DEVELOPMENT OF THE KINETIC-SPECTROPHOTOMETRIC METHOD FOR QUANTITATIVE DETERMINATION OF ZOPICLONE IN TABLETS BY THE PERHYDROLYSIS REACTION

A simple and express method for the quantitative determination of zopiclone in model solutions of the substance and in “Zopiclone” tablets, 7.5 mg, by the kinetic-spectrophotometric method according to 3,3’,5,5’-tetramethylbenzidine oxidation has been developed. It is based on the system of two coupled reaction: 4-methyl-1-piperazineperoxycarboxylic acid generated in zopiclone perhydrolysis reacts with the excess of hydrogen peroxide in the weak alkaline medium with formation of coloured 3,3’,5,5’-tetramethyldiphenylquinone diimine (λmax = 420 nm, рН = 8.4). The reaction is performed spectrophotometrically by measuring the rate of change of the absorbance at 420 nm. The method was used for constructing the calibration graph. The initial rate of the reaction was obtained from the linear site of the slope of the initial tangent to the absorbance-time curve. In the pH range of 8.2-8.5 the rate of the coloured product formation becomes maximum. The calibration graph for zopiclone has a linear dependence in the range of 6-36 mg/l with the limit of detection (LOD) and quantitation (LOQ) of 1.81 and 6.04 mg/l, respectively. For five determinations of 18, 24 and 30 mg/l of zopiclone RSD is 1.81, 1.46 and 1.69%, respectively. The analytical performance of the method was validated statistically with respect to LOD, LOQ, accuracy, precision and linearity for zopiclone estimation in a pure substance and the results were satisfactory. “Zopiclone” tablets compared to the reference method contain 99.83±1.19% of C17H17ClN6O3 (RSD = 0.96% , δ = -0.17%). The assay of zopiclone in the presence of its hydrolysis products without preliminary separation is an important advantage of the method.

A simple and express method for the quantitative determination of zopiclone in model solutions of the substance and in "Zopiclone" tablets, 7.5 mg, by the kinetic-spectrophotometric method according to 3,3',5,5'-tetramethylbenzidine oxidation has been developed.It is based on the system of two coupled reaction: 4-methyl-1-piperazineperoxycarboxylic acid generated in zopiclone perhydrolysis reacts with the excess of hydrogen peroxide in the weak alkaline medium with formation of coloured 3,3',5,5'-tetramethyldiphenylquinone diimine (λ max = 420 nm, рН = 8.4).The reaction is performed spectrophotometrically by measuring the rate of change of the absorbance at 420 nm.The method was used for constructing the calibration graph.The initial rate of the reaction was obtained from the linear site of the slope of the initial tangent to the absorbance-time curve.In the pH range of 8.2-8.5 the rate of the coloured product formation becomes maximum.The calibration graph for zopiclone has a linear dependence in the range of 6-36 mg/l with the limit of detection (LOD) and quantitation (LOQ) of 1.81 and 6.04 mg/l, respectively.For five determinations of 18, 24 and 30 mg/l of zopiclone RSD is 1.81, 1.46 and 1.69%, respectively.The analytical performance of the method was validated statistically with respect to LOD, LOQ, accuracy, precision and linearity for zopiclone estimation in a pure substance and the results were satisfactory."Zopiclone" tablets compared to the reference method contain 99.83±1.19% of C 17 H 17 ClN 6 O 3 (RSD = 0.96% , δ = -0.17%).The assay of zopiclone in the presence of its hydrolysis products without preliminary separation is an important advantage of the method.
A simple kinetic method of the zopiclone determination by its perhydrolysis product with p-phenetidine as a chromogenic substrate was previously proposed [1].The assay of zopiclone in the presence of its hydrolysis products and sensitivity is a distinctive advantage of this method.
The aim of this work was to develop a new kinetic method of the quantitative determination of zopiclone using the indicator reaction of 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by hydrogen peroxide in the weak alkaline medium.
To create and maintain the pH required 0.2 M phosphate buffer with pH 8.4 prepared according to Green was used.For this purpose 12 g of NaH 2 PO 4 was dissolved in 450 ml of DDW and 50.6 ml of 1.9 mol/l NaOH was added, pH was monitored potentiometrically.
Preparation of TMB working solution with the molar concentration of 1×10 -2 mol/l.Dissolve 0.313 g of the accurately weighed portion of TMB in a 100 ml volumetric flask in 50 ml of 96% ethanol.Dilute to the volume with DDW at 20°C and shake thoroughly.
Preparation of zopiclone working standard solution (WSS), 0.3 mg/ml (с = 7.7×10 -4 mol/l).Dissolve 0.0300 g of the zopiclone substance in 96% ethanol in a 100 ml volumetric flask, dilute the solution to the volume with the same solvent and mix thoroughly.
All the chemicals and reagents were of analytical grade and the solutions were freshly prepared.ISSN 1562-7241 All spectrophotometric measurements were made on a SF-46 spectrophotometer (LOMO, USSR) with 1 cm matched quartz cells.The pH of the solutions was monitored by a glass electrode of ESL 43-07 type (the reference electrode -a silver/silver chloride electrode of EVL-1M3.1 type) on a laboratory I-130 ionometer ("Analytpribor" Research and Production Association).
The method of obtaining the data for the calibration curve.Transfer consistently 10 ml of 0.2 mol/l buffer solution (pH = 8.4) into a 25 ml volumetric flask adding from 1 to 5 ml of 0.3 mg/ml WSS, 6.0 ml of 1×10 -2 mol/l TMB solution, 2 ml of 5,6 mol/l H 2 O 2 , dilute to the volume with DDW at 20°C and shake thoroughly for 30 sec.Measure the optical density of the solution obtained at 420 nm vs. blank solution (without the substance to be examined).Control the time from the moment of mixing the solution with the stop-watch.
The results obtained were processed according to the recommendations of the International Union of Pure and Applied Chemistry (IUPAC) [3] and the State Pharmacopeia of Ukraine (SPhU) [2] using mathematical statistics methods.Accuracy verification was performed by "input-output" analysis of the model solution.The content of the active substance in "Zopiclone" tablets, 7.5 mg, was determined by the method of standard.

Results and Discussion
The reaction involved in the present study is based on the perhydrolysis reaction of zopiclone with the excess of hydrogen peroxide in the weak alkaline medium forming 4-methyl-1-piperazinepercarboxylic acid (PA).Then PA formed reacts with TMB to give coloured 3,3',5,5'-tetrametyldiphenoquinone diimine derivative, which exhibits absorption maxima at 420 nm, рН = 8.4 (Fig. 1).
The present study was devoted to the involvement of this colour reaction in the determination of zopiclone.
It has been shown that hydrolytic cleavage products that are present do not interfere the zopiclone determination.The maximal rate of the coloured product formation was observed in the рН range of 8.2-8.5.The initial rate of the reaction was obtained from the linear site (3-10 min) of the slope of the initial tangent to the absorbance-time curve (Fig. 2).
As can be seen from Fig. 2, the initial rate increases with the increase of zopiclone concentration.Since the concentration of TMB and hydrogen peroxide is much higher than the analyte concentration, the course of the indicator reaction became of the pseudo first order with respect to zopiclone, therefore, its perhydrolysis is the limiting stage of the process.The calibration curve obtained by plotting the conditional initial rate of the reaction versus the final concentration of zopiclone under the optimum conditions showed a linear relationship in the range of 6-36 mkg/ml (Fig. 3).
1. Chemistry of zopiclone perhydrolysis and coupled TMB peroxyacid oxidation.The method of least square was used to estimate the regression characteristics of the calibration curve obtained (Tab.1).
The results of the analysis of zopiclone in model solutions and in tablets are shown in Tab. 2 and Tab. 3, respectively.
The assay for the active substance in tablets.Shake carefully the accurately weighed portion of the tablet powder equivalent to 7.5 mg of zopiclone with 10 ml of 96% ethyl alcohol, filter to a 25 ml volumetric flask, dilute to the volume with the same solvent, wash the precipitate and mix thoroughly.
Transfer consistently 10 ml of 0.2 mol/l buffer solution (pH = 8.4) into a 25 ml volumetric flask adding 2 ml of the test zopiclone solution and then continue as when plotting the calibration curve.Similarly perform the experiment with The zopiclone content in one tablet, X (g), was calculated by the formula: where С st -is the concentration of zopiclone in WSS, mg/ml; tgα -is the tangent of the angle slope in the experiment with the test solution of zopiclone, min -1 ; tgα stis the tangent of the angle slope in the experiment with WSS, min -1 ; 25 -is the volume of the volumetric flask, ml; m w -is the amount of zopiclone in a tablet, g; m -is the avarage tablet weight, g.CONCLUSIONS 1.A simple and express kinetic spectrophotometric method for determination of zopiclone has been proposed.
2. The assay of zopiclone in the presence of its hydrolysis products without preliminary separation is an important advantage of the method described.
3. The analytical method for quantitative determination of zopiclone in the substance has been validated statistically according to the criteria of LOD, LOQ, accuracy, precision and linearity.The results obtained are satisfactory; LOQ is 6.04 mg/l.

Table 1
The data of regression analysis tgα = b × С + a where С is the concentration of zopiclone, mg/ml, tgα is the conditional initial reaction rate, min -1 . *

Table 2
Metrological characteristics of the results of the kinetic determination of zopiclonein model solutions (n = 5, Р = 0.95)

Table 3
Metrological characteristics of the results of the kinetic determination of zopiclonein tablets (n = 5, Р = 0.95)