A review on sample preparation and chromatographic determination of acephate and methamidophos in different samples

1 Introduction

Acephate (O,S-Dimethyl acetylphosphoramidothioate) (Table. 1) is an organophosphate insecticide, introduced by Chevron Chemical in 1971 (Magee, 1973) and first time registered for use by the United States Environmental Protection Agency in 1973. It has been observed that the production of acephate increased quickly from last 5 years, surprisingly in India, 10% increase in production of technical grade was observed within one year (Standing Committee on Chemicals & Fertilizers, 2012-13).

Table 1 Chemical structure and physicochemical properties of acephate and methamidophos (Roberts & Hutson, 1999; Tomlin, 2006).

Name	Chemical structure	Molecular weight (g/mol)	Solubility g/L	Log KOW	Henry’s law constant (atm m3/mol)
Acephate		183.16	790 in water at 25 °C	0.13 at 25 °C	5.1 × 10−13 at 25 °C
Methamidophos		141.13	200 in water at 25 °C	−0.65 at 25 °C	8.64 × 10−10 at 25 °C

It is an insecticide registered for use on food crops, agricultural seed and non-bearing plants, institutions and commercial buildings including public health facilities, sod, golf course turf, ant mounds, and horticultural nursery plants.

In soil, plants and insects approximate half-life period of acephate is 3–6 days, although in some soils the half-life may be increased to more than 13–60 days due to variation of properties (physical, chemical and biological) of soils (Antonious and Snyder, 1994; Bouchard and Lavy, 1982; Chuanjiang et al., 2010; Yen et al., 2000). Acidic nature of soil is responsible for long life spam of acephate in soils ((Antonious and Snyder, 1994; Bouchard and Lavy, 1982; Chuanjiang et al., 2010). It has been observed that, after the decomposition acephate generally converts into highly toxic methamidophos (O,S-dimethyl phosphoramidothioate) (Table 1) and methamidophos is also an efficient organophosphorus insecticide (Yen et al., 2000; Chuckwudebe, 1984). Methamidophos is the major metabolite of acephate. It is toxic, not only for insects but also in various components of the environment such as nontarget animals, plants, waters, and soils (Wang et al., 2013) (see Tables 2–4).

Physicochemical properties of acephate and methamidophos are signifying that both are hydrophilic and having low soil sorption with an order acephate > methamidophos (Roberts & Hutson, 1999; Tomlin, 2006). Consequently, their runoff through water medium can produce potential water contamination (Roberts & Hutson, 1999; Tomlin, 2006). Acephate leads to contamination of groundwater much more readily than methamidophos under normal environmental conditions. Studies highlight that wet soils are more conducive for the contamination of the aquatic environment with acephate and its metabolites (Chai et al., 2009; Wang et al., 2013).

2 Mode of action and toxicity

Acephate and methamidophos inhibit acetylcholinesterase enzyme (AChE) in nervous system tissues, although, acephate itself is a weak acetylcholinesterase inhibitor as compared to its decomposed product methamidophos (Wilson et al., 1990; Spassova et al., 2000). The toxicity of acephate and methamidophos varies with application of enantiomeric compound of them (Wang et al., 2013). Both acephate and its metabolite methamidophos have the stereogenic phosphorus atom. The enantioselective bioactivity of enantiomers of acephate and methamidophos has been observed during past years. The R-(+)-enantiomers of acephate and methamidophos were found to be more potent to houseflies than the optical antipodes and racemates, whereas the S-(−)-enantiomers were more toxic for German cockroaches soils (Miyazaki et al., 1988; Wang et al., 2006, 2013). Another experiment showed that (−)-methamidophos was about 8.0−12.4 times more potent to the bovine erythrocytes and Electrophorus electricus than its (+)-form, but the (+)-enantiomer was 7.0 times more toxic to D. magna in 48 h tests (Lin et al., 2006). It was also concluded that D-(+)- methamidophos should be responsible for the organophosphate-induced delayed poly-neuropathy when racemic methamidophos is given to hens (Lotti et al., 1995).

Acephate and methamidophos are extremely toxic to aquatic invertebrates, birds and mammals (Tomlin, 2006; Vyas et al., 1996; Mahajna et al., 1997). The most likely route of exposure to acephate and methamidophos for the public is via residues in food (WHO, 2003, 2005). The prolonged or continued use of acephate and methamidophos in plant protection may lead to significant dermal exposure with an impact on cholinesterase, genotoxicity and cardiotoxicity activities (Farag et al., 2000;Spassova et al., 2000; Padungtod et al., 1998).

Immense toxicity of acephate and methamidophos has been observed in environment including birds (Zinkl et al., 1981; Vyas et al., 1996), animals (Singh and Drewes, 1987), fishes (Szeto et al., 1979), soils and its microorganisms (Wu et al., 2010; Lo, 2010; Battu et al., 2009). Studies have revealed that acephate and methamidophos can persist on soils (Zhang et al., 2005; Battu et al., 2009), fruits and vegetables (Antonious and Snyder, 1994; Bouchard and Lavy, 1982; Chuanjiang et al., 2010; Zhang et al., 2008), in dietary products (WHO, 2003, 2005; Nougadère et al., 2012), cereal and other cash crops (Hiemstra & Kok, 2007; Antonious and Snyder, 1994; Bouchard and Lavy, 1982; Chuanjiang et al., 2010). Both, acephate and methamidophos having high leaching capacity, contaminate agriculture soils by leaching behavior (Chai et al., 2009, 2010). Studies have revealed that the concentrations of acephate and methamidophos in the rat tissues vary from 0.2 to 1.1 ppm. The highest concentrations were found in kidney (4.1–12 ppm), testes (2.4–3.9 ppm) and brain (2.1–2.5 ppm). There was no tendency for methamidophos to accumulate in blood, liver, muscle, fat or heart (Farag et al., 2000; Spassova et al., 2000; Padungtod et al., 1998; WHO, 2003, 2005). But, acephate and methamidophos are found highly toxic to bees and other beneficial insects. The LD₅₀ for acephate was 1.2 μg/honeybee. The LD₅₀ of methamidophos was 1.37 μg/honeybee (EPA, 2006). Exposure of acephate and methamidophos was checked on farmers by Maroni et al. (1990), acephate was detected in urine, red blood cell (RBC) and milk, but methamidophos was in minute amount. Daily maximum acephate urine concentrations range was found from 3 to 9 mg/L. Urinary concentration of acephate was non-detectable after 48 h of the exposure (Maroni et al., 1990).

3 Detection of acephate and methamidophos

Based on physicochemical properties of acephate and methamidophos, most of the researchers decided to use chromatographic technique for the detection of acephate and methamidophos. Because of low molecular mass, gas chromatography (GC) coupled with different detectors has been used for analysis. Gas chromatography requires less pretreatment as well as cleanup methods and shows low detection limit, but at the same time needs derivatization of acephate/methamidophos. Because the high polarity reverse phase liquid chromatography (RPLC) is a method of choice for the detection of acephate and monocrotophos, it has merit of high degree of selectivity and sensitivity. But, the method requires extensive pretreatment and cleanup method(s). In addition, sometimes methods are pH dependent and require use of buffer to inhibit any change in pH. In this review, we propose analytical procedures and materials used in the determination of the acephate and methamidophos present in different sorbing media such as vegetables, human and animal tissues, blood, serum, soils, etc. Before going in detail about any method of detection, let us first discuss about some of the pretreatment and cleanup methods.

4 Pretreatment procedure

Real-world sample for analysis is always complex. In order to get best result with low interference of unwanted substances, pretreatment of the sample is the prime requirement. Because of their high solubility in water, they (acephate and methamidophos) can reach to plant as well as non-target species through absorption as well as adsorption processes. In order to remove acephate and its degraded product from the complex matrix various researchers have established different pretreatment methods.

In soil, acephate and its metabolites adsorb over silica or alumina. For this purpose, the soil samples are collected in the field, are air-dried, grounded and sieved through a mesh with a grain size of 2 mm. This follows mostly liquid–solid extraction technique (prominently Soxhlet based technique) (Araoud et al., 2010; Montesano et al., 2007; Prasad et al., 2013; Schindler et al., 2009). Because of the polarity factor water or methanol is used as an extractant for acephate, although in the more complex soil media other solvents are also used such as, ethyl acetate, hexane, buffered water and cyclohexane–acetone in ratio (1:1).

After pretreatment different cleanup methods have employed to avoid interference with other organic motifs. Depending on types of interference, different groups incorporated different sorbents for isolation of organic compounds from the extracted solutions, including alumina, Florisil, ion-exchange resins, silica gel/silica-based sorbents (e.g., octadecyl-, octyl-, phenyl-, and diol-bonded silica) and graphitized black carbon (Araoud et al., 2010; Montesano et al., 2007; Schindler et al., 2009). Whole procedure of extraction, cleanup and sample loading is summarized in Fig. 1.

Close

Figure 1

Flow steps of extraction, cleanup and sample loading of organophosphate pesticides including acephate and its metabolites with LC/GC–MS column. (Here, RT is room temperature; LC is liquid chromatography; GC is gas chromatography; and MS is mass).

Export to PPT

4.3

4.3 Determination of acephate and methamidophos in vegetables

The observed residues of acephate and methamidophos have found tremendous increase in their residues in year 2008 than year 2006, as per the study that was done on Applesauce, Bananas, Carrots, Cranberries, Eggplant, Grape, Green Collard, Greens Kale, Orange Juice, Peaches, Plums, Potatoes, Raisins, Spinach, Summer Squash, Sweet Peas, Frozen, Watermelon and Winter Squash. Comparative study was done by using chromatographic methods namely liquid or gas chromatography–mass spectrometry, when 8515 samples were analyzed in year 2006 and 8686 samples in year 2008. Out of all these samples, acephate was detected in 1.11% samples in year 2006, while 5.65% in year 2008. On the other hand, methamidophos was found in 1.02% samples of year 2006, which increased to 4.89% in year 2008. Acephate and methamidophos were analyzed on spinach, summer squash, nectarines, blueberries, collard greens, strawberries, and tomatoes (PDPAS, 2006) (Fig. 2). A straightforward extraction method based on liquid chromatography–mass spectrometry (LC–MS) was reported; in which ethyl acetate was used as extractant and 0.1% acetic acid/water mixture was used as mobile phase without undergoing any cleanup procedure. The method was validated at the 0.01 and 0.5 mg/kg level, for both cabbage and grapes. Recoveries were reported in between 80% and 101% with R.S.D. <11% (n = 5). The limit of quantification was 0.01 mg/kg and limit of detection was in between 0.001 and 0.004 mg/kg (Mol et al., 2003).

Close

Figure 2

Study of acephate and methamidophos in food Grains (PDPAS) during 2008 and 2006.

Export to PPT

Another liquid chromatography-tandem quadrupole mass spectrometry multi-residue method for the simultaneous target analysis of a wide range of pesticides and metabolites in fruit, vegetables and cereals (lettuce, orange, apple, cabbage, grape and wheat flour, etc.) was reported by using acetone/dichloromethane/light petroleum as extraction solvents. As per report recoveries ranged from 70% to 110%, with relative standard deviations (RSD) better than 15%, were obtained (Hiemstra and Kok, 2007). The observed values of recoveries and RSD for acephate and methamidophos in lettuce, orange, apple, cabbage, grape and wheat flour matrix are tabulated below at fortification level 0.01 mg/kg (Hiemstra and Kok, 2007).

A gas chromatography (by using a combination of positive chemical ionization and electron impact ionization modes) tandem mass spectrometry method was employed for the analysis of organophosphates (including acephate and its metabolites) in vegetables, the limits of detection obtained in a range of 0.07 to 4.21 μg/kg. Average recoveries were in between 52% and 114% while the RSD values greater than or equal to 29% in all the cases were reported (Agüera et al, 2002). A simple, fast and inexpensive, gas chromatography–mass spectrometry method was introduced for the pesticide determination by single-phase extraction of sample (10 g) with acetonitrile (10 mL), followed by liquid–liquid partitioning after addition of 4 g anhydrous MgSO₄ + 1 g NaCl. Recoveries were obtained in between 85% and 101% (mostly > 95%) and repeatabilities typically < 5% were achieved for a wide range of fortified pesticides (Anastassiades et al., 2003). Another GC based method was developed for the sensitive determination of acephate and other organophosphate pesticides by using 5% acetone in hexane as extracting solvent and 1-chloro-4-fluorobenzene as an internal standard in fruits and vegetables. The limit of detection by the method was found in between 0.005 and 0.01 mg/kg and the limit of quantification was 0.01 mg/kg (Lal et al., 2008).

5 Conclusion

The future perspective of the study is to check the decomposition of both acephate and its metabolite methamidophos in the presence of different microorganisms, especially in plant growth promoting bacteria. There is only one published information available on microbial degradation of acephate by Pseudomonas sp. Ind01, which uses acephate as a source of carbon to support cell growth and can promote the first step of acephate mineralization in soil microbial communities. Also there is information available on the microbial degradation of methamidophos with species Hyphomicrobium species MAP-1. However, in spite of the significant enantioselective bioactivity of acephate and methamidophos, no present investigations on their environmental behavior have taken their chirality into account. It should be recognized that the enantiomers of acephate and its metabolite methamidophos are independent entities which exhibit significant enantioselective bioactivity or toxicity to the target or nontarget organisms as described above and may be transformed by microbes or enzymes at different rates. Thus, information on the enantioselective degradation and environmental behavior of parent acephate as well as its metabolite methamidophos is essential to evaluate the risk of these two chiral insecticides to human and ecological health. This information cannot be obtained from conventional achiral analysis. However, to our knowledge, there was the only one published information available on the transformation and degradation of the enantiomers of acephate and its chiral metabolite methamidophos in soils (Wang et al., 2013). Study highlighted that degradation of racemates is enantioselective in unsterilized soils but not in the sterilized soils, thus confirming that enantioselectivity is microbial based. So there is a need to pay more attentions on chirality based study of acephate and its metabolite methamidophos on different soils under different conditions such as temperature and pH.

Finally, we have concluded that acephate and methamidophos are distributed throughout the environment because of its high water solubility and probable chances of water runoff. Various methods have been reported to analyze level of acephate and methamidophos in different media such as soil, water, vegetable, serum, urine, etc. Most popular among them is liquid chromatography mass spectrometry (LCMS) or gas chromatography mass spectrometry (GCMS). But, LCMS requires extensive pretreatment and cleanup procedure, while GCMS needs derivatization of the compounds. The enantioselective bioactivity of both acephate and methamidophos in future may produce new method(s) of detection including determination of extent of toxicity of these pesticides. It is desirable to develop new methods based on use of chromophores/fluorophores which should be selective for acephate and its degraded products. Because of high use of acephate and methamidophos these days, their easy and fast analysis is also required. A lot of scope for trend breaking research in this area is needed.

In biochemical aspects, decomposition pathway of acephate with selected rhizobacteria in the presence of trace and heavy metal ions, including the mechanisms is under experimentation. Our future work will address the development of spectrophotometric detection of acephate and its metabolite.