Determination of hydrolisis products of clozapine in biomaterial by uv-spectrophotometry method


UV- spectrophotometry method to identify and quantify hydrolysis products of clozapine has been developed. Factors that affect clozapine oxidation reaction have been studied: type and concentration of oxidizers, pH, salt adding, adding order, temperature, excipients. Adsorption maximum- 308 nm. The method was validated: R2 - 0,9998, RSD - 1,1%.

Key words: UV- spectrophotometry, oxidation products, identification, clozapine, validation.

UV- spectrophotometry is widely used in modern chemical, pharmaceutical and toxicological analysis to identify and quantify substances, drugs and toxins.

The theory of this method described in most literature and national pharmacopeias (Kazakh pharmacopeia, Brit. Ph, Eur. Ph., USP).

It is known that spectrophotometry analysis of colozpine is carried out in 0,1 M hydrochloric acid. Clozapine has two adsorption peaks - 242±2 nm and 290±2 nm.

Clozapine is oxidized easily and metabolism in the body is oxidizing hydrolysis. Therefore, we have studied the oxidation products of clozapine by spectrophotometry method.

Research aim is to create simple and sensitive spectrophotometric method to identify clozapine in substance, tablets and biomaterial.

Materials and methods: Clozapine was obtained from scienTEST bioKIMEX GmbH (Germany). Solvents were obtained from POCH (Poland). High-purity deionized water was used throughout the study.

All containers used in this work were made up of high quality glass and washed with high-purity deionized water.

Chromatographic analysis was achieved using a HPLC system (Shimadzu LC-20A, Shimadzu Corporation, Japan) equipped with a SIL-20Aht autosampler, a DGU 20A5 degasser, a SPD-20A UV detector and CTO-10ASvp column oven. A computer equipped with Shimadzu LC Solution software was utilized to record chromatograms. A XBridge C8 column pack with 5.0 micron particle size of dimethyloctylsilyl bounded amorphous silica (4.6*250 mm i.d., Walters, Ireland) was used for the chromatographic separation. The column temperature was maintained at 37°C. The flow rate was 1.0 mL/min. The UV detector was set at a wavelength of 240 nm.

The proposed method is based on the oxidation of clozapine with potassium bromate in an acidic medium (perchloric acid) and in the presence of surfactant Tween-20 to give yellow product which shows a maximum absorbance at 308 nm.

Spectrophotometric properties of the colored oxidation product, as well as various experimental parameters affecting the development and stability of the colored product were carefully studied and optimized. Studied factors include the type and concentration of the oxidant, pH of the medium and the presence of surfactant, the presence of water-miscible organic solvent, the order of addition of reagents, time required to achieve maximum optical density, the presence of added salts and reaction temperature.

Influence of the type and concentration of the oxidizer. Clozapine reacts with potassium bromate in an acidic medium. At the same time an intense yellow color, which is characterized by the maximum optical density at 308 nm (Figure 1). Test solution - a placebo of potassium bromate, perchloric acid and Tween 20 has a slight absorption of 0.056. On the other hand, oxidation of clozapine to chlorates (ClO3-), iodates (IO3), periodates (IO4-) or persulfates

(S2O82-), gives a colored product of oxidation in the range of 260-1100 nm.

Potassium bromate concentration effect was studied in the range of 3-70 mol/l. The maximum absorbance of the oxidation product (Dmax) increased to 25mol/l and remained almost unchanged up to 70 mol/l. Meanwhile, the time required to reach maximum absorption (Tmax) decreases gradually with the concentration of potassium bromate, reaching 1 min at 30 mol/l (Figure 2).

Values of obtained peaks were stable for 30 minutes at a concentration of potassium bromate and 50 mol/l. However, at higher concentrations, the color stability is significantly decreased. For example, potassium bromate at concentrations of >60 mol/l uptake was very unstable and has reached its maximum value only when mixed, remains nearly constant for a few seconds and then gradually decreased. Thus, the concentration of potassium bromate 40 mol/l was chosen in this study as optimum.

Influence of medium. Clozapine is practically insoluble in water, but it may be dissolved in a water miscible organic solvent such as tetrahydrofuran, acetonitrile, ethanol, methanol, or acidic conditions. Therefore, we investigated the influence of different solvents and acids. The presence of 5% and 10% concentration of THF yielded values of Dmax, which is 16% and 30% lower than those obtained in its absence. Moreover, the concentration of acetonitrile up to 10% has little effect on the values of absorbance. However, ethanol and methanol, even at low concentrations joined with potassium bromate in an oxidation reaction in the presence of clozapine.

Thus, an intense yellow color (due to the formation of free bromine Br2), was obtained even in the absence of clozapine. On the other hand, the presence of hydrochloric acid or nitric acid resulted in a very low optical density of the blank sample with absorption bands at 334 and 302 nm due to the formation of free chlorine CI2 and the presence of nitrate ions NO3-, respectively. Also, the use of phosphoric acid produced very low values of absorption, and the use of perchloric acid or sulfuric acid, which does not react with potassium bromate, gave a high optical density of the sample is not reliable for quantitative measurements. Thus, the dissolution of clozapine was performed in 0.1 mol/l perchloric acid or sulfuric acid.

Effect of acid concentration. The effect of sulfuric acid and perchloric acid in a concentration of 0.01 to 1.00 mol/l was studied. Perchloric acid gives higher Dmax values compared with sulfuric acid (Figure 3). Absorption (Dmax) increases with the acid concentration to 0.55 mol/l and then remains constant up to 1.0 mol/l perchloric acid. However, at concentrations of >0.8 mol/l perchloric acid absorbance values were inconsistent and only reached a maximum value when mixed, remains unchanged for several seconds and then decreased gradually. Thus, the concentration of chloric acid 0.60 mol/l was chosen herein as optimal.


Effect of adding salt. The effect of the addition of 0.01 - 0.30 mol/l ammonium sulfate on the color intensity and stability was studied. These concentrations have virtually no effect on the values of Dmax and T A max.

Effect of type and concentration of surfactant. Surfactants not only increase the solubility, but also increase absorption. Thus, it was tested the effect of certain anionic, cationic and nonionic surfactants. It should be noted that the effect of the cationic surfactants was tested in the presence of 0.6 mol/l sulfuric acid instead of perchloric acid to avoid the formation of poorly soluble perchlorate ions. Table 1 shows that Tween-20 is the best surfactant. Furthermore, the addition of Tween 20 at concentrations of 0.05, 0.07, 0.1, 0.2, 0.3% gave absorbance values 0.605, 0.640, 0.665, 0.670, 0.670, respectively. Thus, the concentration of Tween-20 in 0.2% was selected herein as optimal.







Sodium sulfate

10 mol/l


Sodium benzene sulfanate

10 mol/l



Cetyltrimethyl ammonia sulfate

2 mol/l


Mercetyltrimethyl ammonia sulfate

2 mol/l




0,1 об %



0,1 об %


Table 1 - Effect surfactant on clozapine oxidation

Effect of order of addition of reagents. Different orders of addition of reagents gave almost the same values of Dmax. However, potassium bromate can not be added immediately after the perchloric acid to avoid the formation of a high local concentration of small soluble potassium chlorate. Therefore, potassium bromate has been added as the last reagent, i.e. after addition of the surfactant Tween 20 and diluting with water.

Influence of the reaction temperature. Oxidation of clozapine with potassium bromate was studied at different temperatures (15-50°C). Values of Dmax oxidation product were nearly constant, regardless of temperature. Nevertheless, the time required to reach maximum absorbance Tmax temperature dependent. Tmax values were 420, 270, 150, 65, 30, 10, 5 and 0 seconds at 15, 20, 25, 30, 35, 40, 45 and 50 ° C respectively. Consequently, work was carried out at 35°C.

Effect of adjuvants. Systematic study of the effect of excipients were conducted by adding a certain amount of adjuvant to 10 ml of a 5 mg/ml solution of clozapine, and then, insoluble excipients, optionally


washed and all residues were combined in 20 ml volumetric flask and analyzed by the above mentioned method. The results showed that excipients (lactose, starch, magnesium stearate, talc, povidone, which is usually filled with clozapine drugs) have no significant effect.

Method of clozapine detection in substance by UV-spectrophotometry. Standard solutions of clozapine (60 mg/ml) were prepared in a series of test tubes by 5 ml, to make 0.5 ml of perchloric acid and 0.5 ml of Tween-20 and added with water to solution volume 4.5 ml. The resulting solution was heated to 370°C, to this solution was added 0.5 ml of a solution of potassium bromate and shaken. Measure the absorbance of the resulting solution in a spectrophotometer at a wavelength of 308 nm. Reference solution is purified water.

Method of clozapine detection in tablets by UV-spectrophotometry. Take 10 pills of 100 mg of clozapine and pulverized to a powder. About 0.0010 g (accurately weighed) pounded clozapine tablets introduced into a test tube with a capacity of 20 ml. Further, for extracting clozapine was added 2 ml of perchloric acid and 10 ml of water. The vial was put in an ultrasonic bath, and filtered into a volumetric flask of 100 ml. The residue was dissolved in a test tube and filter were washed thoroughly with several portions of water, the solution volume was adjusted to the mark with water. 0.12 ml of this solution was transferred to series of test tubes by 5 ml, and the procedure followed for the substance.

To use this method for quantitative analysis of clozapine we carried out its validation.

The most important criterion for evaluating any analytical procedure is a proof of its validity, consisting of interconnected system characteristics - specificity, linearity, accuracy and repeatability.

The linear dependence of the methodology describes the ability of obtaining analytical signal directly proportional to the amount of clozapine in the test sample. Linearity is maintained to concentrations of clozapine 12.0 mg/ml. Molar absorption coefficient (e) 1.986004 l/mol-1 cm-1. LOD limit of detection is 0.10 mg/ml. The correlation coefficient of the regression graph R2 is 0.9998.

Linear regression analysis of the data gave the following equation:

A= 0.05636+0.06077. where

A- adsorption in 1 cm cuvete;

0.05636- placebo solution adsorption.

Validation of method was evaluated by regression line. It was determined that the standard deviation of residuals equal to 2.33O0-3. the standard deviation of interception - 2.2800-3 and standard deviation of the angle of inclination - 3368O0-4.

Reproducibility of this technique was performed seven times at concentrations of 0.2; 1.0; 5.0; 9.0 and 12.0 ug/ml. Output is >98.7% with a relative standard deviation of <1.1%. This indicates a high accuracy and reproducibility of the developed method.

Comparison with other oxidants. The performance of the proposed method. compared with those of other existing reagents (Table 4). It can be seen that the proposed method is more sensitive due to the high molar absorption coefficient and a better linear range. Following the recommended procedure. we can easily carry out the analysis of clozapine for 1 min stirring the reactants in an aqueous medium without having to use high temperatures. organic solvents for extraction or dilution.

Table 2 - Comparison of previously proposed and the proposed reagents


Waveleng th. nm

Linearity ug/ml

The molar absorption coefficient tetracyanoquinodimethane



1.6 x 104

Chloranile acid



2 x 103

2.3-dichloro-5. 6-dicyano-n-benzoquinone



6 x 103




1.1 x 104

Phosphomolibden acid




Ce(IV) + 3-methylbenzotiazidone




NaCIO + P-N.N- dimetilfenilendiamin-dihydro chloride




Potasium bromate



1.986 x 104

It can be concluded that the proposed method has high sensitivity (molar absorption coefficient e is 1,986 x 104 cm-1, the limit of detection LOD - 0,10 g/ml). On the other hand, the method is simple, because there is no need for solvent extraction or separation steps prior to the analysis. Excipients have no influence on the analysis process. In addition, the proposed method is accurate and reliable. The above results substantiate the use of the proposed method for the analysis and quality control substance, dosage forms and biological objects containing clozapine.


  1. Clozapine. Typical clinical and pharmacological monograph. E-source. Database of Russian Federation registered pharmaceuticals. - 2009
  2. V.G. Belikov. Pharmaceutical chemistry. Part 1. Medpress inform.- 2007.- p624
  3. USP, USP Monographs: Clozapine, - 2009
  4. O.I. Sokolova. Forensical clozapine determination. Abstract. - М., - 2007. -p18
  5. Tanaka E, Nakamura T, Terada M, et al. Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography. J Chromatogr B, 2007, 854(1-2):116-120.
  6. Zhou ZL, Li X, Li KY, et al. Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B, 2004, 802(2):257-262
  7. Yermokhina T Acute azaleptin poisoning: abstract - М., 2004. - p20.
Year: 2014
City: Shymkent
Category: Medicine