Analysis of Organophosphate and Carbamate Pesticides and Anticholinesterase Therapeutic Agents ANANT V. lAIN University of Georgia, Athens, Georgia
of the sample extract, (iii) concentration of purified extract, (iv) separation from impurities and detection, and (v) confirmation. In this chapter, the various steps are discussed briefly, and recent trends for each step are discussed.
I. I N T R O D U C T I O N Organophosphate (OP) and carbamate (CM) pesticides are an integral part of modern agriculture. The publication of Silent Spring by Rachel Carson led to the withdrawal of many organochlorine (OC) pesticides due to their persistence in the environment. At this time, the use of OPs and CMs soared due to their availability and quick degradation in the environment. Although OPs and CMs degrade quickly and are much less persistent in the environment, they are much more toxic to mammals than OC pesticides. Because of their adverse health affects, various governmental agencies set limits on allowable levels of pesticide residues in foods, animal feeds, and the environment. In order to enforce these allowable levels, pesticides are monitored in various types of samples. Also, humans and animals may be poisoned accidentally or maliciously. Thus, there is a need for analytical methods to determine OPs and CMs in food and feedstuffs as well as biological specimens. By nature, analytical results are variable. The science of trace analysis (analysis at parts per million or below levels) is not as precise as most layman and many scientists view it to be (Rogers, 1986). Once devised, analytical methods are like life-forms, subject to evolution. Natural selection is mediated by analytical chemists, which ensures that only the fittest analytical methods survive. Therefore, an analytical method must be fit for purpose. In order to be able to determine trace levels of OPs and CMs in the environment and biological, food, and feed samples, it is necessary to follow a series of operations. Note that most of the advancements and improvements in the analytical methods have occurred by making necessary changes and improvements in various steps used in analytical methods. The papers by Sawyer (1988) and Seiber (1988) clearly demonstrate this point. The various steps in an analytical method are (i) extraction of the sample, (ii) cleanup and purification Toxicology of Organophosphate and Carbamate Compounds
II. S A M P L E E X T R A C T I O N It is essential to separate the target pesticide(s) from the sample matrix. Traditionally, this has been done by blending a homogeneous ground sample with an organic solvent. Since OPs and CMs are nonpolar to polar compounds, the polarity of extraction solvents varies accordingly. There are three general methods for extracting pesticides from solids with organic solvents: soxhlet extraction, homogenization with a solvent, and ultrasonication of the ground sample with an organic solvent. Homogenization is the preferred method for the extraction of pesticides with solvents in the methods described in the Pesticide Analytical Manual (PAM) of the U.S. Food and Drug Administration (FDA, 1994). Handling of the dry samples usually requires the addition of water to moisten the sample, along with the organic solvents. Sonication of the blended mixture expedites the extraction of target compounds. However, it is not recommended for OPs [SW-846 method 8141A; U.S. Environmental Protection Agency (EPA, 1994)]. Acetone, methylene chloride, hexane, petroleum ether, acetonitrile, and methanol are some of the solvents used for extraction of pesticides depending on the sample matrix and the nature of the pesticide(s). Liquid samples are extracted directly from the sample with organic solvents. Water samples are extracted with organic solvents; these methods use solvents that are immiscible with the sample phase. Ideally, one selectively extracts the target compounds by using a solvent whose polarity is similar to that of the target compound. Volatile 681
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SECTION Vlll 9 Analytical & Biomarkers
solvents, such as ether, ethyl acetate, and methylene chloride, are usually used for the extractions of OPs and CMs. For air samples, a certain amount of air is pumped through a trap tube (OSV-2) that contains an adsorbent such as XAD-2, which efficiently traps OPs and CMs, and then the trap is eluted with organic solvents in the case of OPs and buffer in the case of CMs [National Institute for Occupational Safety and Health (NIOSH), 1994].
A. Current Trends in Sample Extraction Current trends have been to accelerate the extraction of pesticides from the sample matrix by accelerated solvent extraction (ASE) or pressurized solvent extraction (PSE), microwave-assisted solvent extraction (MASE), and super critical fluid extraction (SFE). 1. ACCELERATEDSOLVENT EXTRACTION OR
PRESSURIZEDSOLVENTEXTRACTION ASE or PSE performs extractions at elevated solvent temperatures and pressures to achieve higher extraction efficacy. The process may be compared to pressure cooking. After loading a sample into the extraction cell, the cell is filled with solvent, heated and pressurized, and held at the pressure and temperature for a predetermined time. The clean solvent is pumped into the sample cell, and the sample cell is purged with nitrogen gas. This extract is collected into a collection vial. ASE has been used for the determination of OP and N-methyl CM pesticides in foods (Obana et al., 1997; Okihashi et al., 1997; Richter et al., 2001). ASE has also been used for the extraction of OPs and other pesticides from soils, clays, sediments, sludge, and waste solids (SW-846, method 3545A; EPA, 1994). 2. MICROWAVE-ASSISTEDSOLVENT EXTRACTION This method is similar to ASE, except that heating power is supplied by microwaves. MASE has been used for the extraction of pesticides from soil (de Andrea. et al., 2001). Sun and Lee (2003) report a method for the optimization of MASE as well as supercritical fluid extraction of CM pesticides from soil. Lopez-Avila et al. (1998) studied the stability of OCs and OPs when extracted from solid matrixes with microwave energy. 3. SUPERCRITICALFLUID EXTRACTION In this method, CO2 gas at the critical temperature and pressure is used to extract pesticides and other chemicals from solid samples or liquid samples soaked in an inert powder. CO2 and other gases become fluid (neither gas nor liquid) when temperature and pressure reach a critical point called the supercritical phase. In SFE, this fluid is used to extract target chemicals (OPs and CMs) from solid sampies. Instruments connecting the SFE apparatus with online gas chromatography (GC) have been developed in which the series of steps from extraction to analysis have been
automated. The sample is placed in an extraction chamber through which the supercritical fluid is forced, and the target substances are extracted from the samples and trapped in vials by a small amount of methanol or adsorbent such as florisil or ODS resin. The most common gas used in SFE is CO2. However, supercritical CO2 is very nonpolar, so modifiers are added to improve extraction efficiency for polar compounds. Modifiers for CO2 include methanol, dichloromethane, acetonitrile, and water. Modifiers are mixed with the CO2 using a pump or added directly into the samples. The application and effectiveness of SFE for the extraction of pesticides from various types of samples for pesticide analysis have been demonstrated (Hopper, 1997; Hopper et al., 1995; Kim et al., 1998; Lehotay, 2002; Lehotay and Eller, 1995; Lehotay and Valverdegarcia, 1997; Lehotay et al., 1995). A method for the SFE extraction of methamidophos residue from vegetables has been described (Valverdegarcia et al., 1995). A limitation of SFE is that extraction of fatty foods and animal tissues with SFE extracts the fat along with pesticides. A systemic approach to optimization of SFE of pesticides is presented by Juhler (1997). A review of the usefulness of SFE for pesticide determination is presented by Camel (1998). ASE, MASE, and SFC, and their uses and potential pitfalls, have been discussed in a review (Camel, 2001).
III. C L E A N U P A N D P U R I F I C A T I O N Sample extracts for pesticide analysis from food, feed, biological, and environmental samples are usually complicated mixtures. The other chemical components in the sample are extracted along with the pesticide residue. Usually, the residues are present at the parts per million (ppm) level in the sample, whereas other components are present in very large amounts (Rogers, 1986). The other component chemicals in the sample extract can interfere with GC and highperformance liquid chromatography (HPLC) analysis due to coelution of the impurities with target compounds, and affecting the separation capacity of the analytical column due to overloading by impurities. Furthermore, large amounts of nonvolatile or polar compounds can contaminate GC injection ports and columns. It is therefore necessary to clean up or remove nontarget compounds as much as possible. For OPs and CMs, the usual techniques for purification are liquid-liquid partitioning and column chromatography. For cleanup of OPs and CMs, the adsorption columns used are Florisil, charcoal/Celite, and ODS, in which an octadecyl function is bonded to silica particles (FDA, 1994).
A. Current Trends in Cleanup and Purification 1. SOLIDPHASE EXTRACTION This technique came in vogue with the development of HPLC techniques. This technique is a form of partition
CHAPTER 47 9 Analysis of OPs and CMs chromatography and works according to the partition equilibrium between a solid phase and a solvent. If the solid phase is polar, the solvent used is nonpolar. On the other hand, if the solid phase is nonpolar, such as ODS, the solvent used is polar. The advantage of this system is that it is much faster than the traditional column chromatography and uses much less solvent. Solid phases are even available in the form of disks, which can be used to separate OPs and CMs from water (Chiron and Barcelo, 1993; Mersie et al., 2002). The application of SPE has been described for the extraction of pesticides from vegetables (Casanova, 1996), oranges (Yamazaki and Ninomiya, 1999), water (Van-Hoof et al., 2002), and beef muscle (Kuivinen and Bengtsson, 2002). In the case of water, the samples are percolated through the column or filtered through the disk; the pesticides are retained on the column or disk. The pesticides are eluted with appropriate solvents to be used for determination. In the method of Kuivinen and Bengtsson, the bovine muscle is homogenized with ethyl acetate (EtOAc); the homogenate is centrifuged and filtered through anhydrous sodium sulfate. The fat in the filtered extract is precipitated in methanol by cooling, and the extract is diluted with water and passed through an SPE column (isoelute ENV +). After elution with EtOAc, evaporation, and redissolution, the sample is injected into a GC fitted with a capillary column (DB-1701) and detected with a flame photometric detector. In a variation of SPE, honey samples were mixed with Florisil and anhydrous sodium sulfate and loaded in a column, and the column was eluted with hexane-ethyl acetate (90/10 V/V) (Sanchez-Brunete et al., 2002). The column eluate was analyzed by GC and nitrogen phosphorus detector (NPD), as well as GC and mass spectrometry (MS). 2. GEL PERMEATION CHROMATOGRAPHY Gel permeation chromatography (GPC) separates molecules by size. Compounds are separated when sample mixtures are passed through a column packed with material of known pore size. Larger molecules that cannot penetrate the pore size of the packed material (usually known as a gel) elute faster. GPC is used as a general separation method for semivolatile compounds. The separation ability is poorer than that of other forms of chromatography. Thus, GPC is generally used to remove lipids, proteins, and natural resins from samples. It is a very good technique for removing fat in the analysis of fatty samples for pesticides. The GPC technique for pesticide residue determination has been used since 1972. An automated GPC instrument was introduced by Ault et al. (1979) for chromatographic preparation of vegetable, fruits, and crops for OP residue determination utilizing GC with flame photometric detection (FPD). Sannino et al. (1995) described a multiresidue method for the quantitative determination of 39 OPs in seven fatty foods using GPC as a cleanup technique. A report of a cooperative trial to validate the use of GPC
cleanup for the isolation of pesticide residues from fats and oils was published by the Committee for Analytical Methods (Anonymous, 1992). A multiresidue screen for quantitative determination of 43 OPs, 17 OCs, and 11 CMs in plant and animal tissues, including ingesta, was reported by Holstege et al. (1994). Ali (1989) applied GPC cleanup and HPLC (with postcolumn derivatization) for the determination of CMs in animal tissues. 3. IMMUNOEXTRACTIONS Immunoaffinity sorbents (ISs) are more selective compared to ODS sorbents. The first commercial ISs were introduced for the cleanup of samples for the determination of aflatoxins (Groopman and Donahue, 1988). ISs have been synthesized for a limited number of pesticides and pesticide classes. Immunosorbents are formed by covalently bonding antibodies to an appropriate sorbent. Immunosorbent is packed into a solid phase extraction cartridge or precolumn as a classical extraction sorbent (Bouzige and Pichon, 1998). There is much interest in developing ISs for single analytes, which are particularly difficult to analyze at the trace level because of a lack of available extraction methods, such as acephate and methamidophs. 4. SOLID PHASE MICROEXTRACTION The solid phase microextraction (SPME) technique is used in many areas. SPME combines the use of solid phase extraction and concentration of the eluate from SPE in one step. SPME probes are silica fibers coated with organic polymers (DB 1, DB 1701, etc.). These fibers capture and concentrate pesticides and, of course, some other chemicals. The fibers are sealed in a syringe. The sample can be injected directly into a GC by using an accessory. The needle of the syringe is lowered into the injection port and then fiber is introduced in the injection port. Pesticides are thermally desorbed from the fiber and then separated on the analytical column and detected. A review of the application of SPME to the analysis of pesticides has been published (Boyd Boland et al., 1996). Methods for the analysis of pesticides using the SPME technique coupled with GC or HPLC have been described for water (Sng et al., 1997; Beltran et al., 1998; Aguilar et al., 1998) and in a food plant (Chen et al., 1998), biological samples (Musshoff et al., 1999), and soil samples (Ng etal., 1999; Moder etal., 1999). SPME fibers are also finding use in the analysis of chemical warfare agents (CWAs) in water (Lasko and Ng, 1997).
IV. C O N C E N T R A T I O N The cleaned up sample extract has to be concentrated in order to inject microliter amounts of the sample extract in GC or HPLC systems connected to high-sensitivity detectors used in the determinative step. Large volumes of solvents in the sample extracts overload the analytical column, particularly in GC. Several types
S E CT I 0 N V I I I 9 A n a l y t i c a l & B i o m a r k e r s
of concentrators, such as Kuderna-Danish (K-D), Rotory Evaporator (RE), Turbovap, and N-EVAP, are available for sample concentration. The use of K-D, RE, and Turbovap is described in the Pesticide A n a l y t i c a l M a n u a l (FDA, 1994). Information about the use of N-EVAP nitrogen evaporators for sample concentration is available from Organomation Associates.
V. SEPARATION FROM IMPURITIES, D E T E C T I O N , AND D E T E R M I N A T I O N The cleaned up sample extract still contains many impurities; thus, these impurities have to be separated from the analyte before it is determined. The detection and determination of OPs is usually with GC coupled with selective detectors, such as electron capture detector, NPD, FPD, and mass spectrometric detector (MSD or MS). Large varieties of packed as well as megabore and capillary columns are used with these detectors. CMs are determined with GC coupled to NPD or FPD (in S mode). CMs are effectively determined with HPLC/photo diode array or HPLC/fluorescence. For a trace amount, it is essential to do a postcolumn derivatization (Ali, 1989).
A. Chromatography Chromatographic methods in general are the most common methods used at the determinative step in pesticide analysis. Thus, basic understanding of chromatographic procedures and the detection devices is important for pesticide analysis. According to the International Union of Pure and Applied Chemistry, chromatography is the method used primarily for the separation of components of a sample in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be a solid, a liquid supported on a solid, or a gel. The stationary phase may be packed in a column, spread as a layer, or distributed as a film, etc. The mobile phase may be gaseous or liquid. Thus, there are two movements in the chromatographic system: the mobile phase movement, which is usually at a constant rate, and the movement of the components of the sample. The movement of the components depends on their relative distribution between the stationary phase and the mobile phase. This results in the separation of the components. This separation process coupled with the detection and measuring device completes the chromatographic system.
1. GAS CHROMATOGRAPHY In GC, the basic instrument consists of a sample introduction port (injector), an analytical column in an oven (to separate the components of the mixture), a detector (which can detect the presence of chemicals eluting from the analytical column), and a recording or data handling device. The injector,
oven, and detectors are heated zones; the temperature regulation of these zones depends on the application. Initially, packed columns were used in GC, but these columns had only limited resolving power. With the advent of capillary and megabore columns in which the liquid phase is crosslinked to pure silica capillary tubes, the resolving power of the analytical column increased severalfold. Lawrence (1987) showed the separation of 38 OPs on a DB-17 capillary column used for GC with FPD.
2. HIGH-PERFORMANCELIQUID CHROMATOGRAPHY HPLC is a variant of traditional liquid chromatography. The resolution and separation are improved by using small, uniform particle size columns. These columns require the use of a high-pressure pump that can force the liquid mobile phase through the columns, presumably at a constant rate. The basic HPLC instrument consists of an injector, an analytical column (which may or may not be temperature controlled), and a detector. With the development of hardware for pumps, the performance of liquid chromatography has increased manyfolds. The greatest impediment to the use of HPLC in residue analysis has been the absence of selective detectors, as are available for GC. However, the use of HPLC coupled with the MSD in trace analysis of pesticides is increasing, particularly for polar and thermally labile compounds. The technique of postcolumn derivatization (attaching a fluorophore) and fluorometric analysis has proved very useful for the determination of N-methyl CMs in foods (Holstege et al., 1994) and tissues (Ali, 1989; Ali et al., 1993a,b).
B. Detectors 1. ELECTRONCAPTURE DETECTOR The modem ECD contains a 63Ni source. This source emits high-energy beta particles. When these particles collide with the carrier gas molecules to produce low-energy electrons, the electrons continually collect at the cell anode by applying voltage pulses to the cell electrode. Cell current thus produced is measured and the pulse interval (frequency) adjusted to maintain constant cell current. A standing pulse frequency describes the equilibrium condition that exists when only carrier gas is passing through the cell. When molecules of an electrophilic substance enter the detector, electrons are "captured" to a degree depending on the electron affinity of the substance. The electron supply in the cell decreases, and pulse frequency increases to generate constant current. Change in the frequency required to keep the cell current constant is converted to voltage and is sent to the recording device as the detectors response to the analyte.
2. FLAME PHOTOMETRICDETECTOR When GC column effluent is burned in a hydrogen/air flame, compounds containing phosphorus and/or sulfur
CHAPTER 47 9 Analysis of OPs and CMs produce characteristic emissions. A narrowband pass (interference) filter of appropriate wavelength isolates emissions produced by either phosphorus or sulfur. These emissions can be viewed by a conventional photomultiplier tube; a filter with maximum transmittance at 526 nm permits detection of phosphorus compounds, whereas one with maximum transmittance of 304 nm detects sulfur compounds. A single optical filter and photomultiplier tube may be used, or two filters and photomultiplier tubes can be assembled to permit response to both phosphorus and sulfur compounds simultaneously. A variation of FPD known as the pulsed flame photometric (PFPD) has been introduced. The PFPD uses the time-delayed chemiluminescence produced by the heteroatom in a molecule. It is claimed that PFPD is much more sensitive than FPD. Moreover, several other heteroatoms, such as S, P, N, and Se, can be detected by PFPD (Jing and Amirav, 1998). PFPD has been used simultaneously with MS to enhance pesticide detection capabilities (Amirav and Jing, 1998) 3. NITROGEN PHOSPHORUS DETECTOR The NPD is selective to residues containing nitrogen and/or phosphorus atoms. Modem NPD detectors evolved from the potassium chloride thermionic detector. GC effluent impinges onto the surface of an electrically heated and polarized alkali metal salt source (usually rubidium) in the presence of air/hydrogen plasma; ionization occurs and the flow of ions between plasma and an ion collector is amplified and recorded. Detector response to analytes results from the increased ionization that occurs when compounds containing nitrogen or phosphorus elute from the column. At gas flow rates used for NPD operation, the degree of ionization of compounds containing nitrogen or phosphorus is 10,000 times greater than for hydrocarbons. The mechanisms of enhanced response to nitrogen and phosphorus are not completely clear. However, both gas phase and surface ionization processes have been proposed. ECD, FPD, and NPD are discussed in detail in Chapter 5 of PAM (FDA, 1994).
4. MASS SPECTROMETRIC DETECTOR A mass spectrometer is an instrument that can separate charged atoms or molecules according to their massto-charge ratio. Relative molecular masses of organic compounds and biopolymers can be measured in this way, and the instrument is also capable of generating structural information. The sample is introduced into the mass spectrometer, which is generally kept under high vacuum (5-10 mbar). Compounds are converted into gas phase molecules either before or during the charging or ionization process, which takes place in the ion source. Many types of ionization mode are available: The type of compound to be analyzed and the specific information required determine which ionization mode is the most suitable. Once ionized, the molecule ion may fragment, producing ions of lower mass than the original precursor molecule. These fragment ions are dependent
on the structure of the original molecule. The ions produced are repelled out of the ion source and accelerated toward the analyzer region. Although both positive and negative ions may be generated at the same time, one polarity is chosen and either positive or negative ions are analyzed and recorded. Molecules that do not ionize (i.e., remain neutral) are pumped away and will not be detected. There are various types of ionization techniques, such as electron impact ionization (EI), chemical ionization, fast atom bombardment/ liquid secondary ionization, matrix-assisted laser desorption ionization, and electrospray ionization. Recently, tandem mass spectrometers (MS-MS) have been introduced. In a tandem mass spectrometer, two mass spectrometers are coupled together separated by a collision cell. By coupling together two analyzers, separated by a collision cell, additional information can be obtained. The first analyzer is used to select the ion of interest. This ion is then passed into the collision cell, which usually will have been pressurized with an inert gas such as argon. Collision of the ion with the atoms in the cell can induce dissociation of the ion. This is known as collision-induced dissociation: The original ion is referred to as the "precursor" ion and the dissociated ions are known as "product" ions. Product ions are then analyzed in the second mass spectrometer, thus generating a product ion mass spectrum of the original precursor ion. The tandem mass spectrometer provides much more structural information about the analyte molecule and thus is an excellent confirmation technique. The greatest impediment for the use of mass spectrometers in common laboratories has been their high price. A direct sample introduction device has been used for the GC/MS-MS determination and confirmation of pesticides (Lehotay, 2002). It has been claimed that the sample extracts need not be cleaned up before determination. A method for the deterruination of 37 OPs in human tissue using GC/MS has been described (Russo et al., 2002). A HPLC/MS method for the determination of eight different types of CM pesticides in serum has been reported (Kawasaki et al., 1993). In this method, 1.5 ml of serum is mixed with 1.5 ml of 0.2 M phosphate buffer. The mixture is applied to an Extrelute column and then eluted with 15 ml of methylene chloride. The eluate is evaporated and the residue is dissolved in mobile phase. Carbofuran has been determined in stomach/rumen contents by GC/MS. Stomach/rumen contents are extracted with methylene chloride and cleaned up with GPC, and then the sample is analyzed by GC/MS (Osheim et al., 1985). Detection of pesticides in various types of samples without any cleanup has also been reported (Smith and Lewis, 1988). In this method, the samples are shaken with a suitable solvent and then analyzed by GC/MS. This method is qualitative in nature. 5. ION MOBILITY SPECTROMETRY Ion mobility spectrometry (IMS) has been known for more than 25 years. In IMS, the analyte and impurity molecules
SECTION VllI 9 Analytical & Biomarkers
are ionized by a 63Ni source, which is housed in a shielded chamber. As discussed previously, the 63Ni source gives off high-energy electrons, which collide with sample molecules to produce ions. The aspirated air or any other gas used to introduce the sample in the reaction chamber also produces ions. The ions move through an electric field in the drift tube. Smaller ions move faster compared to larger ions. Thus, this technique is based on the size-to-charge ratio of ions. This technique differs from mass spectrometry, in which the separation is on the basis of mass-to-charge ratio of ions. Also, in IMS the ions are formed at atmospheric pressure, whereas in mass spectrometry ions are formed at very low pressures (5-10mbar). A walk-through portal based on IMS has already been installed at the Statue of Liberty for detecting traces of explosives and other illegal chemicals. Compact units are in use at many airports for the detection of traces of explosives and illicit drugs. The application of IMS for monitoring mevinphos (phosdrin), an OP, has already been demonstrated (Tuovinen et al., 2001). Fematoscan (1999), a company involved in the manufacture of IMS-based detectors, has demonstrated the application of a handheld detector, which is a combination of GC and IMS for pesticide detection, including some OPs.
VI. CONFIRMATION Mass spectrometry is the technique of choice for confirmation of pesticides. In the case of OPs, the sample extract can be run on different columns. Relative retention times (RRt) of a given peak can be compared with a table of RR t for different columns (FDA, 1994). For CMs, the alkylated CMs can be chromatographed using GC with NPD detector. The retention time of peaks can be compared with the retention time of the standard compound. If enough of the residue compound is present, then thin-layer chromatography can be used to confirm the presence of the pesticide (FDA, 1982).
VII. SOURCES OF A N A L Y T I C A L METHODS Several excellent sources of analytical methods for OPs and CMs are available. These sources also contain methods for other classes of pesticides:
1. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) 2. Pesticide Analytical Manual, Vol. 2 (FDA, 1991) 3. Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples (EPA, 1980)
4. NIOSH Manual of Analytical Methods (NIOSH, 1994) 5. Test Methods for Evaluating Solid Wastes (EPA, 1994) 6. Manual of Pesticide Residue Analysis, Vol. 1 [Deutsche Forschungsgemeinschaft (DFG), 1987]
7. Manual of Pesticide Residue Analysis, Vol. 2 (DFG, 1992)
8. A World Compendium. The Pesticide Manual (Tomlin, 1997)
9. Official Methods of Analysis (OMA) of AOAC International [Association of Analytical Communities (AOAC), 2003a]
10. ATSDR Pesticide Profiles [Agency for Toxic Substances and Disease Registry (ATSDR), 1994]
11. Residue Analytical Methods (RAM) (EPA, 2003) 12. Environmental Chemistry Methods (ECM) (EPA, 2004) 13. Scientific reviews
A. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) PAM, Vol. 1, is the most exhaustive and complete compendium of analytical methods for the multiresidue method (MRM) analysis of pesticides in food stuffs. There are six chapters in PAM. The first chapter deals with regulatory operations, which describes what pesticide tolerance is and under which laws and statutes the pesticide tolerances are regulated. It also describes preparation of analytical sample and method application. Chapter 2 provides information about general analytical operations and discusses basic analytical techniques such as column chromatography; solvent selection and evaporation; equipment and procedures for comminuting samples; and procedures for specific commodities, such as crabs, eggs, fish, hay, straw, food, and feed ingredients. Also discussed in this chapter are special reagent preparation, tests and purification for applicable reagents, reference standards for pesticides, quality assurance and quality control, preparation of standard operating procedures, hazardous waste disposal, and safety issues. Chapter 3 discusses the need for MRMs of analysis. When the target analyte (residue compound) is not known, it is essential to use a method that can detect the maximum number of residues. For this purpose, multiclass MRMs are used. This chapter describes multiclass MRMs, their capabilities, and their limitations. Three MRMs are described in detail for nonfatty foods as well as fatty foods. Methods 302 and 303 are for nonfatty foods, and method 304 is for fatty foods. Each method is composed of various modules, such as for extraction, cleanup, identification, and determination. The extraction, cleanup, and detection modules are selected based on the nature of the sample. For example, for method 302 for nonfatty foods, there are 7 extraction modules, 6 cleanup modules, and 19 determinative step modules. Thus, method 302 is not a single method but a combination of various analyrical methods. The validation references for each module are also provided. The methods described in PAM are all validated methods. The extraction modules are described as El, E2, E3, etc.; the cleanup modules are described as C1, C2, etc.; and those for the determinative step are described with a prefix D. The complete description of the determinative step
CHAPTER 4 7 9 Analysis of OPs and CMs
is DG or DL followed by a number. The DG modules refer to GC methods and DLs refer to HPLC methods. It should be noted that the extraction module E1 and cleanup module C1 of method 302 are not same as described for method 303 or any other method. Thus, in order to define a method of analysis, the method number, such as 302, 303, or 304, as well as the extraction module and cleanup module should be specified. The determinative step modules, such as DG1, DG2, or DL, are independent of method number and can be used with sample extract prepared by method 302, 303, or 304. Chapter 4 of PAM discusses methods 401-404. Method 401 is for N-methylCMs, method 402 is for acids and phe-
Nonfatty, high-moisture (>75%) commodities
Fruits and vegetables, moisture >75 %, sugar <5%, fat <2%
Dried egg whites, grains, and other foods with low moisture (<75%); fat < 2%. Fruits and other foods with 5-15% sugar. Fruits and other foods with > 15% sugar
nols, method 403 is for phenylurea herbicides, and method 404 is for benzimidazoles. Again, each method contains various extractions and cleanup step modules. Chapters 3 and 4 also contain the table for recovery data and notes for special situations. For example, the recovery of acephate is complete (>80%) using 302, E l - E 3 extractions, and GC/FPD. For the determinative step, it is recommended that wide-bore capillary DEGS column be used instead of packed column; on the other hand, for azinophos-methyl, although recovery is complete, DEGS column is unsuitable. The applicable methods for various modules applicable to OPs and CMs are shown in Table 1.
Multi Residue Methods for OPs and CMs a Extraction module b
El. Extraction with acetone, liquid-liquid partitioning with petroleum ether/ methylene chloride E5. Extraction with acetone, liquid-liquid partitioning with acetone/methylene chloride; alternative to E1 for relatively polar residues E4. Extraction with water/ acetone, liquid-liquid partitioning with petroleum ether/methylene chloride E6. Extraction with acetone, liquid-liquid partitioning with acetone/methylene chloride; alternative to E4 for relatively polar residues El. Extraction with acetonitrile, partition into petroleum ether with high moisture E2. Extraction with acetonitrile, partition into petroleum ether E3. Extraction with water/ acetonitrile, partition into petroleum ether E4. Extraction with acetonitrile and water, partition into petroleum ether Extraction with heated acetonitrile and water, acetonitrile and water, partition into petroleum ether
Cleanup [email protected]
Determinative module c
DG2/14, DG3/DG 16, DG 4/5/17 DG15 DG12 Residues detectable with element selective detectors
C3. Charcoal/silanized celite column cleanup C4. C- 18 cartridge cleanup
DL1 for N-methyl CMs
C1. Florisil column cleanup, with three ethyl ether/petroleum ether eluants; applicable to all extraction modules
DG2/DG14 for residues with phosphorus; DG4/5/17 for residues with nitrogen
C4. Florisil columns cleanup, with three methylene chloride eluants; alternative to C3; some additional residues are recovered
SECTION Vlll 9 Analytical & Biomarkers
TABLE 1. Method
Fatty foods, animal tissue, fatty fish
El. Extraction of fat with sodium sulfate, petroleum ether
Butter, oils, cheese, milk, egg yolks, dried egg whites, oil seeds, high-fat feed, nuts
E3. Extraction of fat by filtering E4. Extraction of fat with solvents from denatured product E5. Extraction of fat with solvents from feed materials, grains, nuts
Fatty, nonfatty, and variable moisture
E 1. Extraction with methanol E2. Extraction with methanol, reduced sample size for low products
C 1. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with three mixed ether eluants C2. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with three methylene chloride eluants C3. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with petroleum ether and three mixed eluants C4. Acetonitrilepetroleum ether partitioning, Florisil column cleanup, petroleum ether and three methylene chloride eluants C5. Gel permeation chromatography m the preferred technique due to automation C 1. Two-stage liquidliquid partitioning, and charcoal/Celite column cleanup
Determinative modulec DG12/14 for residues with phosphorus
DG4/5/17 for residues with nitrogen
DL 1 for N-methyl CMS; method includes postcolumn hydrolysis and derevatization
aFrom FDA (1994). bExtraction and cleanup modules are dependent on method number, such as 302 and 303. CDeterminativemodules are independent of method number; a given determinativemodule is applicable to all methods. Chapters 5 and 6 of PAM discuss the techniques of GC and HPLC, respectively. The descriptions of GC and HPLC as well as the detectors used with each technique are explained in detail. These descriptions keep the residue analyst in mind and provide many helpful points for the analyst.
B. P A M , Vol. 2 (FDA, 1991) PAM, Vol. 2, contains methods designed for the analysis of commodities for residues of only a single compound (although some methods are capable of determining several related compounds). These methods are most often used when the likely residue is known and/or when the residue
of interest cannot be determined by common MRMs. An updated index for Vol. 2 is available online (http:// vm.c fs an. fda. gov/-- frf/pami 1.html).
C. Manual of Analytical Methods for the Analysis o f Pesticides in Humans and Environmental Samples (EPA, 1980) This publication contains methods for the determination of pesticides in humans and environmental samples. The manual describes the collection, preservation, and storage of samples, as well as cleaning of glassware and preparation, storage, and use of pesticide standards. Section 6 of the
C H A PT ER 4 7 9 Analysis of OPs and CMs manual describes methods for OPs and metabolites in tissues and excreta and metabolites in urine. The manual is somewhat outdated because all the GC methodology is based on packed column. The capillary and megabore column could be used after method validation.
D. NIOSH Manual of Analytical Methods
(NIOSH, 1994) The NIOSH Manual of Analytical Methods (NMAM) is a collection of methods for sampling and analysis of contaminants in workplace air and in the blood and urine of workers who are occupationally exposed. These methods have been developed or adapted by NIOSH or its partners and have been evaluated according to established experimental protocols and performance criteria. NMAM also includes chapters on quality assurance, sampling, portable instrumentation, etc. Methods 5600 and 5601 are applicable to OPs and CMs, respectively. In these methods, air is pulled through a sampler tube (OVS-2), which contains XAD-2 adsorbent, for a prescribed time at a given flow rate. The pesticides are trapped in the sampler tube. The pesticides are extracted from the sampler tube by elution with 2 ml of 90% toluene/10% acetone for OPs and by 2 ml extraction solution (0.2% V/V, 0.1 M aqueous triethylamine phosphate buffer in acetonitrile) for CMs. OPs are determined by GC/FPD, and CMs are determined by HPLC with detection at 200 and 225 nm. Method 5006 deals with the determination of carbaryl (which can also be determined by method 5601). Methods 5012 and 5514 describe the determination of the OPs demeton and EPN, respectively.
E. Test Methods for Evaluating Solid Wastes,
(EPA, 1994) This manual deals with the analysis of many contaminants, including OPs and CMs, in wastes. Method 8414 A is applicable to OPs and method 8318 covers CMs.
F. Manual of Pesticide Residue Analysis, Vol. 1
(DFG, 1987) Part 1 of this manual discusses the preparation of reagents and samples, micromethods and equipment for sample processing, and limits of detection determination. Part 2 deals with cleanup methods. Part 3 includes methods for single residue determination. It includes methods for OPs and CMs such as acephate, methamidophos, aldicarb, chlorthiophos, heptenophos, methomyl, pirimiphos-methyl, pyrazophos, tetrachlorvinphos, and triazophos. Part 4 describes MRMs for OCs and OPs (methods S 10 and S 11); OP insecticides (method S13); dithiocarbamates and thiram fungicides (method S15); OPs with thioester group (method S 16); and OPs, OCs, and nitrogen-containing pesticides.
G. Manual of Pesticide Residue Analysis, Vol. 2
(DGF, 1992) Part 1 contains mass spectrometric EI data for confirmation of results. Part 2 deals with the updated cleanup methods, including SPE with various types of sorbents. Part 3 describes some single residue methods (SRMs). The pertinent methods for OPs and CMs are method 261-378-370 for methomyl, carbendazim, and thiophenate-methyl; method 378 for carbendazim; method 658-344 for cabosulfan and carbofuran; method 522 for fonofos; method 405 for glyphosate; and method 441 for oxamyl. Part 4 deals with MRMs, and a pertinent method for OPs and CMs is method $8, an updated version of method S 19. Method $25 is for methyl CMs. Thinlayer chromatographic methods using the automated multiple development technique are described in part 6.
H. A World Compendium. The Pesticide Manual
(Tomlin, 1997) This publication is a reservoir of references for analytical methods for the determination of SRMs for pesticides and insecticides. This also includes references for the analysis of commercial pesticide products. Pesticides and insecticides used worldwide are listed in alphabetical order. Each entry in the manual deals with a single pesticide and lists the properties, types of formulations in which the product is used, and toxicological properties, as well as reference(s) for analysis.
I. Official Methods of Analysis (OMA) of AOAC International (AOAC, 2003a) This is a compilation of fully validated analytical methods. Every method in OMA has been tested for ruggedness and validated through a multilaboratory collaborative study (a minimum of 8 labs for quantitative methods and 10 labs for qualitative methods), undergone rigorous scrutiny by recognized experts, and met AOAC criteria. These methods are preferred by regulators and are cited in the U.S. Code of Federal Regulations, the Codex Alimentarius, and other regulatory codes throughout the world; they are also routinely accepted in compliance actions and in courts. Chapter 7 of OMA deals with pesticide formulation analysis, and Chapter 10 deals with residue analysis in various types of samples.
J. ATSDR Pesticide Profiles (ATSDR, 1994) ATSDR publishes toxicological profiles for hazardous substances. The toxicological profiles for the pesticides chlorpyrifos, chlorfenvinphos, diazinon, dichlorvos, disulfoton, ethion, malathion, and methyl parathion have been published. Each of these profiles contains a section on analytical methods for the pesticide and its metabolites. The references for various analytical methods are given.
S E CT I 0 N V I I I 9 A n a l y t i c a l & B i o m a r k e r s
Many of the analytical methods used for environmental samples are approved by federal agencies such as EPA and NIOSH. Other methods included are those approved by groups such as AOAC International and the American Public Health Association. Additionally, analytical methods that modify previously used methods to obtain lower detection limits and/or to improve accuracy and precision are referenced.
K. Residue Analytical Methods (EPA, 2003) This is a compilation of residue analytical methods for food, feed, and animal commodities to identify and quantify the pesticide residue of interest, determining the total toxic residue of the pesticide regulated by the tolerance (maximum legal residue level), including significant metabolites and breakdown products. EPA's laboratory has tested most of the methods in the RAM index. When some of these methods were tested, EPA's laboratory clarified certain sections of the methods to improve performance or remove ambiguity. In most of these cases, an addendum was added to the method to explain the necessary clarifications. A few of the methods in the RAM index have not been tested in the laboratory but have undergone extensive review regarding their suitability for collection of pesticide residue monitoring data and for tolerance enforcement. Although most of the methods perform satisfactorily, some may have deficiencies. These methods are very similar to those included in PAM, Vol. 2 (FDA, 1991).
L. Environmental Chemistry Methods (EPA, 2004) Environmental chemistry methods for soil and water are used to determine the fate of pesticides in the environment. The methods identify and quantify the pesticide residue of interest, determining the total concentration of pesticides, including the extractable parent compound and significant metabolites and breakdown products. Although the EPA reviews all analytical methods submitted in support of pesticide registration, only approximately 25% of the currently available environmental chemistry methods have been evaluated in EPA's laboratory. Most of the methods perform satisfactorily, but some have deficiencies, particularly some of the older methods. The sites for ECM as well as RAM are continually updated; thus, the date of reference will change with time.
M. Reviews Two reviews on pesticide residue analysis have been published (Sherma, 1999, 2001). These reviews cover various technological developments and methods published during 1997-1998 and 1999-2000, respectively. Sherma (2003) has also written a review on recent advances in the thinlayer chromatography of pesticides.
VIII. QuEChERS METHOD This method for multiclass multiresidue determination of pesticides was introduced by Anastassiades et al. (2002). QuEChERS stands for "quick, easy, cheap, effective, rugged, and safe." The method is applicable to a wide variety of sample types. The method involves the extraction of the sample with acetonitrile, and then MgSO4 and NaC1 are added to the sample and solvent mixture. The mixture is centrifuged and an aliquot of the upper layer is transferred to another tube, in which primary-secondary amine SPE sorbent and MgSO4 are added, mixed, and centrifuged. The upper layer is used for analysis by GC/FPD, GC/MS, GC/MS-MS, or HPLC (Lehotay et al., 2003). This method has also been applied to fatty foods, such as milk and eggs, and an interlaboratory study for the applicability of the method has been conducted (Lehotay and Mastovaska, 2004). The method has been evaluated by Schenck and Hobbs (2004), and it is also applicable to CMs.
IX. I M M U N O A S S A Y S FOR OPs A N D C M s Several immunoassay test kits are commercially available for the determination of OPs and CMs by the enzymelinked immunosorbent assay (ELISA) technique. A complete description of ELISA is outside the scope of this chapter. Briefly, these test kits are based on the use of antibodies, which bind both pesticide and a pesticide-enzyme conjugate for a limited number of antibody binding sites. Antibodies for pesticide are immobilized to the inside of a small plastic tube (well). Since there are the same number of antibody sites in the test well, and the same amount of pesticide-enzyme conjugate is added to each test well, a low concentration of pesticide in the test sample allows more antibody sites to bind with more pesticide--enzyme conjugate molecules. Thus, a low concentration of pesticide will produce a dark blue color on the addition of enzyme substrate. Conversely, a high concentration of pesticide will allow fewer pesticide-enzyme conjugate molecules to bind with antibody binding sites, resulting in a lighter blue color on the addition of the enzyme substrate. In a variation of the previously discussed scheme, the antibodies are attached to paramagnetic particles. The sample to be tested and the pesticide-enzyme conjugate are added to a disposable test tube followed by the antibody-attached paramagnetic particles. Both the pesticide and pesticide-enzyme molecules compete for the available antibody binding sites. At the end of the incubation period, a magnetic field is applied to the test tube to hold the magnetic particles. The contents of the test tube are decanted holding the magnetic particles. The magnetic particles in the test tube are washed, and a substrate and a chromogen are added to the tube. The color produced is inversely proportional to the concentration of pesticide in the sample.
CHAPTER 47 9 Analysis of OPs and CMs During the past 15 years, there has been a consolidation of vendors producing these test kits. Currently, there is only one vendor, Strategic Diagnostics. These kits are available for several OPs and CMs. Note that none of these kits have been validated to receive the PTM status from AOAC International.
X. OP AND CM SCREENS A qualitative colorimetric test based on the inhibition of AChE can screen the presence of OPs and CMs. The thioesters of OPs have to be activated by a dilute bromine solution. Commercial test kits are available for the colorimetric test from Strategic Diagnostic.
A. Pesticide Detector Ticket for OPs and CMs Detection ticket is based on the inhibition of AChE by OPs and CMs. The ticket consists of two parts, one with AChE immobilized on paper disk and the other part is a paper disk impregnated with a substrate. The package is opened, and the enzyme paper is soaked in an aqueous sample extract. Subsequently, the two parts are put together and held between a thumb and a finger (to provide heat) for 2 or 3 min. If the enzyme part of the ticket turns a blue color, CMs and some of the OPs are not present in the sample. OPs have to be treated with dilute bromine water to activate them for inhibition of ACHE. If the enzyme part of the ticket turns blue, then organothiophosphates are also not present in the sample. On the other hand, if the enzyme part of ticket remains white, then OPs and/or CMs are present in the sample. The pesticide detector ticket is marketed by Neogen. The samples can be extracted with organic solvent(s), and methods for different procedures are available from the company online (www.neogen.com). The ticket can also be used for the detection of nerve agents.
XI. M E T A B O L I T E A N A L Y S I S
phosphate (DMP), dimethyl thiophosphate (DMTP), and dimethyl dithiophosphate (DMDTP). Alkyl phosphates are excreted in urine as sodium or potassium salts. Alky phosphates are excreted rapidly. More than 80% of the total dose of parent pesticide is excreted as alkyl phosphate in urine within 48 hr (Aprea et al., 2002). Traditionally, alkyl phosphates have been analyzed by GC coupled with element selective detectors such as FPD and NPD. The first method for the determination of alkyl phosphates was described by St. John and Lisk (1968). This method used methylation of alkyl phosphates due to their polar nature. The alkali thermionic detector used in the method has been replaced with NPD. Other derivatizing agents, such as diazopentane, triazines, and pentafluobenzylbromide (PFBBr), have also been used. The use of PFBBr is advantageous since DMTP and DETP each produce a single product on derivatization with PFBBr, whereas other derivatizing agents produce two isomers for either DMTP or DETE Medlin (1996) introduced the concept of confirming DETE DEDTP, DMTP, and DMDTP by converting them to DEP and DMP, respectively, by reacting thio and/or dithio analogs with 1% bromine water. The conversion of DETP and DEDTP to DEE and that of DMTP and DMDTP to DMP, is complete when urine is buffered with K2CO 3. Figure 1 shows the complete conversion of
The analysis of OP and CM metabolites for monitoring pesticide exposure has been extensively discussed by Murray and Franklin (1992). Thus, in the following sections, a brief history of analytical methods for OP and CM metabolites is given, and advances in their analysis are discussed.
A. OPs Metabolites One of the advantages of OPs is that they do not persist very long in the environment; they degrade and/or metabolize rapidly. Nearly 70% of all OPs produce one or more of the six dialkyl phosphates (DAPs) on degradation or due to metabolism: diethyl phosphate (DEP), diethyl thiophosphate (DETP), diethyl dithiophosphate (DEDTP), dimethyl
FIG. 1. Chromatograms of rat urine (a) before and (b) after oxidative desulfuration. *Unidentified peaks.
SECTION VIII 9 A n a l y t i c a l & B i o m a r k e r s
DMTP and DETP to DMP and DEE respectively. These chromatograms are from urine samples for which the rats were dosed with a mixture of chlorpyrifos, parathion, and coumaphos. The reaction of organothiophosphates with a dilute solution of bromine is called "oxidative desulfuration." We also found that when reference samples for calibration curve are prepared by adding known amounts of the metabolites (DAPs) to the urine of nonexposed people or animals of the same species, the recovery of incurred metabolites improves drastically. In our experiments with rat urine, the recovery of various alkyl phosphates ranged from 85% for DMDTP to 112% for DEE Drevenkar et al. (1991) used the same approach for the preparation of reference samples for calibration. A thin-layer chromatography procedure for the determination of DETE DEDTP, DMTE and DEDTP has been described (Sherma et al., 1999). These compounds were separated on C-18 chemically bonded silica gel plates and detected by TCQ chromogenic reagent. This method in conjunction with GC/FPD method could be used for further confirmation of these compounds. A method for the determination of DAPs in water has been described (Chang et al., 2000). This method uses a strong anion exchange disk to isolate DAPs from water and DAPs are derivatized with methyl iodide in acetonitrile. Capillary GC with FPD was used for determination. A residue analytical method for the determination of DEP
and DETP in fecal samples has been described (Schenke, 2000). The fecal samples were homogenized in water, and DEP and DETP were subsequently alkylated to pentafluorobenzyl esters by a phase transfer reaction. The determination was carried out with GC/MS (Schenke, 2000). An excellent review of the analytical methods for biological monitoring of pesticide exposure is presented by Aprea et al. (2002). This review provides information regarding sample preparation and analytical procedures. MS or MS-MS coupled with GC or HPLC has been used for the determination of DAPs in urine (Bravo et al., 2002; Hardt and Angerer, 2000; Hernandez et al., 2004; Oglobine et al., 2001). The use of GC/MS or GC/MS-MS requires alkylation of DAPs in order to render them volatile for GC separation. HPLC coupled with MS or MS-MS has the advantage that DAPs need not be alkylated. Furthermore, the use of labeled (isotopic) internal standards compensates for the losses in recovery and improves precision. However, the use of labeled internal standards is very expensive since these compounds have to be custom synthesized. B. S p e c i f i c M e t a b o l i t e s ~
When OPs degrade or metabolize, nonspecific (e.g., DAPs) as well as specific degradation products are formed. The specific metabolites that have been analyzed are listed in Table 2.
TABLE 2. Specific Metabolites m OPs Parent compound
3-Chloro-4-methyl-7hydroxycoumarin 2-Isopropyl-6-methylpyrimidin-4-ol 3-Methyl-4-nitrophenol 5-Chloro- 1,2-dihydro- 1isopropyl- [3H]- 1,2,4triazol-3-one 2-[(Dimethoxyphos phorothi oyl)sulfanyl] succinic acid Malathion monocarboxylic acid Malathion dicarboxylic acid O,S-dimethyl hydrogen phosphorthioate 4-Nitrophenol, also known as p-nitrophenol 2-Diethylamino-6-methyl pyrimidin-4-ol
Olsson et al. (2003) Olsson et al. (2003), Aprea et al. (1999) Olsson et al. (2003)
Olsson et al. (2003)
Ameno et al. (1995) Olsson et al. (2003)
Olsson et al. (2003)
Aprea et al. (2002)
DCA O,S-DMPT PNP
Aprea et al. (2002) Tomaszewska and Hebert (2003) Olsson et al. (2003), Aprea
Olsson et al. (2003)
Diazinon Fenitrothion Isazofos, methyl/ethyl
Methamidaphos/acephate Parathion-methyl/-ethyl Primiphos-methyl
et al. (2002)
CHAPTER 47 9 Analysis of OPs and CMs The methods of analysis for many of the specific metabolites have been reviewed by Aprea et al. (2002). Most of the specific metabolites are conjugate glucurornides or sulfates; thus, enzyme or acid hydrolysis is required to liberate the metabolites. Most of the methods for the analysis of specific metabolites use GC/ECD or GC/MS. The urine sample (after hydrolysis) is extracted with a solvent, such as ether or toluene. The filtration of urine is performed through a Sep-Pak C18 cartridge followed by extraction of the eluate with solvents. Several derivatizing reagents such as BSA [N, O-bis(trimethylsilyl)acetamide], 1-chloro-3-iodopentane, or MTBSTFA [N-(tertbutyldimethylsilyl)-N-methyl trifluoroacetamide] have been used to convert the metabolites into volatile compounds. Tomaszewaska and Hebert (2003) reported a method for the analysis of O,S-dimethyl hydrogen phosphorothioate (O,S-DMPT) in urine. O,S-DMPT is a specific metabolite of methamidophos. The urine sample was extracted with a C18 column, and the sample was lyophilized at low temperature to prevent loss of highly volatile and thermally unstable metabolite (O,S-DMPT). The lyophilized residue was derivatized using MTBSTFA and 1% tert-butyldimethylchlorosilane in acetonitrile. After filtration, the derivatized product was analyzed with GC/FPD (pulse FPD) in the phosphorus mode. The limit of detection for the method is reported as 0.004 ppm, with a mean recovery of 108%. A liquid chromatography/electrospray ionization-tandem mass spectrometry for the analysis of specific metabolites, such as BTA, TCPY, CHMC, IMPY, CIT, MDA, PNE and DEAMPY, has been reported (Olsson et al., 2003). The urine sample (2 ml) was hydrolyzed with glucurornides and loaded on an SPE cartridge (Oasis, HLB, or Waters). The cartridge was washed with 0.8 ml of 5% MeOH in 1% acetic acid and then eluted with 2 ml of MeOH. The wash and eluate were collected in separate tubes. The wash fraction was further cleaned by applying it tO a Chem Elute cartridge and eluted with 14 ml of CHC13. It was evaporated to dryness and reconstituted in 100 lxl of 1% acetic acid. The SPE cartridge eluate was also evaporated to dryness and reconstituted in 100 txl of 1% acetic acid. The reconstituted samples were analyzed by LC/MS-MS. The data for the analysis of acephate and methamidophos were also provided. Acephate and methamidophos are two OPs that do not produce DAPs. Acephate produces methamidophos on degradation; thus, it is considered a metabolite of acephate. Olsson et al. (2003) included both acephate and methamidophos in their analytical scheme for specific metabolites. Chang and Lin (1995) reported a HPLC method for the determination of 3-methyl-4 nitrophenol in urine of rats orally dosed with fenitrothion. Although some of these specific metabolites may be derived from several analogs of the same OP or from other non-OP sources, these are termed as specific metabolites. In combination with the DAPs, these metabolites can pro-
vide information regarding the parent compound. For example, p-nitrophenol may be derived from ethyl/methyl parathion or p-aminophenol. The DAP metabolites will be produced only by parathion. If DMP is found in the same urine sample, it clearly indicates exposure to methyl parathion. C. Specific M e t a b o l i t e s - - C M s
Most of the CMs are phenyl N-methyl esters of carbamic acid. On hydrolysis or metabolism, CMs produce phenols. Phenols are polar compounds and thus have to be alkylated before GC determination with either ECD or NPD. HPLC analysis combined with the appropriate detector appears to be the better choice. Aprea et al. (2002) reviewed various methods for unchanged CMs in blood and urine, as well methods for specific CM metabolites, such as benomyl metabolites carbendazim and methyl-5-hydroxy-2-benzaimidazolecarbamate; 1-napthol; 2-isopropoxyphenol (metabolite of propoxur); and carbofuran phenol, which is a metabolite of several pesticides (carbofuran, benfuracarb, carbosulfan, and furathiocarb).
XII. N E R V E A G E N T A N A L Y S I S Nerve agents such as tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and venom toxin (VX) are extremely toxic OP compounds. Although these agents have been controlled by international treatises, some of them have been used in terrorist attacks, such as the satin attacks in Japan. All these compounds are liquids with high vapor pressure except VX, which has a low vapor pressure and is thus more persistent in the environment. All these compounds are unstable in the environment and degrade to methyl phosphonate alkyl esters, which are stable (Elashvili, 2004). The methyl phosphonate alkyl esters further degrade over time, and in the presence of phosphonate ester hydrolase they degrade to methyl phosphonic acid. Different methyl phosphonic alkyl esters are produced based on the other substituents present in the original nerve agents. For example, satin gives isopropyl methyl phosphonic acid, whereas VX degradation results in ethyl methyl phosphonic acid. Since methyl phosphonic acid and alkyl esters, as well as the parent nerve agents, contain phosphorus, the most logical analytical procedure is GC/FPD-P (FPD in phosphorus mode). The same author used GC/FPD-P for the detection of nerve agents. The nerve agents, their intermediate degradation products (alkyl esters of nerve agents) and the final degradation product, methyl phosphonic acid, and their tri-methyl silylated products were determined with GC/FPD-E This publication provided the retention times of the parent, intermediate methyl phosphonic acid alkyl esters, and methyl phosphonic acid. However, the GC column specifications and other GC conditions were not
SECTION Vlll 9 Analytical & Biomarkers
specified. Harper (2002) described a method for nerve agents in air. The air is pulled through a bed of Hayesep D (known as the PCT tube) for a few minutes at 300-700 ml/min. After collection, the PCT tube is heated and the effluent is introduced onto a GC column coupled with FPD detector. The GC/MS-MS methods for the quantitative determination of nerve agents have been reported (Driskell et al., 2002; Barr et al., 2004). The difference between the two methods is that in the study by Driskell et al., the urine samples were concentrated by forming an azeotrope with acetonitrile, whereas in the study by Barr et al., the acidified urine samples were extracted into ether acetonitrile. The samples were derivatized by methylation with diazomethane and analyzed by GC/MS-MS. A microcolumn LC capillary electrophoresis (CE) with FPD for the screening of degradation products of chemical warfare agents, including nerve agents in water and soil, has been described (Hooijschuur et al., 2001). This study was a proficiency study to test the ability of participating laboratories to unambiguously identify chemical warfare agents and their degradation products. This study contains several references to the LC and CE method with FPD of various nerve agents. A method for the detection of nerve agent metabolites based on GC coupled with an atomic emission detector (AED) has been reported (Creasy et al., 1995). The nerve agent degradation products were extracted from spiked water, wipes, and soil samples. The extracted samples were derivatized with 1% trimethylchlorosilane in bis-(trimethylsilyl) trifluoroacetamide. The GC/AED technique was used for separation, detection, and determination of OP nerve agent metabolites. A miniaturized analytical system for separating and detecting toxic nerve agent compounds based on the coupling of a micromachined capillary electrophoresis chip with thick-film amperometric detection has been described (Wang et al., 2001). "Lab on a chip" technology is utilized in the measurement of CWA degradation products using an electrophoresis microchip with a contactless conductivity detector (Wang et al., 2002a). Wang et al. (2002b) also described a single-channel microchip for fast screening and detailed identification of nitroaromatic explosives or OP nerve agents.
A. Retrospective Detection of Nerve Agents Nerve agents strongly bind to acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE); however, the measurement of either AChE or BuChE does not identify the nerve agent or any other compound that may inhibit any of these esterases. It has been shown that both AChE and BuChE inhibited by sarin can be reactivated by high concentrations of fluoride ions (Polhuijs et al., 1997). Van der Schans et al. (2004) reported a method for the retrospective detection of nerve agents by reactivating BuChE in human plasma. The
plasma samples were treated with KF and passed through a Sep-Pak C18 cartridge. The generated phosphonofluoridates were isolated and analyzed by GC/NPD. They also used GC/MS to confirm the nerve agent(s). Besides the laboratory procedures, many field detectors are available for the detection of nerve agents as well as other CWAs.
B. Detection Paper The detection paper is based on certain dyes being soluble in CW agents. Normally, two dyes and one pH indicator are used, which are mixed with cellulose fibers in an unbleached paper. When a drop of CW agent is absorbed by the paper, it dissolves one of the pigments. Mustard agent dissolves a red dye, and nerve agent dissolves a yellow dye. In addition, VX causes the indicator to turn blue, which together with yellow will become green/green black.
C. Detection Tubes Detection tubes, such as civil defense kit, contain special reagents impregnated on inert supports. Air is sucked through the tube with a special pump. Reaction between the reagents in the tube and CWA takes place, and different colors are produced based on the CWA.
D. Detection Ticket This was discussed in Section X,A.
E. Other Detectors Hill and Martin (2002) presented a review of conventional analytical methods for CWAs. They discussed various sensors, such as surface acoustic wave sensors, electrochemical sensors, spectrophotometric sensors, immunochemical sensors, and IMS detector. For OP nerve agents, FPD and MS are the detectors of choice when coupled with GC or LC. Miniature ion trap mass spectrometer has been described for the detection of nerve agents in the field (Patterson et al., 2002; Riter et al., 2002). A book published by the Institute of Medicine and the National Research Council explains the use of various types of detectors for nerve agents as well as CWAs (IOM, 1999).
XIII. ANALYSIS OF AChE INHIBITOR
THERAPEUTIC AGENTS Some AChE inhibitors, such as donepezil, rivastigmine, galantamine, tacrine, eptastigmine, neostigmine, pyridostigmine, and ambenonium, are used as therapeutic agents. Table 3 shows the compound analyzed, methods Used, and reference for the method.
C H APT E R 4 7 9 Analysis of OPs and CMs
TABLE 3. Methods for AChE Inhibitor Therapeutic Agents
HPLC HPLC HPLC/MS-MS
HPLC Capillary electrophoresis HPLC GC HPLC
GC/MS HPLC/MS HPLC/MS-MS HPLC Capillary electrophoresis
XIV. M E T H O D V A L I D A T I O N There are several types of validation for analytical methods. AOAC is the leading organization that conducts methods validation under its auspices. AOAC conducts the following types o f method validation: performance tested method (PTM), peer verified method (PVM), and official methods of analysis (OMA). AOAC OMAs are referenced in the U.S. Code of Federal Register and are used worldwide by regulated industry, product testing laboratories, and academic institutions. AOAC (formed in 1884 as the Association of Official Agricultural Chemists) is an independent association devoted to promoting methods validation and quality measurements in analytical sciences. It does so by reviewing and validating approved standards methods of analysis, promoting uniformity and reliability in statements of results, and developing and promoting criteria useful for laboratory accreditation and analyst certification.
A. Performance-Tested Methods The AOAC Research Institute maintains a frequently updated list of PTMs. PTMs have been independently tested, rigorously evaluated, and thoroughly reviewed by the AOAC Research Institute and its expert reviewers. None of
Yamamoto et al. (1998) Yasui-Furukori et al. (2002), Lu et al. (2004), Matsui et al. (1999) Zecca et al. (1993), Unni and Becker (1992), Herold et al. (1992) Claessens et al. (1983, 1998) Pokorna et al. (1999) Tencheva et al. (1987) Chan et al. (1980) Ellin et al. (1982), Chan and Dehghan (1978) Abu-Qare and Abou-Donia (2001a, g) Chan et al. (1980), Chan and Dehghan (1978) Sha et al. (2004) Enz et al. (2004) Pommeir and Frigola (2003) Hansen et al. (1998) Vargas et al. (1998)
the pesticide detecting kits have PTM status. The reason appears to be that the kit manufacturer(s) has not yet sought this status.
B. Peer-Verified Programs The AOAC PVM program is intended to provide a class of tested methods that have not been the subject of a full collaborative study. Through a less intensive process, the program provides a rapid entry point for methods that are recognized by the AOAC at a level of validation for methods not otherwise evaluated. The distinguishing aspect of an AOAC PVM is that its performance has been checked in at least one other independent laboratory. It is expected that eventually most PVMs will undergo full interlaboratory collaborative studies and obtain OMA status.
C. Single Laboratory Validation In order for a method to be validated as PTM, PVM, and, finally, OMA, it must be validated in a single laboratory. In international trade and regulatory affairs, only validated methods are acceptable. The method need not be validated under the auspices of AOAC, but evidence is required that the method is suitable for the intended purpose and that similar results are expected from other competent laboratories
SECTION VIII 9 Analytical & Biomarkers
and analysts. In order to gather this information, single laboratory validation is essential. In the pesticide arena, new pesticides are introduced regularly. In the United States, the registrant of a pesticide is required to submit a method for the analysis of the active ingredient as well as its residues on the intended commodity. Often, a situation arises in which a given pesticide is used on another commodity, or it may have been used in the environment illegally. The question then arises whether the original method is fit for a new matrix(s). The other scenario is that an old pesticide no longer in use or withdrawn from use is to be analyzed. It may be that the instrumentation and reagents are no longer available, instruments must be modified, and other unanticipated problems may require the method to return to the development phase. Frequently, a method works satisfactorily in one laboratory but fails to operate in another laboratory in the same manner. Validation is a process and not a result. AOAC (2003a) defines validation as the process of demonstrating or confirming the performance characteristics of a method of analysis, and the performance characteristics of a method of analysis are the functional qualities and the statistical measures of the degree of reliability exhibited by the method under specified operating conditions. The performance characteristics are specificity, the ability to distinguish the analyte from other substances, applicability (the matrices and the concentration range), and reliability. Reliability is the most important characteristic of an analytical method and is expressed in terms of percentage recovery, repeatability, and reproducibility. Repeatability and reproducibility are expressed in terms of relative standard deviation within laboratory (RSDr) and between laboratories (RSDR), respectively. Why worry about between-laboratory relative standard deviation (RSDR) when the method is validated only in a single laboratory? It is true that RSDR cannot be determined in a single laboratory; however, it can be predicted based on the concentration of the analyte (Horwitz, 1982; Horwitz et al., 1980). Based on the data from 100 years' worth of interlaboratory method validation studies conducted under the auspices of AOAC, Horwitz determined that relative standard deviation between laboratories and within laboratory is dependent on the analyte concentration. The relationship is expressed by the equation PRSDR % = 2C -~ where C is expressed as the mass fraction. This equation can be expressed in spread sheet notation as PRSDR % = 2 * C^(-0.15). For example, a concentration of 1 ppm is expressed as 1.000E-6 (1 p,g/g). Thus, for an analyte concentration of 1 ppm, PRSDR % = 2 * (1.00E-07) ^ ( - 0 . 1 5 ) = 16%
Another observation of Horwitz, confirmed by Thompson and Lowthian (1997), is that the precision of analytical methods at any given concentration does not improve with time, despite advances in analytical technology.
D. H O R R A T Value The concept of HORRAT values was introduced by Horwitz et al. (1989). The HORRAT value is the ratio of RSDR obtained from the actual experimental data from interlaboratory data to the PRSDR calculated from the Horwitz formula: HORRATR = RSD R / PRSD R The acceptable values for a good interlaboratory validated method are 0.5-2. Similarly, for intralaboratory work, HORRATr = RSDr/PRSDR Horwitz also noted that within-laboratory variation was in general one-half to two-thirds of the variation between laboratories. Thus, the acceptable values for HORRATr are 0.3-1.3 (1/2 of 1/2 = 0.25 rounded to 0.3; 2/3 of 2 = 1.3). Note that the equation for HORRATr differs from the equation given in the AOAC guidelines (AOAC, 2003a). The error stems from the formula used to calculate these values in the AOAC guidelines. HORRATr values should always be calculated using the formula HORRATr = RSDr/PRSDR, rather than the formula HORRATr = RSDr /PRSDr, since RSDr is more variable than RSDR. (W. Horwitz, 2004, personal communication). This relationship is widely used to predict a reasonable relative standard deviation for a given analyte concentration within a laboratory. AOAC (2003a) guidelines for single-laboratory validation of chemical methods also provide guidelines for acceptable recovery values, as shown in Table 4. Thus, armed with these guidelines it is possible to discern whether the experimental recovery rate, as well as the precision of the method, is within acceptable boundaries. TABLE 4.
Acceptable Recovery Values for Various Analyte Concentrations
Concentration 100% 10% 1% 0.1% 0.01% 0.001% (10 ppm) 0.0001% (1 ppm) 1 ppb
Recovery limits (%) 98-101 95-102 92-105 90-108 85-110 80-115 75-120 70-125
CHAPTER 47 9 Analysis of OPs and CMs
XV. C O N C L U S I O N S A mosquito was heard to complain That a chemist has poisoned his brain The cause of his sorrow Was para-Dichloro-Diphenyl-Trichloroethane (DDT). mAuthor unknown (appeared in "lighter elements" in Today's Chemist at Work) Chemists are always developing new pesticides, and analytical chemists are busy developing methods for the analysis of pesticides. This will continue in the future. With the technological advancements in gas and liquid chromatography as well as detection devices, faster and more reliable analytical methods will be possible. With advancements in the technology of mass spectrometry and data handling units, which are coupled with libraries (NIST, Wiley, TOX, etc.) of a large number of chemical compounds (mass spectral data), it has become easier to identify and confirm pesticides as well as other compounds. Miniature mass spectrometers are being developed and, in time, their size and price shall shrink. The future of analytical methods for OPs and CMs is bright. Analytical methods for the analysis of new specific metabolites of OPs will be highly useful for the retrospective evaluation of OP exposure. Because CMs are the phenyl N-methyl esters of carbamic, they produce mainly phenols as the metabolites. Although phenols are ubiquitous in the environment, a multiphenol method that possibly covers all CM metabolites will be useful. The history of the person or the animal may give an indication if the person or animal was exposed to phenols or CMs. The development of M R M methods for both specific and nonspecific DAPs would considerably aid in the diagnosis of OP and CM exposure.
Acknowledgments My sincere thanks to my wife, Gagan, whose encouragement made this chapter possible. I also thank Anu, Shephali, Ritu, and Amit for helping me with computer problems and collating the references.
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