Chapter 10
Approaches to the Synthesis of Haptens for Immunoassay of Organophosphate and Synthetic Pyrethroid Insecticides 1
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John H. Skerritt and Nanju 1
1,2
Lee
CSIRO, Division of Plant Industry Canberra, Austrailian Capital Territory 2601, Australia Department of Agricultural Chemistry and Soil Science, University of Sydney, Sydney, New South Wales 2006, Australia 2
The major groups of insecticides that have been used in agriculture over the last two decades are the organophosphorus insecticides and synthetic pyrethroids. Immunoassays provide a simple means for analysis of large numbers of samples for selected target compounds. The design and development of haptens for immunoassays of the target compounds together with the performance characteristics of the resulting assays are reviewed.
Although several papers in the initial years of the evolution of agrochemical immunoassay concerned the development of antisera to insecticides such as parathion, malathion and D D T (7), more recently, the application of immunoassays to insecticides has lagged somewhat behind that of herbicides. One factor has been the greater difficulty of analysis of food matrices that are the most important with insecticides, compared with the water matrices that are typically the targets of many herbicide assays. However, it is possible that the greater chemical lability of many insecticide groups, compared with that of other agrochemicals has been a major factor, too. The move away from organochlorine insecticides since the 1960s has led to greater use of organophosphates and synthetic pyrethroids, because of the lower persistence of many of the parent compounds and their metabolites in soil, water and food samples (2). The increased lability of the newer pesticides, which is "designed into" the molecules, brings with it instability in either acid and/or base, and has presented a number of challenges in hapten design. In this review, the focus will be on comparing the chemical routes used to develop the haptens (full synthetic schemes and analyses are usually provided in the original citations) and the properties of the antibodies and assays obtained, namely detection limits, specificity and sensitivity to small increments in target pesticide concentration. 0097-6156/96/0621-0124$16.50/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
10. SKERRITT & LEE
Organophosphate & Synthetic Pyrethroid Insecticides
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Organophosphate Haptens The organophosphorus insecticides have a wide spectrum of insecticidal activities and physico-chemical properties, such as solubilities and vapor pressures, and since the 1970's have become the major insecticides used in horticultural crops and on stored commodities such as cereals. There also are several other uses, such as aerial appli cation in cotton and rice cultures, domestic and industrial use as termiticides, and in animal health applications. They can be divided into six main chemical groups, depending on the number and type of appendages on oxygen and sulfur atoms and the leaving group around the phosphorus atom (2). The four main variants range from having four oxygen atoms bonded to the phosphorus atom (orthophosphates); three oxygen atoms with a P-S double bond (phosphorothionates); three oxygen atoms, with the S in the bond between Ρ and the leaving group (phosphorothiolates); and phosphorothiolothionates which have sulfur in both the P-S double bond and in the bond between the Ρ atom and the leaving group. The two minor groups are phosphonates and pyrophosphoramides. The two most important classes in agriculture are the phosphorothionates (or thionphosphates, including parathion, chlorpyrifos, diazinon, fenitrothion and pirimiphos) and the phosphorothiolothionates (also known as dithiophosphates and which include malathion, azinphos-methyl and dimethoate). These two groups have also been the subject of the most intensive development of immunoassays. The structures of some key organophosphorus insecticides are shown in Figure 1. Immunoassays also have been developed for some other cholinesterase-inhibiting compounds, including carbamate insecticides such as carbaryl (3,4), aldicarb (5,6) and methomyl (7), and the potential chemical warfare agent, soman (8,9). Soman (methylphosphonofluoridic acid, 1,2,2-trimethylpropyl ester) does not fit into one of the six classes shown above as it has only two P - 0 bonds, with the Ρ atom also being bonded to a methyl group and a fluorine atom. Phosphorothionates. Although other organophosphates (with the exception of limited work on malathion) were ignored, a substantial proportion of the earlier pesticide immunoassay papers concerned themselves with development of haptens for the analysis of parathion and its active metabolite, paraoxon (Figure 2). All of the initial studies utilized derivatives of the diethyl ester (parathion), although we have more recently developed a pair of haptens based on parathion and methyl parathion using similar chemical routes and points of coupling. While parathion is primarily used in broadacre agriculture and is the target of analysis because of high human toxicity (10), methyl-parathion is much less toxic and is applied directly to stored products as a protectant treatment in countries such as India. Parathion Haptens Coupled Through the Aromatic Nitro-Group. The simplest approach to the development of pesticide antibodies is to derivatize the pesticide itself to create a means of coupling it to protein. With aromatic nitro compounds such as parathion or fenitrothion, this can involve reduction to a substi tuted aniline and either diazotization (Figure 2 (7)), or coupling through a spacer arm attached to the aromatic amine via amide bond formation (Figure 2 (2)). The initial
In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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IMMUNOASSAYS FOR RESIDUE ANALYSIS
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Downloaded by UNIV OF PITTSBURGH on September 8, 2013 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch010
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