos (99.1%) and The injected sample volume was 20 µL. Mobile Phases A and B were Chlorpyrifos (99.8%) were obtained from Sigma Aldrich. Common acetonitrile and Milli-Q water (75:25(v/v)). The flow- rate used was names and structures of the organophosphorus insecticides evaluated kept at 1.2 mL/min. The detector wavelength was 230 nm. The external here are shown in Figure 1. Acetonitrile was purchased from Rankem, standard method was used for this analysis. New Delhi, Analytical grade solvents, dichloromethane and n-hexane, were supplied from Merck Limited, Mumbai, C18-bonded silica (50 Method validation µm) from phenomenex (Torrance, CA, USA), Florisil (60-100 mesh) from Fluka Chemie GmbH CH-9471 Buchs, AR grade sodium sulphate Method validation ensures analysis credibility. In this study, from Merck Limited, Mumbai and honey was purchased from local the parameters accuracy, precision, linearity and limits of detection market. They were brought to the laboratory and stored in plastic (LOD) and quantification (LOQ) were considered. The accuracy of bag under refrigerator condition until they were processed in the the method was determined by recovery tests, using samples spiked at laboratory. concentration levels of 0.01 and 0.1 mg/kg. Linearity was determined by different known concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 µg/ Standard stock solutions mL) were prepared by diluting the stock solution. The limit of detection The organophosphorus insecticide standard stock solutions were (LOD, µg/mL) was determined as the lowest concentration giving individually prepared in acetonitrile at a concentration level of 100 µg/ a response of 3 times the baseline noise defined from the analysis of mL and stored in a freezer at -18°C. The stock standard solutions were control (untreated) sample. The limit of quantification (LOQ, µg/mL) used for up to 3 months. Suitable concentrations of working standards was determined as the lowest concentration of a given herbicide giving were prepared from the stock solutions by dilution using acetonitrile, a response of 10 times the baseline noise. immediately prior to sample preparation. Results and Discussion Sample preparation Specificity Representative 2.0 g portions of honey fortified with 10 µL of Specificity was confirmed by injecting the honey control. There were working standard solution. The mixture was then gently blended in the no matrix peaks in the chromatograms to interfere with the analysis of herbicide residues shown in Figure 1 & Figure 2. Furthermore, the retention times of Monocrotophos, Triazophos, Phosalone, Profenofos Cl O O and Chlorpyrifos were constant at 3.8 ± 0.2, 4.7 ± 0.2, 7.2 ± 0.2, 8.8 ± S O (CH O) P-O H 3 2 N N 0.2 and 12.6 ± 0.2 min. C C OP(OCH2CH3)2 N CH SP(OCH CH ) CH3 CONHCH 2 2 3 2 3 N S Linearity

Monocrotophos Triazophos Phosalone Different known concentrations of organophosphorus insecticides (0.01, 0.05, 0.1, 0.5, 1.0, 2.0 µg/mL) were prepared in O O CH2 -CH3 S acetonitrile by diluting the stock solution. The standard solutions P N S Cl OP(OCH2 CH3 )2 Br O CH2-CH2-CH3 were injected and recorded the peak areas. A calibration curve has Cl been plotted of concentration of the standards injected versus area Cl Cl observed and the linearity of method was evaluated by analyzing six

Profenofos Chlorpyrifos solutions. The peak areas obtained from different concentrations of organophosphorus insecticides were used to calculate linear regression Figure1: Names and structures of five organophosphorous insecticide evaluated. equations. These were Y= 132256.12X+84.23, Y=105266.62X+32.18, Y=115461.52X+12.93, Y=123968.33X+25.12 and 150918.15. +51.36

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uV uV 3.0 Detector A :230nm Detector A :230nm 3.0 2.5 2.5 2.0 2.0 1.5

1.5 1.0

1.0 0.5

0.0 0.5 Chlorpyrifos/12.601 Triazophos/4.730 MonocrotophosO/3.838 Profenofos/8.890 0.0 2.5 5.0 7.5 10.0 12.5 min Phosalone/7.132 0.0

Figure 2: A representative chromatogram obtained from control honey sample. 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 3: Representative chromatogram at fortification level of 0.01 µg/mL. with correlation coefficients of 0.9998, 0.9999, 0.9996, 0.9999 and 0.9997 for Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos, respectively. A calibration curve was show in Figure 2. Calibration curve of Organophosphorous Insecticides Accuracy and precision 350000 300000 Recovery studies were carried out at 0.01 and 0.1 µg/mL fortification levels for Monocrotophos, Triazophos, Phosalone, Profenofos and 250000 Phosalone Chlorpyrifos in honey. The recovery data and relative standard 200000 Triazophos deviation values obtained by this method are summarized in Table 1. 150000 Prefenofos These numbers were calculated from five (6) replicate analysis of in µV-Sec Area 100000 Monocrotophos given sample (Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos) made by a single analyst on one day. The repeatability of 50000 Chlorpyrifos method was satisfactory (RSDs<3%) Figure 3. 0 0 0.5 1 1.5 2 2.5 Detection and quantification limits Concentration in mg/L The limit of quantification was determined to be 0.01 µg/mL. The Figure 4: Representative Calibration curve of organophosphorous insecticides. quantitation limit was defined as the lowest fortification level evaluated at which acceptable average recoveries (87-96%, RSD<3%) were For- Recovery (%) achieved. This quantitation limit also reflects the fortification level at tification Replica- Monocro- Triazo- Phosalone Profeno- Chlor- which an analyte peak is consistently generated at approximately 10 Concen- tion tophos phos fos pyrifos times the baseline noise in the chromatogram. The limit of detection tration in µg/mL was determined to be 0.01 µg/mL at retention time of the peak of R1 85 87 90 89 88 interest Figure 4. R2 89 88 86 88 85 Storage stability R3 91 91 90 85 85 0.01 R4 87 86 87 90 86 A storage stability study was conducted at -20 ± 1°C with honey R5 87 85 86 86 89 samples spiked with 0.5 µg/mL of Monocrotophos, Triazophos, R6 88 87 86 88 91 Phosalone, Profenofos and Chlorpyrifos Samples were stored for a Mean 88 87 89 89 87 period of 30 days at this temperature. Monocrotophos, Triazophos, RSD 2.32 2.37 2.22 2.07 2.77 Phosalone, Profenofos and Chlorpyrifos contents were analysed before R1 95 91 96 93 90 storing and at the end of storage period. The percentage dissipation R2 98 92 92 92 93 observed for the above storage period was only 2% for Monocrotophos, R3 96 91 94 92 91 Triazophos, Phosalone, Profenofos and Chlorpyrifos showing no 0.1 R4 95 93 93 94 95 significant loss of residues on storage. The results are presented in R5 95 94 94 95 93 Table 2. R6 93 92 93 92 92 Mean 95 92 94 93 92 Conclusions RSD 1.71 1.60 1.46 1.36 1.90 This paper describes for the first time a fast, simple sensitive Table 1: Recoveries of the organophosphorous insecticides from fortified honey analytical method based on MSPD with HPLC-UV was developed and control sample (n=6). validated for the simultaneous determination of five organophosphorus The mobile phase acetonitrile and Milli-Q water yields good insecticides residues in honey. The MSPD extraction procedure of the separation and resolution and the analysis time required for the described method is very simple and requires no sample preparation chromatographic determination of the five organophosphorus or pre-treatment, providing adequate clean-up of the matrix. Whole insecticides are very short (around 15 min for a chromatographic run). honey extracts are very clean, with no interfering peaks at the retention time of the target compounds, indicating good selectivity of the Satisfactory validation parameters such as linearity, recovery, proposed method. precision and very low limits were obtained and according to the

Page 4 of 4

Fortified Storage Repli- Recovery in % Acknowledgement concen- Period cation Mono- Triazo- Phosa- Profeno- Chlorpy- The authors are thankful to the Management and Dr. P. Balakrishnamurthy, tration in Days croto- phos lone fos rifos Director, IIBAT, for providing necessary facility to conduct the experiment. in µg/ phos mL References R1 96 94 92 91 92 1. Lu C, Knutson DE, Fisker-Andersen J, Fenske RA (2001) Biological monitoring R2 93 92 94 93 93 Survey of organophosphorus pesticide exposure among pre-school children in R3 96 93 95 91 95 the seattle metropolitan area. Environ Health Perspect 109: 299-303. 0 R4 95 94 94 95 95 2. Barker SA(2000) Applications of matrix solid-phase dispersion in food analysis. R5 94 92 96 92 93 J Chromatogr A 880: 63-68. R6 94 92 91 92 91 3. Zuin VG, Yariwake JH, Langas FM (2003) Analysis of pesticide residues in 0.5 Mean 95 93 94 92 93 Brazilian plants. Braz Chem Soc 14: 304-309. RSD 1.28 1.06 1.99 1.63 1.72 4. Hopper ML (1999) Automated one-step supercritical fluid extraction and clean- R1 90 88 90 88 89 up system for the analysis of pesticide residues in fatty matrices. J Chromatogr R2 92 90 89 89 90 A 840: 93-105. R3 90 89 90 90 91 5. Feride Koc (2008) Determination of aldicarb, propoxur, carbofuron, carbaryl 30 R4 91 91 91 91 92 and methiocarb residues in honey by HPLC with post-column derivatization R5 94 90 92 89 91 and flurescence detection after elution form a florisil column. Journal of food R6 93 91 92 89 89 and drug analysis 16: 39-45. Mean 92 90 91 91 90 6. Korta E, Bakkali A, Berrueta, Gallo B, Vicente F (2002) Study of an accelerated RSD 1.78 1.30 1.34 1.16 1.34 solvent extraction procedure for the determination of acaricide residues in honey by highperformance liquid chromatography-diode array detector. J Food Table 2: Storage stability Details (n=6). Prot 65: 161-166. 7. Tsipi D, Triantafyllou M, Hiskia A (1999) Determination of organochlorine SANCO guidelines [8,9]. For all of the organophosphorus insecticides pesticide residues in honey, applying solid phase extraction with RP-C18 the sensitivity of the method was good enough to ensure reliable material. Analyst 124: 473-475. determination levels lower than the respective MRLs. Therefore, the 8. Guidance document on pesticide residue analytical methods. SANCO/825/00 proposed analytical procedure could satisfactorily be useful for regular rev. 8.1, 6/11/2010. monitoring of organophosphorus insecticide residues on a large 9. SANCO Guidelines (2009) Method validation and quality control number of honey samples. procedures for pesticide residues analysis in food and feed. Document NO. SANCO/10684/2009.

Volume 1 • Issue 6 • 2012 Nageswara Rao et al., 1:6 http://dx.doi.org/10.4172/scientificreports.327 Open Access Open Access Scientific Reports Scientific Reports Research Article OpenOpen Access Access Simultaneous Determination of Toxic Organophosphorus Insecticide Residues in Honey Followed by Matrix Solid-Phase Dispersion Coupled to High-Performance Liquid Chromatography with Ultraviolet Detection Tentu Nageswara Rao*, Atmakuru Ramesh and Tentu Parvathamma Department of Analytical Chemistry, International Inisitituate of Biotechnology and Toxicology (IIBAT), Padappai, Kancheepuram 601301, Tamil Nadu, India

Keywords: Matrix solid-phase dispersion; Organophosphorus Organophosphorus compounds are mainly highly and moderately insecticides; HPLC-UV toxic to humans and warm-blooded animals. Upon entering into the body they affect the cholinergic synapses of the central nervous system Introduction and the peripheral nerve muscle connections [1]. A molecule of an organ phosphorous substance contains two kinds Many compounds of this group have an acute and moderate chronic of groups, the electronegative group ‘x’ forming a relatively unstable toxicity that manifests itself with the frequent introduction of toxic and anhydride bond with phosphorous and an alkoxy group having a sublethal doses. The high activity of phosphatases, carboxyesterases strong bond with phosphorous. The hydrolysis of organophosphorus and amidases in the organisms of warm blooded animals result in the compounds by phosphatases is the main metabolic way leading to a rapid decomposition of organophosphorus compounds into non toxic complete loss of toxicity in the organisms of humans, warm blooded water soluble products which is excreted through urine. The dermal animals, insects and plants. toxicity of organophosphorus insecticide is not high but toxicants like The toxic action of organophosphorus compounds on insects is due methyl-parathion are highly toxic when they get into the skin. to inhibition of the activity of acetylcholinesterase in the cholinergic Several methods have been used for the determination of synapses of the nervous system. (The space between nerve cells, organophosphorus insecticide residues using solid-phase extraction called a synapase). When organophosphorus compounds enter the (SPE), solid-phase micro extraction (SPME), supercritical fluid organism of an insect, the activity of the acetylcholinesterase sharply extraction (SFE), Single drop micro extraction (SDME) and matrix drops. Indications of poisoning appear very rapidly in hyperactivation, solid-phase dispersion (MSPD). However, none of the published next paralysis and then leading to death. Most organophosphorus researches to date have reported the simultaneous analysis of chemical insecticides have a high initial toxicity, and pests perish during the first classes such as Monocrotophos, Triazophos, Phosalone, Profenofos and few hours after treatment. Chlorpyrifos in honey. Substances of this group are good for controlling larvae and The matrix solid-phase dispersion (MSPD) technique was adult individual of pests but their ovicidal action is weak, which is developed by Barker in 1989 [2]. It has advantages over conventional associated with low permeability of egg shells. Oil solutions of some techniques because it employs small amounts of sample and solvent, organophosphorus compounds penetrate quite well into the eggs of and the extraction procedure consists of only a few experimental insects and mites, causing their death. steps. MSPD evolved from the solid-phase extraction (SPE) technique, modified for application to solid and semi-solid matrices. The MSPD The activity of an embryon’s cells and its growth are not violated but acetylcholine accumulates in the embryon. The larvae die owing to the inability of moving when it leaves the egg. This is why plantings *Corresponding author: Tentu Nageswara Rao, Department of Analytical should be treated with oil formulations of such a kind not long before Chemistry, International Institute of Biotechnology and Toxicology (IIBAT), Kancheepuram, Tamil Nadu, India, E-mail: raoau@ rediffmail.com the larvae hatch from the eggs. Received August 18, 2012; Published September 12, 2012 Upon perennial use of organophosphorus insecticides, specific Citation: Rao TN, Ramesh A, Parvathamma T (2012) Simultaneous Determination resistance appears in insects or mites. Specific resistance to of Toxic Organophosphorus Insecticide Residues in Honey Followed by Matrix Solid- organophosphorus compounds appears more rapidly in the species of Phase Dispersion Coupled to High-Performance Liquid Chromatography with Ultravio- insects that produce several generations in a single season. But the most let Detection. 1:327. doi:10.4172/scientificreports.327 dangerous is the development of resistance in phytophagous mites. The Copyright: © 2012 Rao TN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted economical harm form this phenomenon is very great especially in use, distribution, and reproduction in any medium, provided the original author and cotton-growing regions. source are credited.

Volume 1 • Issue 6 • 2012 Citation: Rao TN, Ramesh A, Parvathamma T (2012) Simultaneous Determination of Toxic Organophosphorus Insecticide Residues in Honey Followed by Matrix Solid-Phase Dispersion Coupled to High-Performance Liquid Chromatography with Ultraviolet Detection. 1:327. doi:10.4172/ scientificreports.327

Page 2 of 6 procedure is based on the use of a sorbent, which acts as an abrasive in Extraction procedure order to produce a modified “opening” of the solid matrix, facilitating the extraction process when using a suitable solvent for eluting the 2.0 g of honey sample was weighed out and homogenized with analytes. The use of MSPD for organophosphorus insecticides recovery 2.0 g of C18 –bonded silica for 5 min. The homogenized sample depends on the solubility of the organophosphorus insecticides in was transferred to an MSPD column consisting of a 20 mL capacity the eluting solvent, as well as the interactions between the matrix polyethylene syringe containing 2.0 g florisil and 2.0 g of anhydrous components, sorbent and eluent [3]. Due to the lack of literature sodium sulfate. The elution was performed under vacuum with 20 mL reports concerning the use of MSPD as an extraction technique for of n-hexane-dichloromethane (1:1). The eluent was collected into a 50 organophosphorus insecticides belonging to different chemical classes mL glass tube and then evaporated under gentle stream of nitrogen, from plants, soil water and food products, this paper presents an MSPD with the water bath temperature set at 40-45ºC. Residue was dissolved method for determination of residue of organophosphorus insecticides with 5 mL of acetonitrile. in honey [4-7]. So, the present research considered five different Chromatographic separation parameters chemical classes, namely Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos which analysis by high-performance The HPLC-UV system used, consisted Shimadzu high performance liquid chromatography with ultraviolet detector (HPLC-UV). liquid chromatography with LC-20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed Phase C18 analytical Experimental column of 250 mm×4.6 mm and particle size 5.0 µm (Phenomenex) Standards, reagents and samples Column temperature was maintained at 30°C. The injected sample volume was 20 µL. Mobile Phases A and B were acetonitrile and Milli-Q Certificated analytical standards of Monocrotophos (99.4%), water (75:25(v/v)). The flow- rate used was kept at 1.2 mL/min. The Triazophos (98.2%), Phosalone (99.2%) Profenofos (99.1%) and detector wavelength was 230 nm. The external standard method was Chlorpyrifos (99.8%) were obtained from Sigma Aldrich. Common used for this analysis. names and structures of the organophosphorus insecticides evaluated here are shown in Figure 1. Acetonitrile was purchased from Rankem, Method validation New Delhi, Analytical grade solvents, dichloromethane and n-hexane, Method validation ensures analysis credibility. In this study, were supplied from Merck Limited, Mumbai, C18-bonded silica (50 the parameters accuracy, precision, linearity and limits of detection µm) from phenomenex (Torrance, CA, USA), Florisil (60-100 mesh) (LOD) and quantification (LOQ) were considered. The accuracy of from Fluka Chemie GmbH CH-9471 Buchs, AR grade sodium sulphate the method was determined by recovery tests, using samples spiked at from Merck Limited, Mumbai and honey was purchased from local concentration levels of 0.01 and 0.1 mg/kg. Linearity was determined market. They were brought to the laboratory and stored in plastic bag by different known concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 µg/ under refrigerator condition until they were processed in the laboratory. mL) were prepared by diluting the stock solution. The limit of detection Standard stock solutions (LOD, µg/mL) was determined as the lowest concentration giving a response of 3 times the baseline noise defined from the analysis of The organophosphorus insecticide standard stock solutions were control (untreated) sample. The limit of quantification (LOQ, µg/mL) individually prepared in acetonitrile at a concentration level of 100 µg/ was determined as the lowest concentration of a given herbicide giving mL and stored in a freezer at -18°C. The stock standard solutions were a response of 10 times the baseline noise. used for up to 3 months. Suitable concentrations of working standards were prepared from the stock solutions by dilution using acetonitrile, Results and Discussion immediately prior to sample preparation. Specificity Sample preparation Specificity was confirmed by injecting the honey control. There were Representative 2.0 g portions of honey fortified with 10 µL of no matrix peaks in the chromatograms to interfere with the analysis working standard solution. The mixture was then gently blended in the of herbicide residues shown in Figure 1 & Figure 2. Furthermore, the mortar for 30 min, to assess the homogeneity of the sample. The sample retention times of Monocrotophos, Triazophos, Phosalone, Profenofos was allowed to stand at room temperature for one hour, until analysis. and Chlorpyrifos were constant at 3.8 ± 0.2, 4.7 ± 0.2, 7.2 ± 0.2, 8.8 ± 0.2 and 12.6 ± 0.2 min.

Cl Linearity O O S O (CH O) P-O H 3 2 N N Different known concentrations of organophosphorus C C OP(OCH2CH3)2 N CH SP(OCH CH ) CH3 2 2 3 2 insecticides (0.01, 0.05, 0.1, 0.5, 1.0, 2.0 µg/mL) were prepared in CONHCH3 N S acetonitrile by diluting the stock solution. The standard solutions

Monocrotophos Triazophos Phosalone were injected and recorded the peak areas. A calibration curve has been plotted of concentration of the standards injected versus area

O O observed and the linearity of method was evaluated by analyzing six CH2 -CH3 S P N S Cl OP(OCH2 CH3 )2 solutions. The peak areas obtained from different concentrations of Br O CH2-CH2-CH3 Cl organophosphorus insecticides were used to calculate linear regression Cl Cl equations. These were Y= 132256.12X+84.23, Y=105266.62X+32.18,

Profenofos Chlorpyrifos Y=115461.52X+12.93, Y=123968.33X+25.12 and 150918.15. +51.36 with correlation coefficients of 0.9998, 0.9999, 0.9996, 0.9999 and Figure 1: Names and structures of five organophosphorous insecticide evaluated. 0.9997 for Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos, respectively. A calibration curve was show in Figure 2.

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Accuracy and precision Calibration curve of Organophosphorous Insecticides

Recovery studies were carried out at 0.01 and 0.1 µg/mL fortification 350000 levels for Monocrotophos, Triazophos, Phosalone, Profenofos and Chlorpyrifos in honey. The recovery data and relative standard 300000 deviation values obtained by this method are summarized in Table 1. 250000 Phosalone

These numbers were calculated from five (6) replicate analysis of 200000 Triazophos given sample (Monocrotophos, Triazophos, Phosalone, Profenofos and 150000 Prefenofos

Chlorpyrifos) made by a single analyst on one day. The repeatability of in µV-Sec Area method was satisfactory (RSDs<3%) Figure 3. 100000 Monocrotophos

50000 Chlorpyrifos Detection and quantification limits 0 The limit of quantification was determined to be 0.01 µg/mL. The 0 0.5 1 1.5 2 2.5 Concentration in mg/L quantitation limit was defined as the lowest fortification level evaluated at which acceptable average recoveries (87-96%, RSD<3%) were Figure 4: Representative Calibration curve of organophosphorous insecticides. achieved. This quantitation limit also reflects the fortification level at which an analyte peak is consistently generated at approximately 10 Fortifica- Recovery (%) times the baseline noise in the chromatogram. The limit of detection tion Con- was determined to be 0.01 µg/mL at retention time of the peak of centration interest Figure 4. in µg/mL Replica- Monocro- Triazophos Phosa- Profeno- Chlorpy- Storage stability tion tophos lone fos rifos R1 85 87 90 89 88 A storage stability study was conducted at -20 ± 1°C with honey R2 89 88 86 88 85 samples spiked with 0.5 µg/mL of Monocrotophos, Triazophos, R3 91 91 90 85 85 Phosalone, Profenofos and Chlorpyrifos Samples were stored for a 0.01 R4 87 86 87 90 86 period of 30 days at this temperature. Monocrotophos, Triazophos, R5 87 85 86 86 89 Phosalone, Profenofos and Chlorpyrifos contents were analysed before R6 88 87 86 88 91 storing and at the end of storage period. The percentage dissipation Mean 88 87 89 89 87 observed for the above storage period was only 2% for Monocrotophos, RSD 2.32 2.37 2.22 2.07 2.77 Triazophos, Phosalone, Profenofos and Chlorpyrifos showing no R1 95 91 96 93 90 significant loss of residues on storage. The results are presented in Table 2. R2 98 92 92 92 93 R3 96 91 94 92 91 0.1 R4 95 93 93 94 95 uV R5 95 94 94 95 93 3.0 Detector A :230nm R6 93 92 93 92 92 2.5 Mean 95 92 94 93 92

2.0 RSD 1.71 1.60 1.46 1.36 1.90

1.5 Table 1: Recoveries of the organophosphorous insecticides from fortified honey control sample (n=6). 1.0

0.5 Fortified Storage Repli- Recovery in % concentra- Period in cation Monocro- Triazo- Phos- Profe- Chlor- 0.0 tion in µg/ Days tophos phos alone nofos pyrifos 0.0 2.5 5.0 7.5 10.0 12.5 min mL Figure 2: A representative chromatogram obtained from control honey sample. R1 96 94 92 91 92 R2 93 92 94 93 93 R3 96 93 95 91 95

uV 0 R4 95 94 94 95 95 Detector A :230nm R5 94 92 96 92 93 3.0 R6 94 92 91 92 91 2.5 0.5 Mean 95 93 94 92 93

2.0 RSD 1.28 1.06 1.99 1.63 1.72 R1 90 88 90 88 89 1.5 R2 92 90 89 89 90

1.0 R3 90 89 90 90 91 30 R4 91 91 91 91 92 0.5 R5 94 90 92 89 91 Chlorpyrifos/12.601 Triazophos/4.730 MonocrotophosO/3.838 Profenofos/8.890 0.0 Phosalone/7.132 R6 93 91 92 89 89

0.0 2.5 5.0 7.5 10.0 12.5 min Mean 92 90 91 91 90 RSD 1.78 1.30 1.34 1.16 1.34 Figure 3: Representative chromatogram at fortification level of 0.01 µg/mL. Table 2: Storage stability Details (n=6).

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Conclusions References This paper describes for the first time a fast, simple sensitive 1. Lu C, Knutson DE, Fisker-Andersen J, Fenske RA (2001) Biological monitoring Survey of organophosphorus pesticide exposure among pre-school children in analytical method based on MSPD with HPLC-UV was developed and the seattle metropolitan area. Environ Health Perspect 109: 299-303. validated for the simultaneous determination of five organophosphorus insecticides residues in honey. The MSPD extraction procedure of the 2. Barker SA(2000) Applications of matrix solid-phase dispersion in food analysis. J Chromatogr A 880: 63-68. described method is very simple and requires no sample preparation or pre-treatment, providing adequate clean-up of the matrix. Whole honey 3. Zuin VG, Yariwake JH, Langas FM (2003) Analysis of pesticide residues in extracts are very clean, with no interfering peaks at the retention time Brazilian plants. Braz Chem Soc 14: 304-309. of the target compounds, indicating good selectivity of the proposed 4. Hopper ML (1999) Automated one-step supercritical fluid extraction and clean- method. up system for the analysis of pesticide residues in fatty matrices. J Chromatogr A 840: 93-105. The mobile phase acetonitrile and Milli-Q water yields good 5. Feride Koc (2008) Determination of aldicarb, propoxur, carbofuron, carbaryl separation and resolution and the analysis time required for the and methiocarb residues in honey by HPLC with post-column derivatization chromatographic determination of the five organophosphorus and flurescence detection after elution form a florisil column. Journal of food insecticides are very short (around 15 min for a chromatographic run). and drug analysis 16: 39-45. Satisfactory validation parameters such as linearity, recovery, 6. Korta E, Bakkali A, Berrueta, Gallo B, Vicente F (2002) Study of an accelerated precision and very low limits were obtained and according to the solvent extraction procedure for the determination of acaricide residues in honey by highperformance liquid chromatography-diode array detector. J Food SANCO guidelines [8,9]. For all of the organophosphorus insecticides Prot 65: 161-166. the sensitivity of the method was good enough to ensure reliable determination levels lower than the respective MRLs. Therefore, the 7. Tsipi D, Triantafyllou M, Hiskia A (1999) Determination of organochlorine pesticide residues in honey, applying solid phase extraction with RP-C18 proposed analytical procedure could satisfactorily be useful for regular material. Analyst 124: 473-475. monitoring of organophosphorus insecticide residues on a large number of honey samples. 8. Guidance document on pesticide residue analytical methods. SANCO/825/00 rev. 8.1, 6/11/2010.

Acknowledgement 9. SANCO Guidelines (2009) Method validation and quality control The authors are thankful to the Management and Dr. P. Balakrishnamurthy, procedures for pesticide residues analysis in food and feed. Document NO. SANCO/10684/2009. Director, IIBAT, for providing necessary facility to conduct the experiment.

Volume 1 • Issue 6 • 2012