Recent Techniques Applied for Pesticides Identification and Determination in Natural Products and Its Impact to Human Health Risk

Based on the compilation of the British Crop Protection Council, approximately 860 active substances are formulated in pesticide products currently (Tomlin, 2003). These substances belong to more than 100 substance classes. Benzoylureas, carbamates, organophosphorous compounds, pyrethroids, sulfonylureas, or triazines are the most important groups. The chemical and physical properties of these pesticides may differ considerably. There are several acidic pesticides; others are neutral or basic and some compounds contain halogens, others phosphorous, sulfur, or nitrogen. These heteroatoms may have relevance for the detection of pesticides in natural products. Pesticides such as polychlorinated biphenyls PCB’S organochlorines and organophosphates are found in various parts of the environment in quite small concentrations, but they accumulate and thus become a threat to human health and life. Maximum residue levels (or tolerances) have been established for pesticides in foodstuffs and drinking water in most countries to avoid any adverse impact on public health, and to insist on good agricultural practice. For these reasons a large number of researchers are involved in the surveillance of maximum residue levels or in the identification and quantification of pesticide residues in environmental matrices. A lot of these pesticides were registered in Egypt or most frequently detected in fruits and vegetables in Egyptian market as well as in Europe and USA. To control local, imported and exported food, multi-residue analytical methods are preferred to reduce the workload. In this study, simple and reliable multi-residue method of analysis for determination of pesticide residues in different agricultural products was developed. In this method different pesticide groups, e.g. organophosphates, moderately polar organochlorines, benzimidazoles, N-methylcarbamates and phenoxy acids could be analyzed in one multiresidue method using Liquid Chromatography tandem mass spectrometry (LCMS/MS) and fulfill the Codex and EU regulations. Grape, green beans even vegetable samples were extracted by shaking with acetonitrile .Phase separation was induced by shaking with buffer―salt mixture consisting of magnesium sulfate, sodium chloride, disodium hydrogen citrate sesquihydrate and trisodium citrate dihydrate .The sample was centrifuged and an aliquot of the clear solution dried by shaking with magnesium sulphate . The extract was centrifuged and an aliquot of the clear solution evaporated, re-dissolved in methanol/water buffer solution and injected into LC-system (Afify, 2010) .Quantitation and

(Whatman no.1) fitted on Buchner flask, the blender jar is rinsed with 50 ml acetone and filtered again on the same funnel, the extracted volume is recovered. A 40 ml sample extract is transferred to 500 ml separator funnel, 50 ml petroleum ether and 50 ml dichloromethane are added and shake vigorously for 2 min, transfer the lower aqueous layer to graduated cylinder, the upper organic layer is transferred by passing through anhydrous sodium sulphate supported on washed cotton in funnel on receiving flask, about 2g sodium chloride is added to the aqueous phase and shake vigorously for 1 min until most sodium chloride dissolved, transfer it to the same separator funnel, 50ml dichloromethane is added and shake for 1 min, lower dichloromethane layer is filtered through sodium sulphate, the water layer is taken and the last dichloromethane partitioning s t e p i s r e p e a t e d , s o d i u m s u l p h a t e i s r i n s ed with 25ml dichloromethane, the received solution is evaporated using rotary evaporator to about 2ml at 35-40 °C, continued evaporation by air just to dryness, the residue was re-dissolved in 10 ml [Methanol: ammonium format buffer 10 mM pH 4 (1:1) and filtered through 0.45 µm syringe filter, the clear filtrate was injected directly into LC/MS/MS system.

Ethyl acetate method by Banerjee et al., (2007)
Green beans sample (50g) was add with 10 ml ethyl acetate in 50 ml PTFE centrifuge tube and blended for 1 min. An aliquot of 4 ml was evaporated using rotary evaporator at 40 o C just to dryness. The residue was re-dissolved in 4 ml [Methanol: ammonium format buffer 10 mM pH 4 (1:1) and filtered through 0.45 µm syringe filter. The clear filtrate was injected directly into LC-MS/MS system.

Choosing of pesticides
The 150 chosen pesticides used in this investigation were collected and identified with type of pesticides, chemical class, Field of use and KOW logP as shown in the following .HCl N(CH 2 ) 3 NHCO 2 (CH 2 ) 2 CH 3 CH 3 CH 3  Table 2. Chemical structure of seventeen selected pesticides:

Risk ⁄ safety assessment
The insecticide residues concentrations found in the analyzed potatoes were compared with the tolerance limits established by Codex Alimentarius Commission and the Egyptian Organization for Standardization and Quality Control (EOS), respectively. The dietary intake of insecticides was estimated and compared with the WHO-ADIs, (Tomlin, 2004) as cited by Mansour et al., (2009) as follow: Estimated dose (mg/kg) = Residues (mg/kg) Food item x daily potato consumption (kg) / Body weight (kg)

LC-MS/MS 3.1.1 LC-MS/MS analysis
Separation was performed on a C18 column ZORBAX Eclipse XDB-C18 4.6 mm x 150 mm, 5 μm particle size .The injection volume was 25 μl .A gradient elution program was at 0.3 ml/min flow, in which one reservoir contained 10 mM ammonium format solution in methanol-water 1:9 and the other contained methanol .The ESI source was used in the positive mode, and N2 nebulizer, curtain, and other gas settings were optimized according to recommendations made by the manufacturer; source temperature was 300 o C, ion spray potential 5500 V, decluster potential and collision energy were optimized using A Harvard Apparatus syringe pump by introducing individual pesticide solutions into the MS instrument to allow optimization of the MS/MS conditions. The Multiple Reaction monitoring mode MRM was used in which one MRM was used for quantitation and other was used for confirmation . www.intechopen.com

GC -measurements with different detectors 3.2.1 GC-NPD parameter
GC-NPD analyses were run on HP 6890 series gas chromatograph equipped with nitrogen phosphorous detector (NPD). Data acquisition, processing, and instrumental control were performed by the Agilent ChemStation software. A split/split less (S/Sl) inlet was used with 1.8 mm id liner. Analytes were separated in an Agilent HP-Pass 5 capillary column, 25 m length ,0.32 mm id, 0.52 µm film thickness. The inlet operating temperature is 225 o C, injection volume 1 µL. The nitrogen carrier gas flow was maintained at a constant flow of 1.3 ml/minute. N 2 make up gas flow rate 8 ml/minute for the NPD and H 2 with flow rate of 4.5 ml/minute. The oven temperature program was 90 o C for 2 minute, programmed to 150 o C at 20 o C/minute, and then to 270 o C at 6 o C/minute, it was kept at this temperature for 15 minute. Detector temperature was maintained at 280 o C with H 2 flow of 3.5 ± 0.1 ml/minute and air flow of 100-120 ml/minute.

GC-ECD parameter
GC-ECD analyses were run on HP 6890 series gas chromatograph equipped with electron capture detector (ECD). Data acquisition, processing, and instrumental control were performed by the Agilent ChemStation software. A split/split-less (S/Sl) inlet was used with 1.8 mm id liner. Analyte samples were separated in an Agilent HP-Pass 5 capillary column, 25 m length, 0.32 mm id, and 0.52 µm film thicknesses. The inlet operating temperature is 225 o C, injection volume 1 µl. The nitrogen carrier gas flow was maintained at a constant flow of 1.3 ml/minute. The oven temperature program was 90 o C for 2 minute, programmed to 150 o C at 20 o C/minute, and then to 270 o C at 6 o C/minute, it was kept at this temperature for 15 minute. Detector temperature was maintained at 300 o C.

GC-MSD parameter
GC-MSD analyses were run on an Agilent 7890 series gas chromatograph (Agilent Technologies, Santa Clara, CA) interfaced to an Agilent 5975 mass selective detector (MSD). Data acquisition, processing, and instrumental control were performed by the Agilent MSD ChemStation software (E.0200.493 version). A split/split less (S/Sl) inlet was used with 1.8 mm id liner. Analyte samples were separated in an Agilent HP-5MS capillary column (5% biphenyl/95% dimethylsiloxane), 30 m, 0.25 mm id, 0.25 µm film thickness. The inlet operating conditions were injection volume, 1 µl, flow rate 1.3 ml/minute; the temperature program was set at 79 o C for 0.25 minute, programmed to 300 o C at 10 o C/minute, and kept at this temperature for 2 minute. The helium carrier gas flow was maintained at a constant pressure of 17.296 psi. The oven temperature program was 70 o C for 1 minute, programmed to 150 o C at 50 o C/minute, then to 200 o C at 6 o C/min, and finally to 280 o C at 16 o C/minute; it was kept at this temperature for 5 minute. Electron impact mass spectra in the full-scan mode were obtained at 70 eV; the monitoring was from m/z 50 to 400. The ion source and quadrupole analyzer temperatures were fixed at 230 and 150 o C, respectively.

Recovery tests on grapes
The method recoveries for 150 pesticides were tested by performing 6 replicates of spike grapes at different concentration levels; 0.01, 0.05 and 0.1 mg/kg. The average recoveries and relative standard deviation on each level were calculated (Table 3). The precursor ion, product ion (1) and product ion (2) and retention time will be included in the tables. The injection of 25 µl of acetonitrile into LC system leads to non-symmetrical peak shapes, so that acetonitrile was evaporated and re-dissolved in methanol-water solution. This step improved the pesticide peak shapes and lowered the matrix effect due to precipitation of some insoluble substances. The recovery of most pesticides (143 pesticides) is in the range 70%-110%. The recoveries of 7 pesticides (Chlorfluazuron, L-Cyhalothrin, Deltamethrin, Diafenthiuron, Flufenoxuron, Lufenuron and Pymetrozine) are lower than 70% due to the evaporation of acetonitrile and re-dissolving in methanol-water solution as reported by Afify et al., (2010). The conclusions stated that the proposed method using acetonitrile extraction followed by LC-MS/MS determination is simple, rapid and reliable satisfactory recoveries and repeatability observed .The described method requires little amount of solvents and sample and could be used in controlling levels of pesticides from different classes in natural products samples.

Recovery tests on green beans
The optimized LC-MS/MS parameters and the best extraction procedures (QuEChERS) were used to study the method performance by carrying out recovery tests of pesticides at different levels on green beans samples. Six replicates of recovery tests were done at concentration levels 0.01 mg/kg, 0.05 mg/kg and 0.1 mg/kg on grapes and green beans,   (4) showed that the 150 pesticides could be determined at concentration 0.01 mg/kg with accepted recovery and precision. The recovery of most pesticides (143 pesticides) is in the range 60%-120%, as cited for grapes. The recoveries of the same 7 pesticides (Chlorfluazuron, L-Cyhalothrin, Deltamethrin, Diafenthiuron, Flufenoxuron, Lufenuron and Pymetrozine) are lower than 60% due to the evaporation of acetonitrile and re-dissolving in methanol-water solution mixture as approved in the recovery tests of pesticides in grapes  .0n the other hand recovery test of some pesticides exceeds 100 at concentration at 0.01 mg/kg ( Flumetsulam, Fluroxypyr Imazalil), at concentration of 0.05 mg/kg ( Butachlor, Pirimiphosmethyl, Metsulfuronmethyl, Parathionethyl, Methoxyfenozide) and at concentration of 0.1 mg/kg (Fenoxaprop -Pethyl).

Optimization of sample extraction
Different types of extraction procedures were tested as described in materials and methods using three method (e.g. Luke, QuEChERS and ethyl acetate according to Luke et al. (1975), Anastassiades et al., (2008): and Banerjee et al., (2007). Extraction was done on green beans sample at spiking level of 0.5 mg/kg . Blank samples, standard in solvent and standard in matrix were injected in parallel to spike samples and in the same run. Due to suppression effect of these types of matrices (decreasing in signal intensity) standard prepared in matrix were used for recovery calculations, the results of recovery tests on green beans samples using the different three methods were discussed by the compound with recovery less than 60%  As shown in Table (5) propamocarb-HCl is an example for high polar pesticides which had a low recovery in the extraction by ethyl acetate (1 %) and not completely recovered in the partitioning step in Luke method (11%) .On the other hand the solubility of the pesticides in the different methods are different depending on its polarity which could seen in the results of the recovery test of the three methods such as Pymetrozine and Fenpropathrin pesticides The same results were observed by D´ıez et al. (2006) that Luke was significantly more effective for the extraction of non-polar and medium-polar compounds, but the best recoveries for polar compounds were achieved by QuEChERS and ethyl acetate methods. QuEChERS was the only method that provided an overall recovery value of 60-70% for none, medium and polar compounds, also Kruve et al. (2008) reported in his comparison between Luke method and matrix solid-phase dispersion (MSPD) that the best recoveries were obtained with the QuEChERS method. Therefore the QuEChERS extraction method was found to be better than Luke method and ethyl acetate method because of higher recovery, less solvent and short time of analysis were observed.

Comparison of pesticides chromatograms using GC-NPD, ECD, MSD and LC-MS/MS
The chromatograms of the 150 pesticides injected into GC systems with three different detectors ECD, NPD and MSD ( Fig. 4.a,b,c) were used to compare between GC efficiency and LC-MS/MS (Fig. 5.a) in separation and sensitivity. The total ion chromatogram for the 150 pesticides injected into LC-MSMS system illustrate in (Fig. 5a). It looks that the pesticide peaks are not resolved but in fact due to the high selectivity of the MS/MS system the peaks can be resolved easily (Fig. 5 b, c,d).  It is observed that the pesticide peaks showed in (Fig. 4.a,b,c) by using ECD,NPD and MSD are not resolved and have very low sensitivity while in (Fig. 5a) separation of the 150 pesticides could be analyzed by single chromatographic run of 33 minutes and each MRM could be separated as single peak in a chromatogram by LC-MS/MS system as shown in (Fig 5, a, b, c and d) for fenpropathrin, dichlofuanid and fenhexamide pesticides as studied by . It is clear that although dichlofuanid (Fig. 5.c) has the same molecular weight of fenpropathrin ( Fig. 5b) (absence of cross talk) and has the same retention time of fenhexamide (Fig. 5d), but it is easily resolved from both compounds. These results were supported by Applied Biosystems (2004) (Application Note: Mass Spectrometry) for fenoxycarb 302/88 and methomyl 163/88 that are measured using the same product ions (but with different precursor ions) they are completely separated, (Publication 114AP30-01).

Optimization of mobile phase
A modified multi-residue method for analysis of 150 pesticide residues in green beans using liquid chromatography-tandem mass spectrometry by using three methods as described in material and methods ;QuEChERS as described by Pya, et al., (2008), Luke et al., (1975) method and Ethyl acetate method by Banerjee et al., (2007). The extracts solution of three methods were re-dissolved in methanol ,buffer solution (1:1) 10 mM in pH 4 as modification to increased injection volume to 25 µl without losing our good peak shape. Stabilities of tested pesticides in five different calibration mixture pH for two weeks were studied. Quantitation and identity confirmation was attained by using atmospheric pressure electrospray positive ionization LC-MS/MS in multiple reactions monitoring (MRM) mode. The signal intensity in LC-MS/MS can be influenced by the mobile phase composition. In order to optimize the signal intensity, standard mixtures in methanol were injected into the LC-MS/MS, using different mobile phase compositions. Four different buffer constituents were tested: ammonium format (0.1, 1, 5, and 10 mM) at three pH (3, 3.5 and 4). Evaluation was done by recording the MS/MS signal for each pesticide with a calculation based on 5 mM, pH 4. The mobile phase during this test was composed of 50% buffer constituent in water and 50% methanol. Generally results showed that there is no variation in signal more than 4% between all of tested mobile phase except the 10 mM in pH 4 which had increasing in 26 compounds more than 15% as shown in the following  Table 6. Comparison between pesticides sensitivity using 10 mM buffer compared to 5 mM buffer.
SE: Signal enhancement in 10 mM buffer compare by 5 mM buffer. Pesticides which had high matrix effect suppress its standard signals in the compounds with intensity increased up to 38 % (Tetraconazole) . However, when analyzing different samples, which themselves can influence the signal by altering the mobile phase composition, it is important to use a buffer with a sufficient buffering capacity to stabilize the system. Therefore, higher ionic strength contributes to a more stable system, both for retention and signal. By using ammonium format buffer 10 mM with pH 4, the results of 26 pesticides out of 150 pesticides compounds has increased in its sensitivity. These results approved by Jansson et al. (2004) reported that the best signal response was obtained with pH ranging from 4.0 to 4.2 and that the buffer strength of 10 mM was chosen as a compromise on 57 pesticides. Finally the use of ammonium format mobile phase 10 mM in pH 4 represented the most suitable condition for the separation and sensitivity of tested pesticides, which should be considered during determination of pesticides residues.

Effect of pH on tested pesticides stability
Standard solution of the 150 pesticides was prepared at concentration 0.5 µg/ml and kept in freezer for 15 days at -20± 2 o C and compared to fresh prepared standard solution, the stability of these pesticides at different pH showed by storage recovery in (Fig. 6) and Table (7) also the degradation of pesticides with decreasing more than 10% in different pH were measured  The pesticides which had lost more than 10% of their concentration were showed by degradable percentage in Table (8), for example triflumizole which showed a degradation of 56% followed by 50 % for Fenoxaprop-ethyl at pH 3 . The result was in agreement with the US-EPA (EPA Pesticide Fact Sheet 10/91) studies on triflumizole which showed that hydrolysis studies of phenyl-labeled Carbon 14 triflumizole (radiochemical purity greater than 99%), at 5 ppm, degraded in sterile aqueous 0.01 M buffered solutions with half-lives of 7 to 15 days at pH 5, greater than 30 days at pH 7 and pH 3 to 17 days at pH 9 when incubated in the dark at 25± 2 o C. Fenoxaprop-ethyl showed degradation of 50% which is in agreement with the study done by Zablotowicz et al. (2000) stated that stability was pH sensitive in acidic buffered solutions; that is, below pH 4.6, rapid nonenzymatic hydrolysis of the benzoxazolyl-oxy-phenoxy ether linkage occurred, forming 6-chloro-2,3dihydro-benzoxazol-2-one (CDHB) and ethyl 4-hydroxyphenoxypropanoate or 4hydroxyphenoxypropanoate. Due to high sensitivity, high duty cycle and simple cleaning of the interface of the API 4000 QT, method development and recovery tests were done using methanol/buffer in pH 4 as calibration mixture solution, using this instrument. www.intechopen.com

Optimization of MS/MS 7.2.1 Optimization for precursor ion (parent) and product ion (daughter)
Pesticide standard solutions were prepared in methanol/ ammonium format buffer (1/1) at concentration level of 0.1-0.5 µg/ml and injected individually to optimize for parent ion (MS1 scanning & MS2 static) by scanning at different declustering potential (DP). The optimum DP, which gave the highest sensitivity, was used and changing the collision energy (CE) to optimize for the daughter ion (MS1 static & MS2 scanning). The standard solutions were injected directly into LC/MS/MS system without analytical column, the protonated ions were chosen in ESI+ (MW+1) mode. The compounds which gave accepted intensity with the optimized DP and CE were divided into 3 mixtures and injected into LC/MS/MS system in presence of analytical column using Multiple Reaction Monitoring mode (MRM, MS1 scanning, MS2 scanning) at the optimum DP and CE were used. Optimization of six pesticides will be discussed as an example. In this chapter we will discuss the optimization of Acetamiprid pesticide and the detailed results of the five remaining pesticides (Lambada-Cyhalothrin, Malathion, Methomyl Propargite and Tetraconazole) were described by El-Gammal (2010).

Calculation of isotopic distribution
The analyst software is used to calculate the isotope distribution, the expected nominal molecular weight of 222.1 for the parent compound also isotopic mass of 224.1 of 33% abundance due to the presence of one chlorine atom (37Cl) (Fig. 7). Fig. 7. Expected isotopic distribution of acetamiprid as calculated by the Analyst software.

Optimization of the precursor ions
The injection of individual standard of acetamiprid showed in (Fig. 8) and running Q1 scan (MS1 scanning & MS2 static). It is clear that the parent compound has gained a proton to give molecular ion mass at 223 (M+1), also isotopic molecular ion mass at 225 of 33% abundance due to the presence of one chlorine atom ( 37Cl).

Optimization of the declustering potential
Q1 scanning (MS1 scanning & MS2 static) of acetamiprid while changing the declustering potential from 0 to 240 volts to get the optimum DP. It is clear that the optimum DP for acetamiprid is 49 volts (Fig. 9). Fig. 9. Optimization of declustering potential (DP). www.intechopen.com

Optimization of the daughter ions
The fragmentation of acetamiprid in the collision cell and the Quadra poles Q1 scanning and Q3 scanning (MS1 scanning & MS2 scanning) (Figs 10, 11). www.intechopen.com The following table (9) showed the molecular weight, the calculated molecular weight related to isotopic distribution, isotopic elements of six pesticides compound with their masses, declustring potential (DP) which was very important for the tuning of parent ion and collision energy (CE) necessary for fragmentation .  Table 9. Molecular weight and nominal molecular weight related to isotope distribution.

Pesticide
The conclusion from Table (9) showed that every pesticide compound needs this tuning to get the best conditions for highest sensitivity. It is clear also that each pesticide has different DP and CE to get the best sensitivity; these parameters have been collected to build up the acquisition method for the 150 pesticides 8. Risk assessment based pesticides contamination 8.1 Impact of pesticides contamination to human risk Human milk the major source of infant food have been studied in detailed about the distribution of pesticides residues in all over 26 Governorate of Egypt . Different types of pesticides have been identifies in milk and the results described as follows:

Chlorinated insecticides levels in human milk
The data in Table 10 shows that the main detected organochlorine insecticides and their metabolites were DDE and lindane .DDT and endosulfan I residues were also detected in some milk samples .Endrin was only detected in one of the milk samples in New valley, while aldrin was not detected in any of the milk samples .However, from the 60 human milk samples, 51% of the samples were free from any detectable DDT level a fact which may suggest that there were no recent sources of pollution by intact DDT (Saleh et al., 1996a(Saleh et al., ,b , 1999.

Hexachlorocyclohexanes HCH isomers
δ-HCH lindane was detected in 95 %of the analyzes human milk samples .The lowest levels were found in governorates between Cairo and Assiut and in Suez 0.00-10.00 ppb while the higher levels 10.00-33.00 ppb (were found in the Delta area and in Alexandria . www.intechopen.com The higher levels could be a reflection of the use of lindane in agriculture and in the control of cattle ecto -parasites .Also, this might be due to the human consumption of large quantities of polluted fatty fish (table 10). Kucinski, (1986) have pointed out the presence of organochlorine residues including lindane in different food stuffs meat, dairy products, grain and drinks. Residues of some organochlorine pesticides OCPs, such as HCB and heptachlor as well as some organophosphorus pesticides OPPs, such as methamidophos,thiometon, profenofos, phorate and pirimiphos-methyl were found in a number of potatoes samples produced under different condition (convention, C; organic, O) at concentration levels exceeding their MRLs as reported by Mansour et al., (2009). The results in table (10) proved that pesticides residue in human milk product depends mainly on the regional area .Pesticides residues do its effect and shows its impact factor through creation a lot of diseases as described by the transportation of the pesticides in biological system to reach its biological function .Transportation of pesticides were carried out through protein binding with the major protein in serum like serum albumin as well as other protein exists in liver such as of α-Synuclein Fibril protein Formation and other organs as described by Afify et al., (2000) and Afify ( 2010) .Parkinson's disease involves intracellular deposits of α -synuclein in the form of Lewy bodies and Lewy neurites .The etiology of the disease is unknown; however, several epidemiological studies have implicated environmental factors, especially pesticides . Here we show that several *No .of collection samples Table 10. Distribution of the main organochlorine insecticide residues in Egyptian Mother's milk pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in αsynuclein and significantly accelerate the rate of formation ofα synuclein fibrils in vitro . They propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of α -synuclein, accounting for the observed conformational changes and leading to association and subsequent fibrillation .These observations suggest one possible underlying molecular basis for Parkinson's disease .α-Synuclein, a relatively abundant brain protein of 140 amino acids and of unknown function, was first identified in association with synaptic vesicles Maroteaux et al., 1988. α-Synuclein belongs to the class of proteins known as natively unfolded; i.e., the purified protein at neutral pH is substantially disordered (Uversky et al., 2001a,b) . Regarding potatoes, risk assessment based on their contamination levels from pesticides presented in Tables 12 and 13 a daily potato consumption of 0.06 kg for an adult person of 60 kg body weight (WHO, 2003) yielded the estimates . Comparing the estimated dietary doses for the studied pesticides with their Acceptable Daily Intake-ADI; JMPR (Tomlin, 2004), revealed that only phorate residues either in (C) potato (0.001mg/kg b.w/d) or in organic potato, (0.0013 mg/kg b.w/d) pose risks to human health due to consumption of such potatoes since the estimated dietary doses accounted to 2.22 and 2.68 times the WHO-ADI for this pesticide (0.0005mg/kg b.w/d), respectively table (12) .

Pesticides binding to individual proteins
In vitro Binding of three pesticides Trichlorphenol, Fenvalerate and α-Endosulphan to Rat Serum Transferrin and Albumin for Bio-monitoring of Pesticides Pollution were carried out according to Afify et al., (2000). The results of the electrophoresis separation of the protein subunits of rat serum treated with different pesticides concentration 5, 10, 15 and 20 PPm (Table 13) showed that these pesticides have high affinity to albumin as well as high molecular weight proteins .The increase in the intensity of transferring protein was occurred with trichlorophenol and α-endosulphan .On the other hand, the intensity of the albumin fraction was decreased with fenvalerate, while it is markedly increased with trichlorophenol and α-endosulphan . The individual incubation of each pesticide with transferrin,, albumin or prealbumin showed that trichlorophenol and α -endosulphan was found to cause aggregation of transferring by 49.l and 43.9%, respectively, while fenvalerate was found to cause marked disintegration of transferrin as compared to controls .The albumin fraction was significantly decreased with the three pesticides .The Pre-albumin was found to markedly increased in its Intensity by 44.8 and 57.3 %with Trichlorophenol 5 ppm and α-endosulphan 15 ppm, respectively. The results concluded that several proteins have responded to pesticides treatment including the known serum proteins, transferrin, albumin, pre-albumin and small molecular weight proteins (Table 14) .However, some of the small molecular weights proteins have been identified as results of pesticides binding which require further characterization .Therefore, the detection of serum proteins after electrophoresis is considered a very good diagnostic parameter for bio-monitoring of pesticides pollution as studies by Saleh et al., (1996b); Afify et al., (1997). Investigation was carried out to determine if there are any changes among serum proteins which could be used as a biomarker for pesticides pollution .In addition, during the transport of the pesticides with carrier proteins in blood throughout the organs, do complex cause destruction in macromolecules .The data in table (14) of the present study revealed that the incubated pesticides have, high affinity to the proteins binding sites (Saleh et al., 1996b;Afify et al., 2000). Similar, observations have been recorded for particle mediated uptake of chlorinated pesticides by human, rat and insect lipoprotein (Shalsky and Guthrie, 1975;Larsen et al., 1994) and by serum albumin and α -globulin in rat and rabbit (Shakoori et al., 1996) . The binding of pesticides to proteins is correlated to the binding of DNA .DNA was considered the most important leader of the genetic code in human (Hemminki, 1986 )which may induce genetic, risks) (Ehrenberg et al., 1974.). Therefore, the binding of pesticides to the macromolecules of rat serum protein could be serve as biomarker in the monitoring of pesticide (Hemminki, 1986). Pahler et al .(1999) showed that the accumulation of some proteins such as alpha 2 macro -globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat .These chemicals have been shown to bind to alpha 2 macro -globulin and this binding was found to impair the renal degradation of the protein, resulting in lysosome overload, cell death, increased cell proliferation and, presumably renal tumor formation . The present study proved that the major proteins transferrin and albumin are the main sites for the three studied pesticides .The data of incubation of the three pesticides with transferrin and albumin were showed that the destruction of transferrin and albumin with the three pesticides produced a similar but not identical protein profile and the prealbumin was found to represent the major one as recorded by Altland et al . (1981). Dissociation into small MW proteins has been demonstrated in case of in vitro incubation with the tested pesticides .These results are in agreement with the results obtained by prolonged exposure of proteins to pesticides ) Nilsson et al., 1975) .The changes in the binding of serum acute phase proteins such as transferrin and albumin with some chemicals has been used to detect or identify human breast cancer (Heys et al., 1998). Insecticides have been shown to bind to blood protein especially organochlorine compounds which are extensively bound to blood lipoproteins (Shalsky &Guthrie, 1975, 1977 . Dutta et al .(1992) revealed that malathion an organophosphorus pesticide has profound effect on serum protein as other parameters .Therefore, the detection of the prealbumin as well as small MW proteins after electrophoresis is considered a very good diagnostic marker for pesticide pollution .In conclusion the induced destructed proteins by pesticides in-vivo and in vitro may be utilized as biomarkers reliable for pesticides monitoring ( Saleh et al., 1996b;Afify et al., 2000 ).

Conclusion
To improve agricultural productivity and control pesticide residues in food and environment; three different methods of extraction for pesticides were applied and methods based on chromatographic separation HPLC with mass spectrometric detection(LC-MS/MS tandem spectroscopy) considered useful methods for determination of pesticide residues in natural products under different types of farming production . Therefore this chapter evaluates the capabilities of mass spectrometry (MS) in combination with liquid chromatography (LC) for the determination of multi-residue pesticides extracted with three different methods. LC-MS/MS using electrospray ionization (ESI) are identified as techniques most often applied in multi-residue methods for pesticides at present in most labs . Therefore, applicability and sensitivity obtained by LC-MS/MS is evaluated for each of the selected pesticides. A modified multi-residue method for analysis of 150 pesticide residues in green beans and grapes using liquid chromatography-tandem mass spectrometry were evaluated and compared for a wide range of physicochemical properties followed by LC-MS/MS detection. GC systems with three different detectors GC-ECD, GC-N P D a n d G C -M S D w e r e u s e d t o c o m p a r e b e tween its efficiency and LC-MS/MS in separation and sensitivity.
Multi-residue method of determination of 150 pesticides is developed at 0.01 mg/kg limit of determination which fulfills the EU MRLs for organic agricultural products and baby foods. Grapes and green beans were selected not only for their wide consumption in the local market but also because they are promising exporting products to the international markets. The mass spectrometric parameters were optimized to give the best sensitivity, two MRM's were chosen for quantification and conformation of pesticides. The selected MRM's were based on the optimized declustring potential and collision energy which help improve pesticides selectivity and justification. Risk associated with consumption of foods contaminated by pesticides has stimulated research to find out their impact to human health risk .Therefore Human milk samples were analyzed for pesticides residues along 26 of Egypt Governorates as well as pesticides residues in potatoes produced under different farming condition. In vitro binding of three pesticides e.g. Trichlorphenol, Fenvalerate and α-Endosulphan to rat serum proteins were studied to evaluate their binding and predict biomarker molecules. The book offers a professional look on the recent achievements and emerging trends in pesticides analysis, including pesticides identification and characterization. The 20 chapters are organized in three sections. The first book section addresses issues associated with pesticides classification, pesticides properties and environmental risks, and pesticides safe management, and provides a general overview on the advanced chromatographic and sensors-and biosensors-based methods for pesticides determination. The second book section is specially devoted to the chromatographic pesticides quantification, including sample preparation. The basic principles of the modern extraction techniques, such as: accelerated solvent extraction, supercritical fluid extraction, microwave assisted extraction, solid phase extraction, solid phase microextraction, matrix solid phase dispersion extraction, cloud point extraction, and QuEChERS are comprehensively described and critically evaluated. The third book section describes some alternative analytical approaches to the conventional methods of pesticides determination. These include voltammetric techniques making use of electrochemical sensors and biosensors, and solid-phase spectrometry combined with flow-injection analysis applying flow-based optosensors.

How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following: Abd El-Moneim M.R. Afify (2011). Recent Techniques Applied for Pesticides Identification and Determination in Natural Products and Its Impact to Human Health Risk, Pesticides in the Modern World -Trends in Pesticides Analysis, Dr. Margarita Stoytcheva (Ed.), ISBN: 978-953-307-437-5, InTech, Available from: http://www.intechopen.com/books/pesticides-in-the-modern-world-trends-in-pesticides-analysis/recenttechniques-applied-for-pesticides-identification-and-determination-in-natural-products-and-it