A Catalytic Kinetic Spectrophotometric Determination of Organophosphorus Pesticides in Vegetable Samples

Um método cinético catalítico espectrofotométrico, simples, seletivo e sensível para a determinação de quantidade traço de pesticidas organofosforados foi proposto. O método é baseado no efeito catalítico de pesticidas organofosforados (malationa, dimetoato e forato) na oxidação de LCV (leuco cristal violeta) por iodato de potássio em meio de ácido clorídrico, produzindo um corante violeta. O corante apresenta absorção máxima em 592 nm. O método de tempo fixo foi usado por 15 min. O sistema obedece a lei de Beer no intervalo de 0,02-0,2, 0,032-0,32 e 0,03-0,3 μg mL, para malationa, dimetoato e forato, respectivamente. Parâmetros analíticos importantes como tempo, temperatura, concentração do reagente, acidez, etc, foram otimizados para reação completa. A sensibilidade de Sandell e absortividade molar para o sistema foram 0,0002, 0,0004, 0,0004 μg cm e 1,2 × 10 , 5,21 × 10, 6,3 × 10 L molcm, respectivamente. O método proposto foi aplicado satisfatoriamente à determinação de pesticidas organofosforados, em micro concentrações, em amostras vegetais.


INTRODUCTION
Organophosphorus pesticides are potentially hazardous substances widely used in agriculture due to their high insecticidal activity [1]. Organophosphates are organic esters of phosphoric acid, thiophosphoric acid and other phosphoric acids [2]. These pesticides are toxic for mammals due to the inhibition of the acetyl cholinesterase (AChE), an enzyme necessary for the normal function of the central nervous system [3][4]. The pesticide residue causes serious health hazard, its ill effect can be reduced to minimum by detecting and then controlling it [5]. A number of methods have been developed in the last few years for the detection of organophosphorus pesticides. The most widely used methods are gas chromatography (GC) [6][7][8], high-performance liquid chromatography (HPLC) [9][10], gas chromatography-mass spectrometry (GC-MS) [11], immune assay and fluorescence [12][13] and chemiluminescence (CL) [14][15][16].
Different methods like HPLC, GC and GC-MS have been reported but despite the precision and accuracy of these methods, analyses are restricted to laboratory facilities, are time-consuming and expensive due to its analytical cost, limiting the operation of these instruments to highly qualified laboratory persons while spectrophotometry is considered the most convenient analytical technique because of its inherent simplicity, low cost, and wide availability in most of the laboratories.
Some spectrophotometric methods have also been reported in which most of the methods involve the determination of organophosphorus pesticides by total phosphorous measurements based on the formation of molybdenum blue using various reducing agents [17][18][19] and some are based on the oxidation of pesticide and determination of unconsumed oxidant by bleaching of dye [20]. Some of the methods suffer from interference, poor sensitivity, instability of color or involve extraction whereas other suffers from blank absorption or longer time required for the color development. To overcome these drawbacks a selective and sensitive method has been proposed for the determination of the studied organophosphorus pesticides.
In the present study a validated, rapid, sensitive, selective kinetic spectrophotometric method for the determination of organophosphorus pesticides is developed. The developed method is based on the catalytic effect of organophosphorus pesticides on the oxidation of LCV by potassium iodate in acidic medium. The proposed method is specific for thion compounds containing P=S bond like Malathoin, Dimethoate, Phorate. Now days these pesticides are increasingly being used in agriculture.

EXPERIMENTAL Apparatus
A systronics spectrophotometer 166 was used for spectral measurements. pH measurements were made with systronics digital pH meter 335.

Reagents
All chemicals used were of analytical grade, double deionized water has been used for preparation of solutions.

RESULTS AND DISCUSSION
LCV (Leuco crystal violet) is a compound which can be oxidized with potassium iodate at very slow reaction rate. It has been reported that organophosphates have an induction effect on iodine azide reaction [22], similarly in the proposed method organophosphorus pesticides act as a catalyst in the reaction between LCV and potassium iodate in the presence of hydrochloric acid medium. An organophosphorus pesticide increases the rate of this reaction at ultra trace level, therefore by measuring the increase in absorbance of CV (Crystal violet) which is the oxidized product of

Spectral characteristics and method validation
The absorption spectra of final colored product showed a maximum absorbance at 592 nm (Fig. 1). The reagent blank had negligible absorbance at this wavelength. Beer's law was obeyed over the concentration range of 0.02-0.2, 0.032-0.32 and 0.03-0.3 µg mL -1 for malathion, dimethoate and phorate respectively (Fig. 2). The curve was linear with different slopes for malathion, dimethoate, phorate and has a good correlation. The molar absorptivities and Sandell's sensitivity of malathion, dimethoate and phorate are given in table 1. The slope, intercept, and the correlation coefficient were calculated by least square regression analysis ( Table 1) Table 1).

Effect of acid concentration
The effect of various acids of same concentration such as sulphuric acid, acetic acid, nitric acid has been studied. The results show that hydrochloric acid gives greater sensitivity. The effect of hydrochloric acid concentration on obtaining maximum sensitivity was investigated with 4.7×10 -3 mol L −1 potassium iodate, and 6.7×10 -4 mol L −1 LCV for catalyzed and uncatalyzed reaction at 35 0 C (Fig. 3 ).

Effect of potassium iodate concentration
The effect of potassium iodate concentration in the reaction rate was studied with 2 mol L −1 hydrochloric acid and 6.7×10 -4 mol L −1 LCV at 35 0 C. The results show that by increasing the potassium iodate concentration up to 4.7×10 -3 mol L −1 the sensitivity increases, whereas a greater amount of reagent decreases sensitivity. Thus 4.7×10 -3 mol L −1 potassium iodate was selected throughout the study (Fig. 4).

Effect of Leuco crystal violet concentration
The influence of LCV concentration on the reaction rate was studied in the range of 1×10 -4 to 10×10 -4 mol L −1 with 2 mol L −1 hydrochloric acid and 4.7×10 -3 mol L −1 potassium iodate at 35 0 C. The results show that by increasing the LCV concentration up to 6.7×10 -4 mol L −1 , the sensitivity increases, whereas a greater amount of reagent decreases sensitivity, thus 6.7×10 -4 mol L −1 LCV was selected for the study (Fig. 5).

Effect of temperature and time
The effect of temperature on the catalytic reaction was studied in the range of 10 0 C to 70 0 C with the optimum of the reagent concentrations. The results showed that as the temperature increases up to 35 0 C the sensitivity increases, whereas higher temperature values decreases the sensitivity (∆A s -∆A b ). Therefore, 35 0 C was selected for further study and 15 min time was suitable for the study of catalytic reaction (Fig.   6).

Interference studies
The effect of foreign ions and pesticides expected to exist in field samples of organophosphorus pesticide were studied by adding known amount of different foreign ions to the test solution containing 0.1 µg mL -1 organophosphorus pesticide.
The method was found to be free from most of the interferents. The tolerance limits shown in table 2 are the concentration of interfering species that may cause ≤ (±) 2% variation in the absorbance value.

Determination of organophosphorus pesticides in real vegetable samples
Vegetable samples were collected from agricultural field where organophosphorus pesticide (malathion) was sprayed (1 to 5 pints per acre). The samples were weighed, macerated with ethanol and then filtered through a thin cotton cloth. The filtrate was centrifuged at 1850 g for 10 min. An aliquot of supernatant was taken and evaporated then residue is dissolved in 0.1% acetic acid and analyzed as described above. The filtrate which was greenish yellow due to the presence of organic matter from plant was passed through a silica gel column (10 × 1 cm) to remove chlorophyll and other interfering materials. The column was washed with 10 mL of 0.1% acetic acid.
Washings were collected and analyzed as recommended above by the proposed as well as the conventional ascorbic acid method [21] and the results are shown in table   4.
Ascorbic acid method involves the reaction of orthophosphate with molybdate in acid solution, which forms a yellow-colored phosphomolybdate. The phosphomolybdate complex is then reduced by ascorbic acid, causing a characteristic molybdenum blue species which is measured at 700 nm.

CONCLUSION
The proposed method is more sensitive, simple and selective as compared with other spectrophotometric methods for determination of organophosphorus pesticides. The 100 reported spectrophotometric methods are indirect, lengthy, time consuming, suffer from interference, poor sensitivity, instability of color, blank absorption or involve extraction. The rapid color development, stability and easy availability of the reagent and freedom from a large group of interfering species are some advantages of the method. As the presented method is based on catalytic effect on rate of reaction it can be further applied for simultaneous determination of these pesticides by applying initial rate and partial least square analysis method.