Analysis of Toxicants by Gas Chromatography

Chromatography is an analytical technique which has been used for isolation, purification and separation of organic and inorganic compounds including qualitative and quantitative estimation of compounds. Basically, there are two types of chromatography. One is liquid chromatography and other is Gas chromatography. The chromatography was discovered in 1906 and gas chromatography was developed by Tshett in 1950s. Generally, in GC the mobile phases are gases such as helium (He), or Nitrogen (N2). GC depends upon temperature programming and boiling point of the compounds. The volatile organic compounds (VOCs), poly-aromatic hydrocarbon and pesticides have been analysed by gas chromatography-mass spectrometric technique. In this chapter, we present a review of existing analytical methodology for the environmental and biological monitoring of exposure to toxicants. In an effort to provide a more concise exploration of existing toxicants bio-monitoring methodology that is relevant today. In addition, as much of the toxicants analysis work involving effective dose measurements is its infancy (adduct measurements) or do not provide sensitive indicators of exposure, All these methods are in use but some form of chromatography, but the detection systems range from simple UV absorbance detection to sophisticated mass spectrometric analyses. These methods possess limits of detection (LODs) that span a wide range; some are suitable for only occupational or forensic applications while those with LODs near or lower than the low-μg/ l are useful for detecting incidental environmental exposures. In addition, these methods have been used to measure toxicants and/or their metabolites in a variety of matrices including urine, serum, breast milk, saliva, and post-partum meconium.


Introduction
Chromatography is an analytical technique which has been used for isolation, purification and separation of organic and inorganic compounds including qualitative and quantitative estimation of compounds. Basically, there are two types of chromatography. One is liquid chromatography and other is Gas chromatography. The chromatography was discovered in 1906 and gas chromatography was developed by Tshett in 1950s. Generally, in GC the mobile phases are gases such as helium (He), or Nitrogen (N 2 ). GC depends upon temperature programming and boiling point of the compounds. The volatile organic compounds (VOCs), poly-aromatic hydrocarbon and pesticides have been analysed by gas chromatography-mass spectrometric technique. In this chapter, we present a review of existing analytical methodology for the environmental and biological monitoring of exposure to toxicants. In an effort to provide a more concise exploration of existing toxicants bio-monitoring methodology that is relevant today. In addition, as much of the toxicants analysis work involving effective dose measurements is its infancy (adduct measurements) or do not provide sensitive indicators of exposure, All these methods are in use but some form of chromatography, but the detection systems range from simple UV absorbance detection to sophisticated mass spectrometric analyses. These methods possess limits of detection (LODs) that span a wide range; some are suitable for only occupational or forensic applications while those with LODs near or lower than the low-μg/ l are useful for detecting incidental environmental exposures. In addition, these methods have been used to measure toxicants and/or their metabolites in a variety of matrices including urine, serum, breast milk, saliva, and post-partum meconium.

Gas chromatography equipped with different detectors for analysis of Arial pollutants
Generally, five types of detectors are coupled with GC (GC-ECD, GC-NPD, GC-FID, GC-PID and GC-MS). The analysis of compounds on GC depends upon nature of compound, 135 conducting a risk based assessment which can be derived from pre-existing site assessment data, thereby decreasing the cost and time required to obtain the additional samples and analytical information, and (2) unique TPH distributions can be critically evaluated with mass spectra in order to ascertain the nature and potential source of the compounds present in the TPH mixtures.
A gas chromatography-mass spectroscopic method in electron ionization (EI) mode with MS/MS ion preparation using helium at flow rate 1 ml min −1 as carrier gas on DB-5 capillary column (30 m × 0.25 mm i.d. film thickness 0.25 μm) has been developed for the determination of benzene in indoor air. The detection limit for benzene was 0.002 μg ml −1 with S/N: 4 (S: 66, N: 14). The benzene concentration for cooks during cooking time in indoor kitchen using dung fuel was 114.1 μgm −3 while it was 6.6 μgm −3 for open type kitchen. The benzene concentration was significantly higher (p < 0.01) in indoor kitchen with respect to open type kitchen using dung fuels. The wood fuel produces 36.5 μgm −3 of benzene in indoor kitchen. The concentration of benzene in indoor kitchen using wood fuel was significantly (p < 0.01) lower in comparison to dung fuel. This method may be helpful for environmental analytical chemist dealing with GC-MS in confirmation and quantification of benzene in environmental samples with health risk exposure assessment (Sinha et al., 2005).
The benzene and toluene was quantified using the GC-MS/ MS technique from indoor air. The geometrical mean (GM) of benzene exposure for cooks during cooking hours in an indoor kitchen using mixed fuel was 75.3 μg/m 3 (with partition) and 63.206 μg/m 3 (without partition), while the exposure was 1.7 μg/m 3 for open type. The benzene exposure was significantly higher (p < 0.05) in an indoor kitchen with respect to open type using mixed fuels. Concentration of benzene (114.1 μg/m 3 ) for cooks in an indoor kitchen with partition using dung fuel was significantly higher in comparison to open type kitchen. (Sinha et al., 2005). The toxic compounds are toluene, xylene, ethylbenzene, 2-4dimethyl heptanes ,cyclohexane propanol, 1-(methyl ethyl) cyclohexane, propyl benzene, 1-ethyl-3-methyl benzene, heptyl cyclohexane, 1-methyl-3propyl benzene, diethyl benzene, 4-ethyl octane, naphthalene and 3-methyl decane were present in thinner. All these compounds were confirmed using retention time parameter, molecular ion peak and other characteristic peak using National Institute of Science Technology (NIST) library search. The percentage composition of thinner has been also explained (Sinha and Zadi 2008). A GC-MS method is also very useful for measurement of main urinary metabolites of benzene, namely phenol, catechol,hydroquinone, 1,2,4-trihydroxybenzene, t,t-muconic acid and S-phenylmercapturic acid. Measurement of urinary benzene was performed via headspace solid phase microextraction of 0.5 mL of urine specimens followed by GC-MS. A number of methods including GC-FID and GC-MS are available for analysis of benzene from ambient environment but the more sensitive procedure of Pre-concentration on charcoal followed by GC-MS analysis is preferred. GC-MS technique being used for the qualitative and quantitative analysis for methyl tertiary butyl ether (MTBE) and benzene in gasoline. In this method, the internal standard was used to improve precision, standard deviation and ion extraction/ monitoring for the measurement of specific highly volatile organic compounds in air pollution samples (Carmichael et al., 1990). A carbotrap tube (2 mm) was used to determine volatile organic compounds in ambient air. Such compounds were desorbed and thermally analyzed with GC-MS With the use of GC-MS technique about 54 toxic hydrocarbons were quantified in the ambient air of Tehran. Polyurethane foam (PUF) cartridge samples were analyzed for dioxins and furans as per EPA test method TO-9 (Keen and Doug project No 46310001131). This gas chromatography high resolution mass spectrometric (GC-HRMS) method was also used for the analysis of poly chlorinated dibenzofurans in ambient air.

Chromatography equipped with different detector for analysis of pesticides in food samples
GC-MS/MS method was used for the analysis of organochlorine pesticides (OCs) residues ( -HCH, -HCH, -HCH, -HCH, p,p-DDE, p,p-DDD, p,p-DDT, -endosulfan,endosulfan, and endosulfan sulphate) in carbonated drinks (Miller and Miline 2008). Furthermore, a method was reported (Paya et al., 2007) for the analysis of pesticide residues using a quick, cheap, effective, rugged, and safe (QuEChERS) multi-residue method in combination with gas chromatography and tandem mass spectrometric detection (GC-MS/MS). A mixture of 38 pesticides was quantitatively recovered from spiked lemon, raisins, wheat and flour using GC-MS/MS, while 42 pesticides were recovered from oranges, red wine, red grapes, raisins and wheat flour using GC-MS/MS for determination (Paya et al., 2007). Several GC methods have also been developed for pesticide residual analysis in different food (Kumari and Kathpal 2009;Kumari et al., 2006;kumara et al., 2002) commodities (e.g., vegetables, fruits and other products of food).
A sensitive method for the quantification of 11 pesticides in sugar samples to the µgkg _1 level has been developed. These pesticides are often used in an agricultural context. A simple solvent extraction followed by selective analysis using a gas chromatography-mass spectrometric method was used. This method was accurate (> 99%) as it possesses limits of detection in the 0.1-ug kg -1 range, and the coefficients of variations are less than 15% at the low μgkg -1 end of the method's linear range. The percent recovery of all the pesticides at the lowest levels of detection ranges from 82% to 104%. This method was used for the quantification of pesticides in sugar samples collected from different factory outlets from different parts of India. In this study, 27 refined sugar samples were analysed in which one sample showed a detectable level of the chlorpyrifos. This study showed that Indian sugar is free from the commonly-used pesticides at the low µgg -1 levels (Sinha et al., 2011). The reported method for the analysis of pesticides in food samples is tabulated in Table-1. However, recently, a method has been reported (Li Wu, Chen & Zhang 2010) detailing the percentage recoveries, RSD, and LOD in vegetable sample. The reported RSD for all the pesticides used in this study ranged from 2.8% to 10.4% and the recovery ranged from 75% to 11%. The reported LOD in vegetable samples ranged from 3 to 10μgkg -1 for all pesticides. Also, an LC-MS/MS method has been used for the analysis of pesticides in fruit and vegetable samples (Camino sanchez et al., 2010). In this method, QuEChERS was used for the extraction. The reported mean percentage recoveries mostly ranged between 70% and 120%, and RSD were generally below 20% at the lower spiked level. The CEI with quantum dot fluorescence detector was used for analysis of vegetable samples (Chen & Fung 2010 (Kamel, Qian, Kolbe & Stafford, 2010). This method was based on solid phase extraction followed by UPLC-MS/MS chromatographic separation and a full-scan mass spectrometric detection. The recovery for all pesticides ranged from 70% to 120% with LOD ranged from 1 to 2000μgkg -1 . The HPLC method was also used for analysis of pesticides in vegetables samples (Lin et al., 2010). In this method liquid extraction was used. In the vegetable samples, the observed recoveries ranged from 77% to 99% and LOD ranged from 0.5 to 3μgkg -1 while GC-NPD was used for analysis of vegetable samples reported mean percentage recoveries mostly ranged between 70% and 96%, and RSD were generally below 15% (Srivastava, Trivedi, Srivastava, Lohani & Srivastava 2010). The LOD values ranged from 1 to 9μgkg -1. However it should be noted that HPLC and GC-NPD are not confirmatory techniques.

Gas chromatography equipped with different detector for analysis of pesticides in biological samples
A sensitive GC-MS method in MS/MS ion preparation was developed for quantitative estimation and qualitative determination of chlorpyrifos in human blood samples. Dissociation energy effects on ion formation of chlorpyrifos and sensitivity of this analytical method was well demonstrated. Chlorpyrifos was extracted using methanol/hexane mixture from 0.2 ml human blood, deactivated with saturated acidic salt solution. The extract was then re-concentrated and analyzed by electron impact (EI) MS/MS gas chromatography-mass spectrometer. The MS/MS spectra of chlorpyrifos ion were recorded on different dissociation energy (30-100 V) to establish the structural confirmation and to demonstrate the effect of this energy on sensitivity, S/N ratio and detection limit for quantification of chlorpyrifos, which is reported for the first time. At different exciting amplitude (30-100 V), different behaviors of base peak, sensitivity, S/N ratio and detection limit of this method were observed for quantification of chlorpyrifos. The mass spectra recorded at dissociation energy <70 V, in between 70-80 V and >80 V correspond to the m/z was used for the quantification of chlorpyrifos. The detection limit for quantification was 0.1 ng/ml with S/N: 2 in human blood. The linear calibration curve with the correlation coefficient (r > 0.99) was obtained. The percentage recoveries from 95.33% to 107.67% were observed for chlorpyrifos from human blood. The blood samples were collected at different time intervals. The concentration of chlorpyrifos in Poisoning case was 3300, 3000, 2200, 1000, 600, 300 ng/ml on day 1, 3, 6, 8, 10 and 12, respectively. On the 12th day of exposure of chlorpyrifos, 90.9% reduction in concentration was observed. On day 14 th , the chlorpyrifos was not detected in the blood of the same subject. Thus, the present study is useful for detection of chlorpyrifos in critical care practices and also provides tremendous selectivity advantages due to matrix elimination in the parent ion isolation step in blood sample analysis for chlorpyrifos . The effect of dissociation energy on ion formation and sensitivity of triazofos in blood samples was studied. Six millilitres hexane was used for the extraction of triazofos from 2mL serum samples. The extract was reconstituted in 1mL hexane and analyzed by GC-MS/MS in electron impact MS/MS mode. The structure, ion formations, nature of base peak and fragmentation schemes were correlated with the different dissociation energies. The new ion was obtained at mass to charge ratio 161 (100%), which was the characteristic ion peak of triazofos. On using different exciting amplitudes, different behaviours of fragmentation schemes were obtained. The effect of dissociation energy on sensitivity of the analyte was also demonstrated. The mass spectra recorded at different exciting amplitudes <50V in between 50-60V and >60V correspond to the m/z 161 (100%), 77 (100%) and 119 (100%), respectively. The maximum sensitivity of analyte in blood sample was obtained on using 50V dissociation energy. Additionally, the effect of current on sensitivity of the method was also demonstrated. In all conditions, the new characteristic ion at m/z 161 was obtained and used for quantification of triazofos in blood samples with maximum sensitivity. The limit of detection and quantification was 0.351 and 1.17 ngmL −1 , respectively, with 99% accuracy. The observed correlation coefficient was 0.995. The inter-day percentage recoveries from 83.9% to 111% were obtained below 9.38 percentage RSD. This present method gives combined picture of confirmation and quantification of triazofos in critical care practices and also provides tremendous selectivity advantages due to matrix elimination in the parent ion isolation step in blood sample analysis for triazofos in poisoning emergency cases (Sinha 2010). A number of families in a rural area of Jabalpur District (Madhya Pradesh), India, were affected by repeated episodes of convulsive illness over a period of three weeks. The aim of this investigation was to determine the cause of the illness. The investigation included a house-to-house survey, interviews of affected families, discussions with treating physicians, and examination of hospital records. Endosulfan poisoning was suspected as many villagers were using empty pesticide containers for food storage. To confirm this, our team collected blood and food samples, which were transported to the laboratory and analyzed with GC-ECD. Thirty-six persons of all age groups had illness of varying severity over a period of three weeks. In the first week, due to superstitions and lack of treatment, three children died. In the second week, symptomatic treatment of affected persons in a district hospital led to recovery but recurrence of convulsive episodes occurred after the return home. In the third week, 10 people were again hospitalized in a teaching hospital. Investigations carried out in this hospital ruled out infective etiology but no facilities were available for chemical analysis. All persons responded to symptomatic treatment. The presence of endosulfan in blood and food samples was confirmed by GCMS. One of the food items (Laddu) prepared from wheat flour was found to contain 676 ppm of -endosulfan. Contamination of wheat grains or flour with endosulfan and its consumption over a period of time was the most likely cause of repeated episodes of convulsions, but the exact reason for this contamination could not be determined. This report highlights the unsafe disposal of pesticide containers by illiterate farm workers, superstitions leading to delay in treatment, and susceptibility of children to endosulfan . Mass spectrometry method has been used with capillary gas chromatography in electron ionization mode to identify the presence of , and endosulfan sulphate in human serum samples. The fragmentation ions were obtained due to ring cleavages, rearrangement and remote charge process. Solvent extraction procedure was used for isolation of compounds from serum sample. A detection limit as low as 100 ppb for , and endosulfan sulphate could be easily achieved for confirmation. For endosulfan and endosulfan sulphate the m/z values were obtained at intervals of M +2 . The m/z values of , endosulfan are identical but they differed in retention time (Sinha et al., 2004). Therefore, the spectra reported might serve as reference spectra for identification of different type of organo-chlorine pesticide in human serum samples. The GC-MS has been employed to quantitatively detect at low ppb levels of and endosulfan in human serum, urine and liver but this failed to separate , -isomers. GC equipped with a mass detector was used to measure the levels of endosulfan in human blood at ppb level.
Although some OC pesticide metabolites are monitored in urine, they are most commonly measured as the intact pesticide and/or its metabolite in whole blood, serum, plasma, or other lipid-rich matrices. These methods and the specific pesticides measured are outlined in Table 2. Although some pesticide metabolites are monitored in urine, they are most commonly measured as the intact pesticide and/or its metabolite in, whole blood, serum, plasma, or other lipid-rich matrices. Typically, serum or plasma is extracted using a liquid partitioning or solid-phase extraction (SPE) and the extract is analyzed using capillary gas chromatography (GC) with electron-capture detection (ECD) ( Table-2). These methods are reliable and use affordable instrumentation. However, GC-ECD analyses have a higher potential for detecting interfering components than do more selective analysis techniques.
Other methods for analysis of serum extracts include mass selective detection (MSD aka MS) [ Table-2] and high-resolution mass spectrometry with isotope dilution quantification. These analyses are typically more selective and sensitive than GC-ECD analyses; however, the high cost of instrumentation and isotopically labeled standards and the complex operation and maintenance of these instruments often preclude their routine use in most laboratories.
Typically, serum or plasma is extracted using a liquid partitioning or solid-phase extraction (SPE) and the extract is analyzed using capillary gas chromatography (GC) with electroncapture detection (ECD) ( Table-2). A sensitive and accurate analytical method was developed for quantifying 29 contemporary pesticides in human serum or plasma. These pesticides include organophosphates, carbamates, chloroacetanilides, and synthetic pyrethroids among others and include pesticides used in agricultural and residential settings. This method employs a simple solid-phase extraction followed by a highly selective analysis using isotope dilution gas chromatography-high-resolution mass spectrometry. This method is very accurate, has limits of detection in the low pg/g range and coefficients of variation of typically less than 20% at the low pg/g end of the method linear range (Barr et al., 2002).

Conclusion
In this chapter, we present a review of existing analytical methodology for the biological and environmental monitoring of exposure to pesticides has been presented. A critical assessment of the existing methodology has been done and other areas which need more research have been identified. An effort has been made to use to measure toxicants. There are many detectors which are equipped with Gas Chromatography like Mass, electron capture detector, flame ionization detector, phosphorous nitrogen detector etc. But, in one modern context the mass detector is the best detector which is very useful for confirmation and quantification of all types of compounds. The electron capture detector is only suitable for quantification of organo-chlorine compounds but it is not confirmatory technique. The nitrogen phosphorous detector is suitable for quantification of nitrogen and phosphorous compounds. Other types of detectors except mass detector are not suitable as confirmatory technique.