Spectrophotometric and Potentiometric Determination of Some Organophosphorus Pesticides in Bulk Powder and in their Dosage Forms

 

Abd El-Aziz B. Abd El-Aleem¹, Mamdouh R. Rezk¹, Shaban M. Khalile²,

Omneya K. El-Naggar²*

¹Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

²National Organization for Drug Control and Research (NODCAR), Giza, Egypt.

 

 

ABSTRACT:

Two simple, sensitive and precise methods were developed and validated for determination of diazinon, chlorpyrifos and temefos pesticides in bulk powder and dosage forms. The spectrophotometric method involves the oxidation of these pesticides with potassium iodate in acidic medium with the liberation of iodine and subsequent extraction with cyclohexane followed by measuring the absorbance at λ= 520 nm. Beer`s law is obeyed in the concentration range of 0.05-1.00 mg ml^-1 for diazinon and chlorpyrifos and 0.05-0.6 mg ml^-1 for temefos. The apparent molar absorbitivities of the resulting coloured products are found to be 0.539x10³, 0.368x10³ and 0.474x10³ L mol^-1 cm ^-1 for diazinon, chlorpyrifos and temefos, respectively, whereas Sandell sensitivities are 0.5647, 0.9527 and 0.9841 µg cm ^-2, respectively. The potentiometric method involves the direct titration of the studied pesticides with N-bromosuccinimide in acidic medium and the end point is determined potentiometricallyusing platinum indicator electrode. The ratio of drug: NBS was 1:3 for diazinon andchlorpyrifos and 1:6 for temefos. The proposed methods were successfully applied to the analysis of the studied pesticides in bulk powder and in dosage forms without interference from other additives.

 

 

 

INTRODUCTION:

Organophosphorus pesticides (OPP) are the most widely used pesticides amounting to more than several billion US dollars annually. Their popularity is largely contributed to their favorable characteristics such as biodegradable and short persistence compared to organochlorine pesticides ^[1] OPP protect crops from pests by inhibiting acetylcholinestrase enzyme activity in insects. They are sprayed over crops or soil causing residues to be found in surface and groundwater, fruits, vegetables and in drinking water ^[2] since the majority of the OPP are volatile and thermally stable; they are amenable to gas chromatography analysis.

 

 

The determination of OPP in fruits, vegetables and water using GC with either electron captur detector(ECD) ^[3], flame photometric detector (FPD)^[4,5,6], nitrogen phosphorus detector (NPD) ^[7,8] or mass Spectrophotometry ^[9,10] has been reported. The main objective of this study is to find fast, accurate and sensitive spectrophotometric and potentiometric methods for determination of diazinon, chlorpyrifos and temefos in their pure and dosage forms. The mechanism of oxidation of these OPP with iodate and N-bromosuccinimide is suggested in order to throw more light on the nature of the oxidation products formed.

The chemical structures of diazinon, chlorpyrifos and temefos are shown in Fig.(1)

 

 

Fig.1: The chemical structures of (a) Diazinon, (b) Chlorpyrifos and (c) Temefos.

 

EXPERIMENTAL:

Instrument:

Spectrophotometric method:

All absorbance measurements were made with a double beam UV-1601PC (Shimadzu, Japan) ultraviolet-visible spectrophotometer provided with matched 1-cm quartz cells and Temperature Controller was used for all spectrophotometric measurements.

 

Potentiometric Method:

The potentiometric measurements were performed using electronic potentiometer pH/mV-meter model Hanna HI9321 with combined platinum electrode HI3131 and magnetic stirrer Hanna model HI 300N. Automatic pipette (volac UK) of volumes ranged from 100-1000µl and automatic burette (2ml/0.01 division, bottle 500ml) were used

 

Samples and Reagents:

All chemicals used were of analytical reagent grade. Chemicals (suppliers) were as follows: Diazinon (Wenzhou Lucheng Dongon Dyestuff, Intermediate Plant, China), Chlorpyrifos and Temefos (JiangSuKnaida Agrochemical Co., Ltd, China). The following available commercial preparations were analyzed: Diazinon sand bait labeled to contain 0.5g % diazinon, Insect stop E.C labeled to contain 25 g% chlorpyrifos and Temo larvae labeled to contain 50g % temefos. All the commercial preparations are supplied from Egychem., Egypt. Potassium iodate is supplied from BDH chemicals Ltd, Poole, England. Cyclohexane from sd Fine-chem Limited, Mumbai, India. NBS from Lobachemie Pvt. Ltd, India. Doubly distilled water was used to prepare all solutions.

 

Prepared Solutions:

Potassium iodate solution 1% m/v and sulphuric acid 30% v/v were prepared for spectrophotometric assay. 1 M sulphuric acid and 0.01 M N-bromosuccinimide were prepared for potentiometric assay. 0.01M N-bromosuccinimide was prepared by dissolving 178 mg of NBS in 100 ml bidistilled water and standardized   iodimetrically^[11].

 

Stock Standard Solutions:

For Spectrophotometric Method:

A stock standard solutions (1 mg ml^-1) of diazinon, chlorpyrifos and temefos, were prepared by accurately weighing 10 mg of pure sample of each pesticide, and transferring it separately into  10-mL volumetric flasks, with addition of methanol to make up to volume.

 

For Potentiometric Method:

0.01 M solution of each pesticide was prepared by weighing 304.35 mg, 350.59 mg and 466.46 mg of diazinon, chlorpyrifos and temefos, respectively, transferring them separately into 100-ml volumetric flasks and dissolving in methanol up to the mark.

 

The Pharmaceutical Prepared Solutions:

For Spectrophotometric method:

Diazinon Sample Solution:

A mass of 10 g of Diazinonsand was transferred into 250-ml stoppered conical flask, 30 ml of methanol were added and shaked mechanically for about two hours. The prepared solution was filtered through Whatman No. 42 filter paper into 50-ml volumetric flask and completed to volume with methanol to obtain stock solution with a concentration of 1 mg ml^-1.

 

Chlorpyrifos Sample Solution:

A volume of 0.4 ml of Insect stop E.C solution was transferred into a 100-ml volumetric flask, completed to volume with methanol to obtain stock solution with a concentration of 1 mg ml^-1.

 

Temefos Sample Solution:

A volume of 0.2 ml of Temo larvae solution was transferred into a 100-ml volumetric flask, completed to volume with methanol to obtain stock solution with a concentration of 1 mg ml^-1.

 

For Potentiometric Method:

Diazinon Sample Solution:

A mass of 30.435 g of Diazinonsand was transferred into 250-ml stoppered conical flask, 30 ml of methanol were added and shaked mechanically for about two hours. The prepared solution was filtered through Whatman No. 42 filter paper into 50-ml volumetric flask and completed to volume with methanol to obtain 0.01M stock solution.

 

Chlorpyrifos Sample Solution:

A volume of 1.4 ml of Insect stop E.C solution was transferred into a 100-ml volumetric flask, completed to volume with methanol to obtain 0.01 M stock solution.

 

Temefos Sample Solution:

A volume of 0.93 ml of Temo larvae solution was transferred into a 100-ml volumetric flask, completed to volume with methanol to obtain 0.01 M stock solution.

 

Procedure:

For Spectrophotometric method:

Transfer different portions containing amounts in the range 0.05- 1 mg for diazinon and chlorpyrifos and 0.05-0.6 mg for temefos separately into series of 50-ml stoppered separating funnels. Add 3 ml potassium iodate solution and 3 ml of 30% v/v sulphuric acid then mix well. Add to each funnel 10 ml cyclohexane, shake well for 1 min to extract iodine colour, leave it for about 5 min, transfer the cyclohexane layer quantitatively into 10-ml volumetric flasks and complete the volume with cyclohexane.  Measure the absorbance at 520 nm against cyclohexane as blank.

 

For Potentiometric method:

Transfer 0.2-2 ml of each pesticide solution separately into 100-ml beakers. Add 3 ml 1 M sulphuric acid and dilute with bidistilled water to 50 ml. Titrate potentiometrically, using Pt electrode with 0.01M NBS solution by drop wise addition from automatic burette (2ml, 0.01) with constant stirring. Determine the equivalent point by the plots of E versus V and ΔE/ΔV versus V, where E is the potential (e.m.f.) of the titrating solution, V is the volume of the titrant and ΔE is the change in potential resulting from the addition of ΔV, at definite volume of the titrant.

Determination of the pesticides in dosage forms:

The procedure was done as in the bulk forms using different concentrations of the pesticide as pharmaceutical forms.

 

RESULTS AND DISCUSSION:

Spectrophotometric method:

The concentrations of diazinon, chlorpyrifos and temefos are found to be proportional to the measured absorbance at 520 nm obtained by the formation of iodine from potassium iodate during its reactions with the three pesticides under study in acidic medium Fig. (2). Different experimental conditions affecting the developed colour produced are shown in Fig. (3,4,5 and 6). In order to study the effect of time, samples are assayed and the absorbancies are determined after varying time intervals at room temperature Fig. (3). The results indicate that time intervals of 30 min for diazinon and 15 min for both chlorpyrifos and temefos are required to obtain complete reaction. Fig.(4) Shows the effect of temperature on the colour reaction. Higher absorbancies are obtained at room temperature and hence it is chosen as it gives better reproducibility. Higher temperatures are not preferred owing to the loss of iodine at higher temperatures and consequently low results. An investigation of the type of acid on the formation and extraction of iodine into cyclohexane is shown in Fig. (5). It shows that maximum absorbancies are obtained in case of sulphuric acid. The effect of the volume of 1%m/v potassium iodate was studied and was found that 1.5 mls in case of diazinon and chlorpyrifos and 3 mls in case of temefos optimum for the colour formation and stability as shown in Fig.(4).

 

 

Fig. 2: shows the maximum absorption of (a) Diazinon, (b) Chlorpyrifos and (c) Temefos at λ = 520 nm during their reaction with potassium iodate to give iodine.

 

Fig.3: Effect of time on the reaction between the studied pesticides and potassium iodate

 

Fig. 4: Effect of temperature on the reaction between the studied pesticides snd potassium iodate

 

Fig.5: Effect of type of acid on the reaction between the studied pesticides and potassium iodate

 

Fig 6: Effect of volume of potassium iodate on the formation and extraction of iodine

 

In order to prove the applicability of the proposed method and the reproducibility of the results obtained, five replicate experiments at three different concentrations of the studied pesticides are carried out. Both repeatability and intermediate relative standard deviations are less than 2 %Table (3). The small values of RSD% indicate that the proposed method is highly accurate, precise and reproducible. Applying the continuous variation Fig. (7) and molar ratio Fig.(8) methods under the selected conditions, the ratio between diazinon, chlorpyrifos and temefos : iodate was found to be 1:1 [drug: iodate] in case of diazinon and chlorpyrifos and 1:2 [drug: iodate] in case of temefos.

 

Fig. 7: Continuous variation of the redox reaction between the studied pesticides and potassium iodate

 

Fig. 8: Molar ratio of the redox reaction between the studied drugs and potassium iodate

 

Under these conditions, a linear correlation is obtained between the absorbance and the concentration over the range of 0.05- 1 mg ml^-1 in case of diazinon and chlorpyrifos and 0.05- 0.6 mg ml^-1 in case of temefos. The apparent molar absorbitivities, Sandell sensitivities and the regression line equations for each pesticide are tabulated in Table (1). The accuracy and precision of the method were evaluated by performing five replicate analyses on pure drug solutions at three different amount/ concentration levels (within the working ranges). The RSD (%), an indicator of accuracy did not exceed 0.765, 0.752 and 0.627% for diazinon, chlorpyrifos and temefos respectively, and intraday precision which is also called the repeatability expressed in relative standard deviation (RSD) (%) was within 0.045-0.120 for diazinon, 0.066- 0.128 for chlorpyrifos and 0.093- 0.149 for temefos, indicating the high accuracy and precision of the method. The results of this study are compiled in Table 3. The reproducibility of the method, also called the day-to-day precision or intermediate precision, was assessed by performing replicate analyses on pure drug solutions at three levels over a period of five days preparing all solutions afresh each day. The day-to-day RSD values were less than 1% reflecting the usefulness of the method in routine analysis of the investigated pesticides in quality control laboratories. The performance of the proposed method is assessed by comparison with reported methods ^[12,13,14] using the t-test for the accuracy and F-value for the precision, the calculated values did not exceed the corresponding theoretical values, indicating insignificant differences between results as shown in Tables (4) and (5).

 

 

Table 1: Regression equations and validation parameters for the proposed spectrophotometric method for the determination of diazinon, chlorpyrifos and temefos.

 

*Y=a+bX, where Y is the absorbance, a is the intercept, b is the slope and X is the concentration in µg ml^-1.

**LOD is limit of detection=(3.3Xσ)/ S where _σis the standard deviation of 5 replicate determinations under the same conditions as for the sample analysis in the absence of the analyte and S is the sensitivity, namely the slope of the calibration graph.

***LOQ is the limit of quantification ==(10Xσ)/ S .

Table 2: Determination of diazinon, chlorpyrifos and temefos in pure form by the proposed spectrophotometric method.

Diazinon			Chlorpyrifos			Temefos
Taken mg ml-1	Found*  mg ml-1	Recovery %	Taken mg ml-1	Found* mg ml-1	Recovery %	Taken mg ml-1	Found* mg ml-1	Recovery %
0.300 0.500 0.700	0.304 0.509 0.710	101.33 99.80 100.43	0.300 0.500 0.700	0.298 0.495 0.703	99.33 99.00 100.43	0.300 0.500 0.700	0.302 0.498 0.705	100.67 99.60 100.71
Mean		100.52	Mean		99.59	Mean		100.33
SD		0.769	SD		0.749	SD		0.629
RSD%		0.765	RSD%		0.752	RSD%		0.627

* Average of five determinations.

Table 3: Intra and inter-day validation for the determination of diazinon, chlorpyrifos and temefos using the proposed spectrophotometric method.

 

 

Table 4: Statistical analysis of the results obtained by applying the proposed spectrophotometric  and reported methods ^[12,13,14]for the determination of pure diazinon, chlorpyrifos and temefos.

The theoretical values of t-test= 2.780 and of F value= 6.390 at 95% confidence level

 

 

 

Table 5: Statistical analysis of the results obtained by applying the proposed spectrophotometric and reported methods^[12,13,14]for the determination of diazinon, chlorpyrifos and temefos in dosage forms.

The theoretical values of t-test= 2.780 and of F-value=6.390 at 95% confidence level

 

 

Potentiometric Method:

N-bromosuccinimide (NBS) was one of the most important positive bromine containing compounds. The usefulness of the reagent was increased by the variety of experimental conditions under which it could be used to bring about particular type of reactions. NBS undergoes various reaction types extending from oxidation, addition, substitution of allylic and benzylicbromination. The oxidation reactions of NBS generally involves the abstraction of hydrogen from C-H, O-H, N-H or S-H bond, though reactions involve the addition of oxygen had been also observed ^[11]. Some methods are based on the fact that NBS readily and quantitatively brominates an aqueous acidic solution of some compounds and is itself irreversibly reduced to succinimide^[15]. NBS is reported to react with some aliphatic tertiary amines to produce secondary amines, with the removal of one of the alkyl group to form an aldehyde ^[11]and substitute it by Br radical. Some of organic compounds containing sulphur, the sulphur atom can be oxidized, as example in phenothiazine derivatives^[16] and tenoxicam and piroxicam^[17]. In this work a potentiometric method is described for the determination of diazinon, chlorpyrifos and temefos pesticides in pure and dosage forms. NBS is found to react quantitatively with the studied pesticides in sulphuric acid medium. Diazinon, chlorpyrifos and temefos are directly titrated potentiometrically in H₂SO₄ medium with 0.01 M NBS as a titrant. The titration curves of diazinon, chlorpyrifos and temefos show well defined S-shaped stoichiometric end point Fig.(9).The determination of the end point from the potentiometric data will help in the calculation of the pesticide concentration. The potential (E_h) jumpat the end point is amount in average of 300 mV/0.1 ml of NBS titrant.

 

Effect of acids:

It is found that, H₂SO₄ medium is chosen as the suitable medium for potentiometric determination of diazinon, chlorpyrifos and temefos more than HCl and HNO₃ acids. The end points are shifted away from the expected value in both HCl and HNO₃ and no sharp reflection in the end point, due to the reducing properties of HCl which consumes more NBS and HNO₃ oxidation power that leads to less consumption of NBS.A study of the stoichiometry of the reaction in different acids with different concentrations revealed that 3 ml of 1 M sulphuric acid give the best results. Also, the jump in the potential at the end point per 0.1 ml NBS titrant is higher in H₂SO₄ medium than HCl and HNO₃ mediums.

 

(a)

 

   

(b)

 

  

(c)

Fig. 9: Typical titration curves used for potentiometric determination of end point of the reaction between NBS and the studied pesticides using Pt electrode (a) 0.5 ml diazinon, (b) 0.5 ml chlorpyrifos, (c) 0.5 ml temefos

 

For stoichiometric study, it is found that; three moles of NBS are required for complete oxidation of each mole of diazinon and chlorpyrifos and six moles of NBS are required for complete oxidation of one mole of temefos.  The method used in these computations is the parametric method (PM₃) described by Stewart ^[18]. The program is running under the molecular orbital calculation package ^[19]. From the molecular orbital calculation study for the structure of the three pesticides in both neutral and cationic forms using semi-empirical molecular orbital calculations and applying parametric method (PM₃) it is clear that:

a)      Diazinon in both neutral and cationic forms may refer to the possible bond rupture P₈ – S₁₅ (bond length 1.923, bond order 1.134), P₈ – O₁₂ (bond length 1.694, bond order 0.749) and P₈ – O₉ (bond length 1.680, bond order 0.798) as shown in Table (6).

 

b)      Chlorpyrifos started its structure decomposition by weaker bond P₈ – S₉ (bond length 1.912, bond order 1.175) followed by bond P₈ – O₁₃ (bond length 1.685, bond order 0.766) then P₈ – O₁₀ (bond length 1.681, bond order 0.757) in neutral form as shown in Table (7).

c)      Temefos: the possibility of bond rupture is for P₈ – S₁₃ (bond length 1.927, bond order 1.326), P₈ – O₁₁ (bond length 1.708, bond order 0.812) and P₈ – O₉ (bond length 1.709, bond order 0.859) for the right side of the structure, and so for the left side of the structure where  P₂₂ – S₂₇ (bond length 1.924, bond order 1.333),  P₂₂ – O₂₃ (bond length 1.706, bond order 0.813) and P₂₂ – O₂₄ (bond length 1.704, bond order 0.862)  as shown in Table (8).

 

 

Table 6: Shows the bond length, bond order and atomic charge of diazinon.

 

 

 

Table 7: Shows the bond length, bond order and atomic charge of chlorpyrifos.

 

 

Table 8: shows the bond length, bond order and atomic charge of temefos.

 

The same order of bond rupture can be detected in cationic forms of the pesticides. This ordering depends upon the rupture of largest bond length and least bond order. From tables 6,7 and 8 it is noticed that most active bonds are P₈–S₁₅ , P₈–O₁₂ and P₈-O₉ for diazinon, P₈–S₉, P₈ – O₁₃ and P₈–O₁₀ for chlorpyrifos, P₈–S₁₃, P₈– O₉ and P₈-O₁₁ for the right side of the temefos structure and P₂₂ – S₂₇, P₂₂ – O₂₃ and P₂₂ – O₂₄ for the left side. This means that the two pesticides diazinon and chlorpyrifos have three centers for the possibility of the reaction with NBS and the two pesticides have a ratio of 1:3 [pesticide: NBS] and that for temefos, which has symmetrical groups in both sides, has six centers for the reaction, so the stoichiometry of the reaction is 1:6 [pesticide : NBS]. This stoichiometric ratio obtained by molecular orbirtal calculations is confirmed by practical data that refer to the number of exchanged electrons between reactants is 6 electrons. From the calculated heat of formation ΔH (Kcal/ mol) and total energy values, the most stable one is temefos as shown in Table (9).

 

Table 9: shows the total energy, ΔH and IE of diazinon, chlorpyrifos and temefos.

 

Potentiometric determination of diazinon, chlorpyrifos and temefos pesticides using NBS titrant:

After the selection of suitable ratio and acid it is also suitable to know the concentration limits of these materials at which these reactions are quantitative and consequently the evolution of applying Pt-electrode and proposed procedures to determine the cited drugs quantitatively via titration with NBS. Table (10) shows the results of studying quantitativeness of the titration between the pesticides at different concentrations and the percent recovery was calculated.

The pesticides can be calculated from these equations:

1 ml 0.01 M NBS ≡ 1.013 mg diazinon

1ml 0.01 M NBS ≡ 1.167 mg chlorpyrifos

1ml 0.01 M NBS ≡ 0.777 mg temefos

 

Diazinon, chlorpyrifos and temefos can be determined in the concentration range of 0.6-6, 0.7-7 and 0.93-9.33 mg ml^-1 for diazinon, chlorpyrifos and temefos, respectively. The relative standard deviation values are found to be 1.406 %, 1.151 % and 1.064 % for diazinon, chlorpyrifos and temefos, respectively as shown in  Table (10) which indicates that N-bromosuccinimide can be used successfully in the determination of diazinon, chlorpyrifos and temefos in pure forms. This method is also applied to diazinon, chlorpyrifos and temefosin their dosage forms. The fact that, the mV values before the end points in the titration curves of pure pesticides and their corresponding dosage forms are almost identicals, provide evidence that, the other excipients that might be present in dosage forms do not affect the titration curves. Table (11) summarizes the results obtained for diazinon, chlorpyrifos and temefos in their dosage forms. The recoveries are in good agreement with the reported methods[12, 13, 14] and the RSD > 2%. Thus, the reproducibility and accuracy is very satisfactory for the analysis of dosage forms as well as pure forms.

 

 

Table 10: Determination of diazinon, chlorpyrifos and temefos in pure form by the proposed potentiometric method.

Diazinon			Chlorpyrifos			Temefos
Taken mg ml-1	Found* mg ml-1	Recovery %	Taken mg ml-1	Found* mg ml-1	Recovery %	Taken mg ml-1	Found* mg ml-1	Recovery %
1.00 2.00 3.00	1.02 2.03 2.98	102.00 101.50 99.33	1.00 2.00 3.00	1.01 1.98 3.03	101.00 99.00 101.00	1.00 2.00 3.00	1.00 2.04 3.01	100.00 102.00 100.33
Mean		100.94	Mean		100.33	Mean		100.78
SD		1.419	SD		1.155	SD		1.072
RSD%		1.406	RSD%		1.151	RSD%		1.064

 

 

 

Table 11: Statistical analysis of the results obtained by applying the proposed potentiometric and reported methods^[12,13,14]for the determination of diazinon, chlorpyrifos and temefos in dosage forms.

Pesticide	Studied method	Reported methods [12,13,14]	n	Student's t-test	F value
Diazinon in Diazinon 0.5g% sand bait	101.09±0.874	100.23±0.730	5	1.689	1.433
Chlorpyrifos in Insect stop 25g% E.C	99.59±0.318	99.42±0.190	5	1.026	2.801
Temefos in       Temo larvae 50g%	100.67±0.429	100.41± 0.522	5	0.829	1.480

The theoretical values of t-test= 2.780 and of F-value=6.390 at 95% confidence level.

 

CONCLUSION:

The two described spectrophotometric and potentiometric methods are simple and selective for the quantitative determination of diazinon, chlorpyrifos and temefosin their pure forms and in their dosage forms without interference from excipients. These methods have linear response in the stated ranges with good accuracy and precision. They can therefore be used for routine analysis by quality control departments.

 

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Received on 11.03.2017        Modified on 05.05.2017

Accepted on 11.06.2017     ©A&V Publications All right reserved