A New Sensitive Spectrophotometric Determination of Cypermethrin Insecticide in Environmental and Biological Samples

Um método espectrofotométrico novo e altamente sensível foi desenvolvido para a determinação do inseticida cipermetrina em níveis de partes por milhão. O método é baseado na hidrólise alcalina de cipermetrina ao íon cianeto, o qual reage posteriormente com iodeto de potássio e leuco cristal violeta. A absorção máxima do corante cristal violeta formado é medida em 595 nm em meio ácido. A lei de Beer é obedecida no intervalo de concentração de 3,0 a 17 μg, em um volume final da solução de 25 mL (0,12-0,68 ppm). A absortividade molar e sensibilidade de Sandell encontradas foram 3,3×10 L mol cm e 0,054 μg cm, respectivamente. O desvio padrão e desvio padrão relativo foram ± 0,001 e 0,22%, respectivamente. O método é simples, sensível e livre de interferências de outros pesticidas e íons. Outros inseticidas piretróides não interferem no método proposto. O método tem sido aplicado satisfatoriamente para a determinação de cipermetrina em amostras ambientais e biológicas.

The aim of the present work is to develop a rapid, low cost, accurate and simple analytical method for the determination of cypermethrin at trace levels.In this paper, a simple and sensitive spectrophotometric method is described for the determination of cypermethrin, where cypermethrin is hydrolysed to give cyanide ion, which further reacts with potassium iodide and leuco crystal violet to produce a crystal violet dye with maximum absorbance at 595 nm in acidic medium.The reagent is selective for cypermethrin, amongst the pyrethroid group.The colour system obeys Beer's law in the range of 0.12-0.68ppm of cypermethrin.The method has been applied to the determination of cypermethrin in various samples of water, vegetables, fruits, foliages and biological samples.

Experimental
Apparatus A Systronics UV-Vis spectrophotometric model 104 with matched silica cells was used for all spectral measurements.A Systronic pH meter model 335 was used for pH measurements.A Remi C-854/4 clinical centrifuge force of 1850 g with fixed swing out rotors was used for centrifugation.

Reagents
All reagents used were of Anala R grade or of the best available quality.Double distilled demineralized water was used throughout.Cypermethrin (Syngenta Crop Protection Private Limited, India): A stock solution of 1 mg mL -1 was prepared in ethanol.Working standard solutions were prepared by appropriate dilution of the stock standard solution with water.A 3% hydrochloric acid aqueous solution was used.A 20% sodium hydroxide aqueous solution was used. 6A 0.1% potassium iodide aqueous solution was used.Leuco crystal violet (Eastman Kodak Co.) [LCV] was prepared adding to a 1 liter volumetric flask 200 mL of water, 3 mL of 85% phosphoric acid and 250 mg of leuco crystal, (4,4',4"methylidynetris(N,N',-dimethylaniline) (CH[C 6 H 4 N (CH 3 ) 2 ] 3 ).It was shaken gently until the dye gets dissolved.The content of the flask was then diluted to 1 liter with water. 20

General procedure: preparation of calibration curve
An aliquot of test solution containing 3.0 to 17 μg of cypermethrin was taken in a 25 mL graduated cylinder and to it 1.0 mL of 20% sodium hydroxide was added.The solution was kept for 10 min at room temperature for complete hydrolysis.Then, 1 mL of 0.1% potassium iodide was added in acidic medium to liberate iodine and then 1 mL leuco crystal violet was added and shaken thoroughly and kept for 15 min for full colour development.The crystal violet dye was produced.The solution was then diluted to the mark with water and absorbance was measured at 595 nm against a reagent blank.

Spectral characteristics
The crystal violet dye formed in the proposed reaction shows maximum absorption at 595 nm (Figure 1).All spectral measurements were carried out against demineralized water as the reagent blank which showed negligible absorption at this wavelength.The colour system obeys Beer's law in the range of 3.0 to 17 µg of cypermethrin per 25 mL of final solution at 595 nm (Figure 2).The molar absorptivity and Sandell's sensitivity were found to be 3.3×10 5 L mol -1 cm -1 and 0.054 μg cm -2 , respectively.

Optimization of conditions
Hydrolysis of cypermethrin to cyanide ion was studied at different temperatures and alkalinity.It was observed that alkaline conditions were required for the hydrolysis (Figure 3).Maximum hydrolysis 6 was observed with 20% sodium hydroxide at temperature range of 30-35 o C as it gave maximum absorbance values, good stability and quantitative results.It was observed that 1 mL of leuco crystal violet was sufficient for complete colour reaction (Figure 4).
The effect of pH on the colour reaction was studied and it was found that constant absorbance values were obtained at pH range of ~ 4.5-5.5 by hydrochloric acid and no buffer solution was required to stabilize the colour.The coloured species remain stable for more then 7 days under optimum conditions.
Precision of the method was checked by the replicate analysis of working standard solution containing 4 μg of cypermethrin in 25 mL final solution over a period of 7 days.The standard deviation and relative standard deviation were found to be ± 0.001 and 0.22% respectively.

Effect of foreign species
The effect of common foreign species and pesticides were studied to assess the validity of the method.Known amounts of foreign species and pesticides were added to the standard solution contained 10 μg of cypermethrin prior to hydrolysis and the solution was analysed by the proposed method .The method was found to be free from interferences of most of the foreign species and pesticides (Table 1).

Application Determination of cypermethrin in vegetables, fruits and foliages
Various samples of vegetables, fruits and foliages each of 25 mg, were taken, collected from agricultural fields, where cypermethrin had been sprayed as an insecticide.The samples were macerated with two 20 mL portions of ethanol-demineralized water (1+1), filtered through a thin cotton cloth and filtrate was centrifuged at 1850 g for 10 min.In case of vegetables and fruits the filtrate was quantitatively transferred into a 50 mL volumetric flask and made up to the mark with 50% ethanol.Aliquots of supernatant were taken in a 25 mL graduated cylinder and then 1.0 mL of 20% sodium hydroxide was added and kept for 10 min at room temperature for complete hydrolysis.Then 1 mL of potassium iodide and leuco crystal violet was added in acidic medium and shaken thoroughly and kept for 15 min for full colour development.The solution  The amount causing an error of ±2% in absorbance value.b Tolerance limit without its removal from the sample.
was then diluted to the mark with water and absorbance was measured at 595 nm against a reagent blank.
In case of foliages, the filtrate was passed through a silica gel column (10×1 cm) filled with 5 mg silica gel, 21 which was found to be sufficient for removal of chlorophyll and other interfering materials present in the extracted sample.The column was washed with 10 mL of 50% ethanol, washings were collected in a 25 mL volumetric flask and aliquots were analysed as recommended above (Table 2).

Determination of cypermethrin in water
River water samples, which received run off water from agricultural field, were collected.These samples were filtered through a Whatman No. 40 filter paper.Aliquots of water samples were taken in a 25 mL graduated cylinder, to it sodium hydroxide was added and analysed as described above (Table 2).

Recovery of cypermethrin in biological samples
The presence of cypermethrin in blood and urine has been reported in detectable concentration. 22,23Thus the method has been applied for the determination of cypermethrin in biological samples.Synthetic samples were prepared by adding known amounts of cypermethrin to these samples and than analysed after deproteination with trichloroacetic acid 24,25 as described above (Table 3).

Conclusions
The proposed method is rapid, simple and sensitive and the reagent described here is sensitive and selective for insecticides containing a nitrile group.The lower limit of detection of the method is about 0.003 μg.
The proposed method has been applied to the determination of cypermethrin in various samples of water, vegetables, fruits, foliages and biological samples.
To check the recoveries, known amount of cypermethrin were added to various samples of vegetables, fruits, foliages and biological samples and then analysed by the proposed method (Tables 2 and 3).  a Mean of three replicate analyses; b Water sample 25 mL, 1 mL aliquot of sample was analyzed, after treatment as described in procedure; c Sample 25 g (sample taken from a field where cypermethrin had been sprayed).

Figure 1 .
Figure 1.Absorption spectra of the dye formed by reaction of cypermethrin and leuco crystal violet.

Table 1 .
Effect of foreign species for cypermethrin(10 ppm)

Table 3 .
Recovery of cypermethrin in biological samples a Mean of three replicate analyses.b Amount of biological sample = 1 mL.

Table 2 .
Determination of cypermethrin in environmental and agricultural samples