• Methamidophos;
  • High-performance liquid chromatography;
  • Chiral separation;
  • Polysaccharide-based chiral phases;
  • Butyrylcholinesterase


  1. Top of page
  2. Abstract
  7. Acknowledgements

Many chiral pesticides are introduced into the environment as racemates, although their pesticidal activity is usually the result of preferential reactivity of only one enantiomer, while the other enantiomer may have toxic effects against nontarget organisms. Methamidophos (O,S-dimethyl phosphoramidothioate), a chiral compound, is an insecticide widely used in agriculture in both developed and developing countries. However, this pesticide has a high toxicity not only to targeted insects but also to human and animals. In the present study, the enantiomers of methamidophos were enantiomerically separated by a semipreparative chiral liquid chromatography at the multimilligram scale on a polysaccharide-based chiral stationary phase and a preliminary evaluation of their in vitro inhibition of plasma butyrylcholinesterase (BChE) of hens was performed. In the present study, our first effort was to resolve the racemic mixture of methamidophos and to that end reversed-phase, normal-phase, and polar organic elution conditions were investigated in four different polysaccharide-based chiral phases. The best performance was achieved on a cellulose tris(3,5-dimethylphenylcarbamate) phase under normal phase. This chromatographic condition allowed the separation of 225 mg of methamidophos enantiomers with a high degree of chiral purity (>98%) in a short analysis time. Significant differences were found between the concentration that causes 50% of enzyme inhibition (IC50) of the three isoforms of methamidophos. (−)-Methamidophos showed an IC50 approximately three times larger than the (+)-enantiomer for plasma BChE of hens. Environ. Toxicol. Chem. 2012;31:239–245. © 2011 SETAC


  1. Top of page
  2. Abstract
  7. Acknowledgements

Chirality is an important concept in various fields of chemistry and biochemistry, and its importance to the biological activity of single enantiomers has long been recognized. In living organisms, which are based on chiral molecules such as proteins and enzymes, and as a result of this stereoselective environment, two enantiomers of a compound behave differently when introduced into a biological system 1. Many chiral pesticides are introduced into the environment as racemates despite the fact that their activity is usually the result of a preferential reactivity to only one enantiomer, while the other enantiomer may have toxic effects against nontarget organisms 2.

Organophosphorus pesticides (OP) are usually esters, thiol esters, or acid anhydrides, derivatives of phosphorus-containing acids. They have become the most widely used insecticides in the world since the 1970s because they inhibit acetylcholinesterase (AChE) in insects 3. Most of these compounds are sold in the racemic form, and the chiral center of these pesticides may be in the atoms of phosphorus, carbon, or sulfur 4.

Methamidophos (O,S-dimethyl phosphoramidothioate) (Fig. 1) is a chiral insecticide, because of an asymmetric center at the phosphorus atom; it is widely used in agriculture 5, 6 to control chewing and sucking insects and spider mites on a variety of crops such as mustards, cotton, tobacco, sugar beets, lettuce, potatoes, and tree fruits 7. However, the toxicity of methamidophos is not limited to target insects; humans and animals also have suffered from a high incidence of acute and delayed toxic effects of this OP. McConnell et al. 8 have observed that methamidophos was the OP most frequently reported as the cause of organophosphate-induced delayed neuropathy in humans in recent years. Another epidemiologic study in Brazil certified methamidophos as one of the most common pesticides currently used in the 11 Brazilian states studied 9. In addition, some authors observed in rice cultures that during storage, residues of methamidophos was more persistent than acephate and had comparatively lower substantial loss 5. However, according to Lotti et al. 10 neuropathic effects were not seen in hens when the methamidophos racemic mixture was administered to these animals with a dose greater than the lethal dose for 50% of population exposed (LD50) and prophylaxis against the cholinergic effects. Thus, measurement of AChE and butyrylcholinesterase (BChE) has been used to indicate acute effects and overexposure, respectively 11. However, the neuropathy target esterase has been used to indicate delayed effects 12. Therefore, the lack of information on the enantioselectivity of methamidophos and the excess of toxic effects on nontarget organisms reinforce the need for performing new toxicity studies with methamidophos enantiomers.

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Figure 1. Chemical structure of methamidophos. The asterisk (*) denotes chiral center.

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Two approaches to obtaining pure enantiomers exist: asymmetric synthesis and resolution of racemic mixtures. An asymmetric synthesis is useful when one of both enantiomers is desired, but the exhaustive time required in developing and adjusting the synthesis to provide high yields can make this approach very impractical 13. Resolution methods using capillary electrophoresis, gas chromatography, and high-performance liquid chromatography (HPLC) are the most efficient analytical tools to achieve chiral separation of enantiomers from a racemic mixture. Among these methods, HPLC is the most frequently employed methodology to obtain small quantities of single enantiomers, which can be recovered following the separation and subsequently used in toxicological experiments 14.

The polysaccharide-based chiral stationary phases (CSPs) are the most useful and versatile chiral columns for the analysis and preparative separation of a wide range of chiral compounds 15. Moreover, these phases have been shown to be effective on multimodal elution (normal, reverse, and organic polar mode), broadening its application for different classes of compounds 16.

Many publications have described the successful analytical separation of methamidophos on polysaccharide-based phases under normal elution 4, 7, 17, 18. Ellington et al. 4 tested several chiral columns based on polysaccharides for the separation of 12 organophosphorus pesticides and enantioresolution was obtained for the enantiomers of methamidophos, crufomate, and trichloronate on a Chiralcel OD® column (cellulose tris(3,5-dimethylphenylcarbamate)) under normal phase. However, Tian et al. 19 did not separate the enantiomers of methamidophos under reverse phase elution. To date, no studies have dealt with chiral separation on the semipreparative scale of methamidophos.

Thus, to the best of our knowledge, no study has addressed the chiral separation of methamidophos on the multimilligram scale. The present study describes an investigation of the resolution of methamidophos on arylcarbamate derivatives of cellulose and amylose, as well as the influence of different mobile phases on enantiomeric separation using multimodal elution. The best performance obtained was scaled up to semipreparative HPLC to obtain multimiligram quantities of (+)- and (−)-methamidophos for further evaluation of in vitro toxicity against plasma BChE in hens.


  1. Top of page
  2. Abstract
  7. Acknowledgements


The preparative HPLC system consisted of a Shimadzu LC-6AD pump, a Rheodyne 7725i injector fitted with a 50 µl loop, and an SPD 10AV variable wavelength ultraviolet (UV)-vis detector with a CBM SCL-10AV interface. Data acquisition was performed using Class-VP software.

The analytical HPLC system consisted of two Shimadzu LC-10 AD pumps, a SIL-10AF autoinjector fitted with a 20 µl loop, and a SPD-M10AVvp UV-vis detector with an SCL-10 AVvp interface. Data acquisition was performed using Lab Solution software. A PerkinElmer 241 polarimeter with a sodium lamp was used to determine the specific rotation of the separated enantiomers of methamidophos in ethanol, at room temperature. All HPLC analyses were performed at room temperature.


All solvents were of HPLC grade, purchased from Merck (methanol, ethanol, and 2-propanol) or Mallinckrodt Baker (n-hexane and n-heptane). The mobile phases were prepared in a volume/volume (v/v) relation and degassed for 10 min in an ultrasonic bath prior to use. The technical racemic methamidophos was generously supplied by Bayer Cropscience. Heparin 25,000 IU/5 ml was purchased from Roche, Brazil; Deltametrin (K-otrine®) from Bayer Cropscience; piperazine citrate (Proverme®) from Tortuga Agrarian Zootechnical. Bovine serum albumin, Coomassie Brilliant Blue G-250, acetylthiocholine iodide, phosphoric acid 85%, and 5,5′-dithiobis(2-nitrobenzoic acid) were purchased from Sigma. Chiralcel OD chiral phase was purchased from Chiral Technologies. All other chemicals were of analytical grade.

Chiral stationary phases

The chiral columns were prepared at the Federal University of São Carlos laboratory as described in other studies 20–22 and consisted of amylose tris[(S)-1-phenylethylcarbamate] (CSP-1) and amylose tris(3,5-dimethylphenylcarbamate) (CSP-2) coated onto APS-Nucleosil (500 Å, 7 µm, 20%, w/w), and cellulose tris(3,5-dimethylphenylcarbamate) (CSP-3) coated onto APS-Hypersil® (120 Å, 5 µm, 15%, w/w). All analytical columns were packed into a stainless-steel 15 × 0.46 cm internal diameter (i.d.) column for analytical separation. In addition, we also tested the commercial Chiralcel OD [cellulose tris(3,5-dimethylphenylcarbamate)] (20 µm) lab-packed into a stainless-steel 15 × 0.46 cm i.d. column (CSP-4). The Chiralcel OD (20 µm) was lab-packed into a stainless-steel 20 × 0.70 cm i.d. column for semipreparative separation (CSP-5). A Shandon HPLC packing pump was employed for all column packing.

Sample preparation

The solutions of methamidophos for analytical and semipreparative separation were prepared by dissolving the appropriate amounts of the pesticide in ethanol, resulting in final concentrations of 6.67 for the analytical system, and 25.0, 40.0, 50.0, and 75.0 mg/ml for the semipreparative system. All solutions were prepared fresh daily.

The analyses were performed on the four polysaccharide-based chiral columns with 16 mobile phases at a flow rate of 1.0 ml/min for analytical or 3.0 ml/min for semipreparative separation. All mobile phases were prepared at a percent (v/v) proportion. Retention factor, selectivity, and resolution of the three chromatographic modes were calculated; no degradation of column performance was observed.

Chromatographic conditions

Methamidophos was detected at λ = 230 nm. The retention factors (k) were determined as k = (tr-t0)/t0, where tr and t0 are retention time and dead time, respectively. The separation factors (α) were calculated as α = k2/k1, where k1 and k2 are the retention factors for the first and second eluting enantiomer, respectively. Resolutions (Rs) were calculated as Rs = 2(t2-t1)/(w1 + w2), where w1 and w2 are the base width of the peaks to the first and second eluting enantiomer, respectively. The dead time (t0) was estimated using 1,3,5-tri-tert-butylbenzene.

Semipreparative chromatographic separations were achieved through multiple injections fitted with a 50 µl loop at a flow rate of 3.0 ml/min. The collected fractions of each enantiomer were rota-evaporated at 30°C; for 30 min, at 90 rpm, and analyzed using the analytical column to determine their enantiomeric purity (EP). The EP was calculated as the ratio between (+ or −)-enantiomer area divided by the sum of the (+)- and (−)-area multiplied by 100 23. To calculate the specific rotation [α]D of each fraction collected the following equation was used: [α]D = α/c · l, where α is specific value found in the polarimeter, c is the concentration of the solution in g/L, and l is the length of the polarimeter compartment in decimeters.

Biochemical analysis

Sample collection

Twelve Isabrown leghorn hens (70–90 weeks, weighing 1.5–2.4 kg) were obtained from the Hayashi farm cooperative of Guatapará (SP, Brazil). Before starting the experiments, the hens were treated to eliminate ectoparasites and endoparasites as described by Emerick et al. 24. After this treatment, the hens were housed, three per cage, in a temperature- and humidity-controlled room (24 ± 2°C and 55% ± 10 RH), with an automatic 12:12 light:dark photocycle, lights on at 7 AM. Purina® feed and filtered tap water were provided ad libitum. All experimental procedures were conducted with the approval of the Research Ethics Committee of the School of Pharmaceutical Sciences of Araraquara (SP, Brazil) in accordance with their guidelines for the care and use of laboratory animals (Resolution 24, 2009).

Blood (0.5 ml) was collected from the axillary vein in a tube containing heparin and centrifuged (10 min; 250 g). From the obtained plasma, 70 µl was diluted with 9.930 ml of deionized water and the amount of protein contained in this solution was determined based on the Bradford method 25. The enzyme activity is given per g of protein.

In vitro experiments

The activity of BChE (µmol/min/g of protein) was determined using the method described by Ellman et al. 26 plus eight different concentrations of methamidophos (ranging from 0.01 to 20 mM in deionized water) and its isoforms as inhibitor. Four readings of each concentration were taken at intervals of 60 s, at 37°C, 450 nm with constant stirring at 600 rpm in a spectrophotometer UV/vis HP 8453 and the absorbance used to calculate the enzyme activity was the average between these four readings per minute.

Inhibitor concentrations capable of inhibiting 50% of enzyme activity (IC50) were determined using the equation of the line graph of the log of percent of activity versus concentration of inhibitor (semilog plots).

Statistical analyses

Differences in biochemical analyses were examined for statistical significance by one-way analysis of variance (ANOVA), followed by Tukey's test for multiple comparisons. The regression values were calculated using the method of least squares. These tests were run in Microsoft Office Excel® 2007 for Windows. The criterion of significance was p < 0.05 for all statistical analyses. All biochemical data means the average of three samples, which were made in replicate (n = 3) and are expressed as mean ± standard deviation (SD).


  1. Top of page
  2. Abstract
  7. Acknowledgements

Analytical HPLC

When chiral compounds are the target molecules, it is desirable to use CSP to develop the HPLC methods because of the direct injection of compounds, which promotes simplicity for the overall method of analysis 27. According to statistics from the American Chemical Society (Journal of the American Chemical Society), more than 90% of the chiral HPLC separations between the years 2005 and 2007 were performed with polysaccharide-based chiral stationary phase 12. Polysaccharides such as cellulose and amylose are among the most abundant optically active biopolymers with perfectly defined structures and can resolve several enantiomers including OPs 28. A number of publications have reported the separation of a large number of OPs on dimethylphenylcarbamate and phenylethyl-carbamate of cellulose and amylose, and the enantioselectivity of these polysaccharide-CSPs is attributed to the degree of steric fit of the enantiomers in the chiral cavity of the stationary phase 29–31. In addition, the mechanism of the interactions between the enantiomers and the CSPs can occur through hydrogen bonding, dipole moments, and π–π interactions 32.

To optimize the chromatographic conditions for the enantioseparation of methamidophos, initially, four analytical columns (15 × 0.46 cm) consisting of amylose tris[(S)-1-phenylethylcarbamate)] (CSP-1), amylose tris(3,5-dimethylphenylcarbamate) (CSP-2), cellulose tris(3,5-dimethylphenylcarbamate) (CSP-3), and a Chiralcel OD [cellulose tris(3,5-dimethylphenylcarbamate)] (CSP-4) were evaluated. The chromatographic parameters such as retention factor (k), enantioselectivity (α) and enantioresolution (Rs) of methamidophos was systematically evaluated for all compositions of mobile phases tested (Table 1). During the initial experiments, it was verified that methamidophos showed a greater solubility in ethanol than in 2-propanol; thus, ethanol was the solvent selected to prepare the samples for injection. In addition, to verify the influence of the mobile phase on the enantioseparation of methamidophos, the polysaccharide-base CSPs (1, 3, and 4) were evaluated under normal-phase, reversed-phase, or polar organic mode.

Table 1. Chromatographic parameters assessed in the analytical separation of methamidophos
Mobile phaseCSP-1CSP-2CSP-3CSP-4
  • *

    Phases not tested.

    CSP = chiral stationary phase (see Materials and Methods); k = retention factor; α = enantioselectivity factor; Rs = resolution.

Heptane/ethanol (90:10)******2.5510***
Hexane/ethanol (80:20)
Hexane/ethanol (90:10)2.301.210.803.29105.431.090.807.601.241.00
Heptane/2-propanol (90:10)***4.141.310.803.781.210.806.191.351.00
Hexane/2-propanol (95:05)***21.7911.0030.001.161.51Retained in 60 min
Hexane/2-propanol (92:08)***8.8911.0010.591.151.256.401.381.56
Hexane/2-propanol (90:10)Retained in 60 min2.2410.803.361.221.004.921.231.25
Hexane/2-propanol (80:20)6.801.200.80******
Hexane/2-propanol (70:30)*********
Hexane/2-propanol/ethanol (85:5:10)*********
Hexane/2-propanol/acetic acid (90:9:1)******3.861.331.133.941.271.25
Methanol/water (50:50)0.1010*********
Methanol/water (30:70)0.1010*********

The initial mobile phase composition tested was a mixture of n-hexane/2-propanol (80/20). The chromatographic parameters k, α, and Rs obtained for CSP-1 and CSP-3 showed poor resolution (Rs < 1.0) and median selectivity (α = 1.20 and 1.23, respectively) for the enantiomers of methamidophos. Different proportions of 2-propanol (30, 10, 8, and 5%) were also evaluated with CSP-1, CSP-3, or CSP-4; among the chromatographic conditions studied, the highest resolutions were 1.51 and 1.56 when n-hexane/2-propanol (95:05) and (98:02) were tested with CSP-3 and CSP-4, respectively. When n-hexane/2-propanol (90:10) and (95:05) were evaluated with CSP-1 and CSP-3, respectively, these chiral phases showed high retention factors for methamidophos. The replacement of 2-propanol by ethanol was also tested; however, it was noticed that the use of ethanol at 10 and 20% helped to decrease the retention factor and did not favor the improvement of the separation between the two enantiomers of methamidophos (Table 1). Likewise, the effect of using n-heptane was also evaluated in place of n-hexane, acid additive (1%) in the mobile phases and a mixture of hexane/2-propanol/ethanol; however, none of these attempts improved the chromatographic parameters for the enantioseparation of methamidophos.

Moreover, reverse-phase mode and polar organic mode were also tested on CSP-1, CSP-3, and CSP-4 and no resolution was observed under these chromatographic modes studied. The analyte has a high polarity and polar mobile phases did not allow sufficient interactions for chiral discrimination between the OP and CSPs. However, in normal mode it was possible to obtain satisfactory enantioseparation when a mixture of hexane/2-propanol (95:05) and (92:08) was tested with CSP-3 and CSP-4, respectively. In summary, the separation factor and resolution obtained for methamidophos in CSP-3 and CSP-4 were satisfactory in one elution mode tested (normal-phase), while CSP-2 demonstrated limited enantioresolution in most of the chromatographic conditions investigated. Both CSP-1 and CSP-2 proved to be inappropriate for chiral separation of methamidophos, while CSP-3 and CSP-4 were able to separate the enantiomers of methamidophos in at least one elution mode. These sequences of chromatograms are illustrated in Figure 2.

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Figure 2. Effects of 2-propanol on the chiral separation of methamidophos (6.67 mg/ml). Chromatographic conditions: cellulose tris(3,5-dimethylphenylcarbamate) coated onto aminopropylsilyl (APS)-Hypersil (120 Å, 5 µm, 15%, w/w) (15 × 0.46 internal diameter) for (A,C,D); Column Chiralcel OD (20 µm) (15 × 0.46 i.d.) for (B); ultraviolet detection at 230 nm; mobile phase: n-hexane/2-propanol (80/20), n-hexane/2-propanol (90/10), n-hexane/2-propanol (95/05); flow rate of 1.0 ml/min; volume of injection: 20 µl.

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These results can be explained by the fact that the analyte enters into the chiral cavity; thus enantioselective interactions occurred, resulting in chiral discrimination. It has been assumed that the separation of racemates on amylose- and cellulose-based CSPs was due to the formation of solute–CSPs complexes through inclusion of the enantiomers into the chiral cavities 13. Nevertheless, in the present study the cellulose-based CSPs presented higher resolution for methamidophos enantiomers and also allowed shorter retention time for the total analysis. Okamoto and Kaida 33 attributed the difference in chiral recognition ability between amylose-CSP and cellulose-CSP to the conformational difference between them. Therefore, the chiral discrimination observed for the enantiomers of methamidophos on CSP-2 and CSP-3 is believed to be due to the same reason.

The mobile phase in chiral chromatography plays a crucial role in the interaction process. It affects not only the retention factor but also the enantioselectivity or enantioresolution 27. Table 1 shows that the retention factor of methamidophos changed greatly using alcohols with higher lipophilicity as mobile phase modifier.

Semipreparative HPLC

We directed our experiments to developing analytical methods that could be easily scaled up to semipreparative loadings and a short time of analysis with high resolution. In Figure 2, while the mobile phase with the best resolution is n-hexane/2-propanol (95/05 v/v), we succeeded in isolating the enantiomers of methamidophos in a semipreparative column with a mobile phase consisting of n-hexane/2-propanol (90/10 v/v), knowing that the larger dimensions of the column would favor the best resolution. Another important factor to consider when performing multimilligram chromatographic separation is the solubility of the related compounds, because this can limit the quantity of injection and thus damage the productivity of the separation. In the present study, the poor methamidophos solubility on the selected mobile phase contributed to the low volume of injection, and it was only possible to inject 3.75 mg of racemate per run (75 mg/ml in 50 µl of injection) (Fig. 3).

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Figure 3. Effects of methamidophos concentration (20, 40, 50, 75 mg/ml) on the load capacity of the chiral chromatographic column. Chromatographic conditions: Column Chiralcel OD (20 µm) (20 × 0.7 internal diameter); ultraviolet detection at 230 nm; mobile phase: n-hexane/2-propanol (90/10); flow rate of 3.0 ml/min; volume of injection: 50 µl.

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The first enantiomer fraction was collected at 2-min time intervals, from the beginning to the end of the first enantiomer peak and at 3 min for the second (Fig. 4). These collected fractions were reinjected in the analytical system (Fig. 5), to evaluate their EP, using the same conditions from the semipreparative system. In addition, to assess the absolute configuration of each enantiomer, the optical activities of the isolated enantiomers were measured using a polarimeter ([α]D ethanol, 25°C). The results of optical rotation were compared with previous published results from Lin et al. 7 and the results obtained in the present study (Table 2) are in agreement with the results observed previously. The (+)-methamidophos, in this elution condition, is the first enantiomer to elute. The EP of the isolated enantiomers also is provided in Table 2. The second enantiomer eluted is always more difficult to isolate with high EP, especially when the enantiomeric resolution is poor.

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Figure 4. Chromatogram of the semipreparative separation of methamidophos enantiomers. Chromatographic conditions: Column Chiralcel OD (20 µm) (20 × 0.7 internal diameter); ultraviolet detection at 230 nm; mobile phase: n-hexane/2-propanol (90/10); flow rate of 3.0 ml/min and volume of injection of 50 µl.

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Figure 5. Chromatograms of the methamidophos enantiomers isolated reinjected in the semipreparative chiral stationary phase column. Chromatographic conditions: column Chiralcel OD (20 µm) (20 × 0.7 internal diameter); ultraviolet detection at 230 nm; mobile phase: n-hexane/2-propanol (90/10); flow rate of 3.0 ml/min; volume of injection: 20 µl. (A) the second fraction isolated, (B) the first fraction isolated and (C) racemate.

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Table 2. Parameters for the semipreparative method of separation of the methamidophos enantiomers
Injected mass (mg)248.0248.0
Obtained mass (mg)221.0229.0
Enantiomeric purity (%)99.598.3
Recovery (%)89.092.0
[α]D, ethanol+45.6−47.8
Yield (mg/h)7.35.6

The chiral stationary phase consisting of Chiralcel OD [cellulose tris(3,5-dimethylphenyl carbamate)] (20.0 × 0.7 cm) was selected to be used with n-hexane/2-propanol (92/08) as mobile phase at a flow rate of 3 ml/min and 230 nm. This selection was based on the best resolution and selectivity obtained during the evaluation of the analytical columns. However, when the semipreparative column was used, the enantiomers of methamidophos were retained more strongly; to adjust the retention factor, the proportion of 2-propanol was increased to 10%. This new composition of the mobile phase still allowed baseline resolution and, additionally, a shorter k was obtained.

Moreover, the load capacity of the chiral semipreparative column was determined by injections of different concentrations of methamidophos in ethanol (25, 40, 50, and 75 mg/L). Figure 3 demonstrates the separation profile obtained for each concentration injected. The sample concentration of 75 mg/ml was selected for all injections performed during the semipreparative separation. The chromatogram of the semipreparative separation is shown in Figure 4. The single enantiomers of methamidophos were seen to be stable for six months because these enantiomers were reinjected and no changes in chromatographic parameters were noted.

Using this method, the racemic mixture of methamidophos (75 mg/ml) was injected in 140 applications of 50 µl each. This method produced 221.0 mg of the first enantiomer and 229 mg of the second enantiomer to elute. These amounts were isolated from 496 mg of racemic methamidophos. Individual fractions of enantiomers isolated were reinjected onto the analytical chiral column to determine their EP The first enantiomer was isolated with 89.0% recovery and an EP of 99.5% while the second enantiomer was isolated with 92.0% recovery and an EP of 98.3%. Chromatograms of each enantiomer of methamidophos obtained after purification are given as examples in Figure 5. The optical rotations of two enantiomers collected were also measured using a polarimeter ([α]D ethanol, 25°C). The recovery, enantiomeric purity, specific rotation, and yield are summarized in Table 2.


The curves of percent inhibition versus concentration of (+/ − )-methamidophos, (+)-methamidophos, and (−)-methamidophos are presented in Figure 6. The IC50 was determined using the equation of the line graph of the log of percent of activity versus concentration of inhibitor 34. Cholinesterase inhibition is used as an indicator to monitor OP exposure, and the primary toxicity of these compounds arises as a result of covalently binding to cholinesterases such as BChE and AChE 35. Toxicological evaluation of OPs is based mainly on the control of AChE as causing the cholinergic syndrome and BChE as a biomarker of exposure. Differentiating the toxicity of methamidophos enantiomers for BChE could be welcome to decrease the side effects of overexposure to nontargets organisms. Starting from this principle, an evaluation was made of the inhibition of plasma BChE in hens. The form (+)-methamidophos showed lower IC50 (Fig. 6), the racemic form showed intermediate IC50, and the (−)-methamidophos showed higher IC50 (Table 3). The regression data of the semi-log plots presented good r-squared values larger than 0.98 for the three isoforms of methamidophos (Table 3).

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Figure 6. Comparison between the curves of percent of inhibition versus the concentration of (+/−)-methamidophos, (+)-methamidophos, and (−)-methamidophos for plasma butyrylcholinesterase (BChE) of hens (n = 3).

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Table 3. Summary of inhibition caused by isoforms of methamidophos in relation to butyrylcholinesterase
InhibitorIC50 (mM)r2
  • **

    Statistically different from the racemate and (−)-methamidophos (p < 0.05).

    IC50 = concentration that causes 50% of enzyme inhibition.

(+)-Methamidophos2.2 ± 0.2**0.9905
(+/−)-Methamidophos4.3 ± 0.40.9896
(−)-Methamidophos6.4 ± 0.30.9932

Lin et al. 7 showed that the (−)-methamidophos was a more potent inhibitor of bovine AChE than (+)-methamidophos. Corroborating these findings, Bertolazzi et al. 34 showed that the (−)-enantiomer of methamidophos has an inhibitory constant for AChE bigger than (+)-enantiomer. Surprisingly, in the present study, methamidophos enantiomers showed a different behavior for plasma BChE in hens, because the (+)-methamidophos presented an IC50 lower than (−)-methamidophos. This inversion between inhibition of two α/β hydrolyses with 54% of identical amino acid sequence 36 may be explained by the fact that the active site gorge of BChE is higher than that of AChE 37. Thus, it is possible to say that the results of enantiomers inhibition on esterases are not easily extrapolated from one hydrolyse to another.


  1. Top of page
  2. Abstract
  7. Acknowledgements

The next step of this work will be to evaluate the inhibition caused by the three isoforms of methamidophos in other blood esterases to verify the acute and delayed toxicity of these isomers in hens (an animal model to study delayed effects of OPs). In conclusion, the cellulose-based CSP demonstrated satisfactory capacity for enantiomeric resolution at the multimilligram scale for methamidophos enantiomers. Both enantiomers of methamidophos were obtained with a high degree of enantiomeric purity (>98%) and high recoveries in a short time of analysis. Significant differences were found between the IC50 of the three isoforms of methamidophos for in vitro inhibition of BChE. The (−)-methamidophos showed an IC50 approximately three times larger than the (+)-methamidophos for plasma BChE of hens.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Financial support for this work was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP Grant 2009/51048-8 and 2007/02872-4.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  • 1
    Kurihara N, Miyamot J, Paulson GD, Zeeh B, Skidmore MW, Hollingworth RM, Kuiper HA. 1997. Chirality in synthetic agrochemicals: Bioactivity and safety consideration. Pure Appl Chem 69: 20072025.
  • 2
    Wang Y, Tai K, Yen J. 2004. Separation, bioactivity, and dissipation of enantiomers of the organophosphorus insecticide fenamiphos. Ecotoxicol Environ Saf 57: 346353.
  • 3
    Johnson MK, Jacobsen D, Meredith TJ, Eyer P, Heath AJ, Ligtenstein DA, Marrs TC, Szinicz L, Vale JA, Haines JA. 2000. Evaluation of antidotes for poisoning by organophosphorus pesticides. Emergen Med 12: 2237.
  • 4
    Ellington JJ, Evans JJ, Prickett KB, Champion WL. 2001. High-performance liquid chromatographic separation of the enantiomers of organophosphorus pesticides on polysaccharide chiral stationary phases. J Chromatogr A 928: 145154.
  • 5
    Kong Z, Dong F, Xu J, Liu X, Li J, Li Y, Tian Y, Guo L, Shan W, Zheng Y. 2012. Degradation of acephate and its metabolite methamidophos in rice during processing and storage. Food Control 23: 149153.
  • 6
    Gubert P, Ávila DS, Bridi JC, Saurin S, Lugokenski TH, Villarinho JG, Fachinetto R, Pereira ME, Ferreira J, Rocha JBT, Soares FAA. 2011. Low concentrations of methamidophos do not alter AChE activity but modulate neurotransmitters uptake in hippocampus and striatum in vitro. Life Sci 88: 8995.
  • 7
    Lin K, Zhou S, Xu C, Liu W. 2006. Enantiomeric resolution and biotoxicity of methamidophos. J Agric Food Chem 54: 81348138.
  • 8
    McConnell R, Téllez ED, Cuadra R, Torres E, Keifer M, Almendárez J, Miranda J, El-Fawal HAN, Wolff M, Simpson D, Lundberg I. 1999. Organophosphate neuropathy due to methamidophos: Biochemical and neurophysiological markers. Arch Toxicol 73: 296300.
  • 9
    Chrismana JR, Koifmanb S, Sarcinellia PN, Moreira JC, Koifman RJ, Meyer A. 2009. Pesticide sales and adult male cancer mortality in Brazil. Int J Hyg Environ Health 212: 310321.
  • 10
    Lotti M, Moretto A, Bertolazzi M, Peraica M, Fioroni F. 1995. Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers. Arch Toxicol 9: 330336.
  • 11
    Sanchez-Hernandez LC, Walker CH. 2000. In vitro and in vivo cholinesterase inhibition in lacertides by phosphonate- and phosphorothioate-type organophosphates. Pest Biochem Physiol 67: 112.
  • 12
    Johnson MK, Glynn P. 1995. Neuropathy target esterase (NTE) and organophosphorus-induced delayed polyneuropathy (OPIDP): Recent advances. Toxicol Lett 82/83: 459463.
  • 13
    Zongde Z, Xingping L, Xiaomei W, Hong Z, Yanping S, Liren C, Yongmin L. 2005. Analytical and semipreparative resolution of enantiomers of albendazole sulfoxide by HPLC on amylose tris (3,5-dimethylphenylcarbamate) chiral stationary phases. J Biochem Biophys Methods 62: 6979.
  • 14
    Nillos MG, Gan J, Schlenk D. 2010. Chirality of organophosphorus pesticides: Analysis and toxicity. J Chromatogr B 878: 12771284.
  • 15
    Okamoto Y, Ikai T. 2008. Chiral HPLC for efficient resolution of enantiomers. Chem Soc Rev 37: 25932608.
  • 16
    Cass QB, Degani ALG, Cassiano N. 2000. The use of a polysaccharide-based column on multimodal elution. J Liq Chromatogr Relat Technol 23: 10291038.
  • 17
    Miyazaki A, Nakamura T, Kawaradani M, Marumo S. 1988. Resolution of biological activity of both enantiomers of methamidophos and acephate. J Agric Food Chem 36: 835837.
  • 18
    Wang P, Jiang J, Liu D, Zhang H, Zhoue Z. 2006. Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography. J Agric Food Chem 54: 15771583.
  • 19
    Tian Q, Lv C, Wang P, Ren L, Qiu J, Li L, Zhou Z. 2007. Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions. J Sep Sci 30: 310321.
  • 20
    Matlin SA, Tiritan ME, Crawford AJ, Cass QB, Boyd DR. 1994. HPLC with carbohydrate carbamate chiral phases: Influence of chiral phase structure on enantioselectivity. Chirality 6: 135140.
  • 21
    Matlin SA, Tiritan ME, Cass QB, Boyd DR. 1996. Enantiomeric resolution of chiral sulfoxides on polysaccharides phases by HPLC. Chirality 8: 147152.
  • 22
    Tiritan ME, Cass QB, Del Alamo A, Matlin SA, Grieb SJ. 1998. Preparative enantioseparation on polysaccharide phase using microporous silica as a support. Chirality 10: 573577.
  • 23
    Gawley RE. 2006. Do the terms “% ee” and “% de” make sense as expressions of stereoisomer composition or stereoselectivity? J Org Chem 71: 24112416.
  • 24
    Emerick GL, Peccinini RG, DeOliveira GH. 2010. Organophosphorus-induced delayed neuropathy: A simple and efficient therapeutic strategy. Toxicol Lett 192: 238244.
  • 25
    Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantity of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254.
  • 26
    Ellman GL, Courtney KD, Andres V, Featherstone RM. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7: 8895.
  • 27
    Francotte ER. 2001. Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers. J Chromatogr A 906: 379397.
  • 28
    Yashima E. 2001. Polysaccharide-based chiral stationary phases for high-performance liquid chromatography enantioseparation. J Chromatogr A 906: 105125.
  • 29
    Ye J, Wu J, Liu W. 2009. Enantioselective separation and analysis of chiral pesticides by high-performance liquid chromatography. Trends Anal Chem 28: 11481163.
  • 30
    Ye J, Zhao M, Liu J, Liu W. 2010. Enantioselectivity in environmental risk assessment of modern chiral pesticides. Environ Pollut 158: 23712383.
  • 31
    Nillos MG, Rodrigues-Fuentes G, Gan J, Schlenk D. 2007. Enantioselective acetylcholinesterase inhibition of the organophosphorus insecticides profenofos, fonofos, and crotoxyphos. Environ Toxicol Chem 26: 19491954.
  • 32
    Wang T, Yadan WC. 1999. Application and comparison of derivatized cellulose and amylose chiral stationary phases for the separation of enantiomers of pharmaceutical compounds by high-performance liquid chromatography. J Chromatogr A 855: 411421.
  • 33
    Okamoto Y, Kaida Y. 1994. Resolution by high-performance liquid chromatography using polysaccharide carbamates and benzoates as chiral stationary phases. J Chromatog A 666: 403419.
  • 34
    Bertolazzi M, Caroldi S, Moretto A, Lotti M. 1991. Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase. Arch Toxicol 65: 580585.
  • 35
    Wang L, Du D, Lu D, Lin CT, Smith JN, Timchalk C, Liu F, Wang J, Lin Y. 2011. Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: A biomarker of exposure to organophosphate agents. Anal Chim Acta 693: 16.
  • 36
    Shenouda J, Green P, Sultatos L. 2009. An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon. Toxicol Appl Pharmacol 241: 135142.
  • 37
    Nicolet Y, Lockridge O, Masson P, Fontecill-Camps JC, Nachon F. 2003. Crystal structure of human butyrylcholinesterase and of its complexes with substrates and products. J Biol Chem 278: 4114141147.