Flow Injection Spectrophotometric Determination of Acetochlor in Food Samples

Acetochlor is an herbicide widely used for controlling grass weeds in various crops.1 Acetochlor was first used in 1994 in the U.S.A.2 and in Europe in 2000.3 As a member of the chloroacetanilide class of broad leaf herbicides, it is applied to the soil as a pre- and postemergence treatment. Acetochlor is mainly absorbed by the roots and leaves, inhibiting photosynthetic electron transport of the host plant.4 The pollution problems associated with acetochlor have increasingly attracted attention. The toxicity of acetochlor, such as inducing sister chromatid exchanges in cultured human lymphocytes,5 mutagenizing germ cells of male rats,6 and altering thyroid hormone-dependent gene expression in Xenopus laevis,7 has been reported. The typical half-life of chloroacetanilide herbicides under natural conditions ranges from 15 to 30 days.3 The presence of acetochlor and its metabolites has become a significant pollution problem, and effective methods for their removal or treatment need to be pursued. Various techniques such as HPLC,8,9LC-MS,3,10 and GC11,12 are used for the determination of acetochlor and its metabolites in soil and water. The aim of the present study was to develop a simple and quick method for the determination of acetochlor in formulations and food samples.

Materials and methods


Figure 1 - Single-channel FI for the spectrophotometric determination of acetochlor. R = coupling coil length, D = detector, W = waste, pp = peristaltic pump.

The flow injection (FI) system comprised a peristaltic pump (Becton Dickinson, Franklin Lakes, NJ) with PTFE (1.19 mm i.d.) and silicon (1.71 mm i.d.) flow tubes, V-450 six-port injection valve (Upchurch Scientific Inc., Oak Harbor, WA), and UNICO UV-2100 UV-VIS spectrophotometer (United Products and Instruments Inc., Dayton, NJ) as the detector. A schematic of the FI system is given in Figure 1.

Reagents and chemicals

All chemicals used were of analytical reagent-grade purity. Sodium nitrite, aniline (Merck, Darmstadt, Germany), concentrated hydrochloric acid (Merck), and ethanol (Merck) were used for this work. Standard reference material was purchased from Dr. Ehrenstorfer GmbH (Augsberg, Germany). A commercial sample containing acetachlor was purchased commercially from a local market.

Solution preparations

  1. Aniline solution (1%): 1% aniline solution was prepared by adding 1 mL of aniline in 50 mL of ethanol and diluted with distilled water up to 100 mL.
  2. Nitrite solution (0.15%): Nitrite solution was prepared by dissolving 0.225 g sodium nitrite in distilled water and diluted up to 100 mL.
  3. Diazotized aniline solution: Diazotized aniline solution was prepared by mixing nitrite solution (0.15%) and aniline solution (1%) in a 3:1 ratio.
  4. Standard acetochlor solution: Acetochlor solution was prepared and hydrolyzed by taking a known volume of standard acetochlor in a beaker followed by the addition of 0.2 M of HCl and a few milliliters of ethanol. This was heated in a boiling water bath for 10 min, cooled, and diluted up to 10 mL with ethanol. Working standard solutions were prepared in the range of 2–0.009 ppm by suitable dilution of the stock standard solution.

Recommended procedure for calibration

  1. Batch procedure. To different concentrations of hydrolyzed acetochlor working standard solution was added 6 mL of diazotizing reagent at room temperature and kept for 10 min, and azo dye was formed. The dilution was made with distilled water up to 10 mL, and absorbance was measured at 400 nm against reagent blank.
  2. FI procedure. The diazotized reagent was continuously pumped as a carrier stream and used as blank. One milliliter of hydrolyzed acetochlor standard solution in the concentration range of 2–0.009 ppm was injected into the carrier stream, and absorbance of the azo dye was measured continuously at 400 nm.

Procedure for the assay of acetochlor in commercial formulations

For the determination of acetochlor in commercial formulations, 0.1 mL of commercial sample was hydrolyzed in the same way as standard. Working standards were prepared by dilution with distilled water. The color was developed by the addition of 6 mL diazotizing reagent and diluted with distilled water up to 10 mL in a volumetric flask, and absorbance was measured at 400 nm. The amount of acetochlor present in each preparation was determined from the calibration plot using a standard curve.

Procedure for assay of acetochlor in food samples

Fifteen grams of homogenized food sample and 20 mL of sugarcane juice were taken in a beaker, and 50 mL of solvent (petroleum ether and acetone, 1:1) was added to it for extraction of herbicides. The mixture was shaken for 2 hr. After equilibration, the sample was filtered and passed through 10 g of sodium sulfate. The filtrate was evaporated on a rotary evaporator up to a volume of 5 mL and hydrolyzed with 0.2 M hydrochloric acid by heating in a boiling water bath for 10 min. Two and one milliliter from the hydrolyzed extract were used for batch and flow injection analysis, respectively. Each sample was analyzed in triplicate.

Percent recovery

A recovery test was performed on control samples and fortified with a known concentration of acetochlor solution. The solvent was evaporated at room temperature for 1 hr. Six replicates of the fortified samples were analyzed.

Results and discussion

Preliminary studies

Figure 2 - Proposed reaction mechanism for spectrophotometric determination of acetochlor.

Figure 3 - Investigation of hydrochloric acid volume for hydrolysis of acetochlor herbicide.

Figure 4 - Investigation of time for hydrolysis of acetochlor herbicide.

Figure 5 - Investigation of nitrite concentration for spectrophotometric determination of acetochlor herbicide.

Figure 6 - Investigation of nitrite volume for spectrophotometric determination of acetochlor herbicide.

Figure 7 - Investigation of aniline concentration for spectrophotometric determination of acetochlor herbicide.

Figure 8 - Investigation of aniline volume for spectrophotometric determination of acetochlor herbicide.

The proposed reaction mechanism for the formation of azo dye based on coupling hydrolyzed acetochlor herbicide with the diazotized reagent is shown in Figure 2. The influence of acidity on the hydrolysis of acetochlor was studied in the range of 0.1–0.5 using hydrochloric acid. The absorbance was increased with increasing acid concentration from 0.1 to 0.2 M of hydrochloric acid solution (Figure 3). Above this range, the absorbance remains constant; therefore, 0.2 M acid was used for further hydrolysis of acetochlor herbicide. The maximum time for hydrolysis was found to be 10 min in a boiling water bath (Figure 4). The effects of reagent concentration were studied for the formation of diazotized compound, and the concentration and volume of nitrite solution were optimized (Figures 5 and 6). It was found that 8 mL of 0.15% nitrite solution was sufficient for the reaction. Aniline was used as a diazotized reagent, and it was found that 4 mL of 1% aniline solution was sufficient for the optimum reaction (Figures 7 and 8).