Optimization of clozapine chromatography conditions in biomaterials

Summary

The method of high performance liquid chromatography to identify and quantify clozapine in tablets and biomaterial has been developed. Optimal phases of acetate buffer (pH 5,5) and acetonitrile (60:40) has been selected. An XBridge C8 column pack with 5.0 micron particle size of dimethyloctylsilyl bounded amorphous silica was used for the chromatographic separation. Retention time for clozapine in all the studies was 10, 40 min. Validation showed its specificity, precision and repeatability (R2 = 0, 9991-0,9999, RSD no more than 3,18%)

Key words: high performance liquid chromatography, identification, assay, validation

It is hard to imagine the analytical diagnostics of psychoactive substances without chromatographic methods of analysis (it takes about 70-80%)

The most reliable method is high performance liquid chromatography (HPLC) which has good validation performance. There are a number of advantages with HPLC: studying a lot of parameters simultaneously, good separation, identification, quantitative analysis etc.

To solve a variety of analytical tasks we need necessary optimal conditions of chromatographic separation. Unlike gas chromatography, where the separation process affects the nature of the stationary phase and the analytes, the composition of the eluent in HPLC (solvent mixture or mobile phase) plays the role of a —third” variable that takes an active part in the chromatographic process, indicating that the selectivity liquid chromatographic system. Therefore, production methods and to identify ways to further optimize it should be based on knowledge of the phases of sorbents used and physical-chemical properties of the analyte and solvent.

Materials and methods: CLZ was obtained from scienTEST bioKIMEX GmbH (Germany). Methanol and acetonitrile were HPLC grade and 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. Mobile phase was a mixture of ammonia acetate (0.03 g/mL, pH 5.5) - acetonitrile (60 : 40, v/v). The flow rate was 1.0 mL/min. The UV detector was set at a wavelength of 240 nm.

The individual stock solutions of CLZ was prepared at the concentration of 0,5 mg/mL by dissolving proper amount of its substance in appropriate mobile phase or methanol , and then stored at -20 °C. A series of CLZ solutions have also been prepared at the concentrations of 1;10;25;50;75;100 ug/mL.

To obtain the concentration of 1 ug/mL stock solution #2 was prepared by diluting the individual stock solution with a proper volume of mobile phase or methanol.

Results and discussion. On the basis of UV-Vis spectrum, two wavelengths of 240 and 294 nm were chosen for the[analysis of Clz. The obtained chromatograms indicated the higher absorption and bigger peak area at 240 nm. Thus, the method was set at the wavelength of 240 nm. A C8 column was used for the separation and short analytical time and better resolution were obtained when compared to a C18 column. In order to get the most appropriate mobile phase, different ratio of acetonitrile-water, methanol-water and different concentration of ammonia acetate or sodium dihydrophosphate in water were studied. Finally, a mixture of ammonia acetate (0.03 g/mL, pH 5.5) - acetonitrile (60: 40, v/v) was chosen as the mobile phase to achieve the best resolution factors.

The affection of flow rate and temperature was studied. Temperature does not bring significant changes either in peak area or retention time. Slowing the flow rate prolongs the retention time and has the impact on peak areas. The proposed method had good analytical performance: the R2 value of the proposed method was 0,9996. The repeatability and intermediate precision were less than 5 % at the lowest concentration of linearity range. The recovery at all concentration levels did not exceed 1.00 %.

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It was deemed appropriate to evaluate the possibility of using the procedure developed for the determination of clozapine in extracts isolated from the objects of biological origin. In this paper we have used the liver of corpses killed by injuries, the urine of living persons who did not use drugs for a long time. Method of determination of clozapine in biological fluids (urine) and biomaterial (liver). The dried residue extracts after liquid-liquid extraction, was dissolved in 20 ml of eluant, 0.5 ml of which was injected into the chromatograph. Chromatograms of clozapine isolated from urine and liver shown in Figure 2.

As you can see in figures 1 and 2, the retention time of clozapine in a procedure developed by us for substance as well as for biological objects coincide and make 10.40 min.

To develop a methodology for the analysis of clozapine in tablets, clozapine tablets 25 mg and 100 mg (“Egis pharmaceuticals” (Hungary) and —Technology” (Ukraine) were taken as a counterpart.

For further studies, we prepared a model mixture of 100 mg of clozapine tablets of the following composition:

Active substance: Clozapine 100 mg. Excipients: lactose monohydrate starch magnesium stearate povidone 25 to obtain the model mixture weighing 200 mg.

The method of analysis of clozapine tablets. Accurately weighed model mixture was dissolved in mobile phase composition of acetate buffer and acetonitrile (60:40) in a volumetric flask of 25 ml, while the drug concentration in the solution is 0.5 mg/ml (solution A).

The stock solution B at the concentration of 0.05 mg/ml of solution A was prepared by diluting with the mobile phase in a volumetric flask of 25 ml.

20 ul of stock solution B introduced into the chromatograph for analysis.

Chromatogram of clozapine tablets is shown in figure 3.

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Retention time in all cases is 10,39±0,01 min.

Quantitative evaluation of clozapine in the samples were determined by the absolute calibration of the dependences of the height and area of the chromatographic peak of the number of clozapine. For this purpose, standard solutions were prepared at a concentration of 1 ug/ml to 100 ug/ml. 20 ul of the resulting solution was injected into the chromatograph and chromatography was carried out under the conditions described above. The results of analysis of model mixture urine samples containing clozapine are shown in table 1.

Table 1 - Clozapine analysis in urine results

Conditions and characreistics

urine

Added clozapine, ug/ml

20

Defined clozapine, ug/ml

19,88

Number of samples

5

standard deviation

0,07

The regression coefficient (A correlation)

0,9997

Average recovery,%

64,2

In order to develop a specific, precise and accurate method for the quantitative assessment of Clz tablets, several methods containing different mobile phases and flow conditions were applied. Finally, previously specified conditions turned out to be appropriate for the analysis and the HPLC method was validated on the basis of ICH Q2, Eur. Ph. and USP guidelines. In order to confirm the suitability of the method it was tested in terms of the following parameters.

Specificity. No interfering signals with the marked peak should be observed. Peak asymmetry factor should be contained in the range of 0.8-1.5 and retention factor (k), calculating on the basis of the following formula, should be in the range of 0.5-20:

k = (to - t) / to

where t is retention time of Clz and to is retention time of the substance that does not interact with the column (potassium dichromate).

The analysis of the placebo sample and the test sample chromatograms revealed no interfering substances with the main peak of Clz. The peak retention time of Clz and potassium dichromate were 10.40 min and 2.85 min, respectively, peak symmetry factor was acceptable and retention factor (k) was 2.6. Therefore, the method is specific.

Limit of detection and quantification.Limit of detection (LOD) and quantification (LOQ) were calculated based on the following equations:

LOD = 3.3 SD / a,

LOQ = 10 SD / a,

respectively, where SD is the standard deviation for injections of the lowest concentrations of a linear curve and —a” is a directional factor of the linear curve (presented in the next chapter).

The analysis of the results enabled to determine the concentration of 0.126 ug/ml as the limit of detection and the concentration of 0.38 ug/ml as the limit of quantification.

Linearity. The correlation coefficient for validated methods should be no less than 0.995, and the average balance of residuals should be no more than 10 %.

Linearity of the method was tested in the range of 1-100 ug/mL, on the basis of six-point calibration curve (1, 10, 25, 50, 75, and 100 ug/ml) (Figure 3). Each sample of specified concentration was analyzed three times and the values were averaged. The following regression equation was obtained: y = 119851x - 30894. The correlation coefficient for the validated method was 0.9996 and no value of the balance of residuals was higher than 10 % (Table 2). Thus, the method is linear in the range of 1-100 ug/mL.

Balance of the residuals

 

1

10

25

50

75

100

peak average

89771 I

1099680

3089045

5961382

8828856

12027034

amount from

           

function

88957

1167616

2965381

5961656

8957931

11954206

difference

814

-67936

123664

-274

-129075

72828

residuals

0,915049

-5,81835

4,170257

-0,004596038

-1,4409

0,609225

Table 2 - Balance of residuals

Accuracy.The relative standard deviation (RSD %) should not be more than 5 %. Percent of recovery should be within the range of 95-105 %.

Studies on accuracy were performed using three concentrations from the linearity range of the method (1, 25 and 100 ug/mL). Each sample of a predetermined concentration was prepared three times using the stock solution according to the complete analytical procedure. Each prepared sample was analyzed in triplicate. For the obtained values standard deviation (SD), relative standard deviation (RSD %) and the percent recovery were calculated. The relative standard deviations for the tested concentrations did not exceed 5 %. However, recovery in the lowest point of linearity range exceeded 105 % (Table 3).

Accuracy

 

1 ug/ml

25 ug/ml

100 ug/ml

Average conc.

1,009544

26,26019

100,7554

SD

0,007222

0,689156

0,214912

RSD (%)

0,71%

2,60%

0,21%

Recovery (%)

100,90%

105,04%

100,75%

Table 3 - Recovery at different values of concentrations

Precision. Repeatability.The test of repeatability was performed at one concentration (1 ug/ml) of the linearity range. Six samples of the same concentration were prepared from the stock solution according to the complete analytical procedure. Each of them was injected in triplicate. For the obtained results the average value, SD and RSD % were calculated (Table 4). The obtained relative standard deviation (RSD %) should be no more than 5 %.

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The relative standard deviation (RSD %) for the study of repeatability was 0.89 %. The method is repeatable in the linearity range.

Intermediate precision. The test of interday precision was performed at the same concentration (1 ug/mL) using the same procedure. The variable was the day and the analyst. For the obtained results (from repeatability and interday precision) the average value, SD and RSD % were calculated (Table 5). The obtained relative standard deviation (RSD %) should be no more than 5 %.

Stability.Stability was examined in three measuring points, in time t0, t24 and t48 using one concentration of 25 ug/mL. The incubation temperature of samples was 25 °C. Samples at the measuring points were analysed in triplicate (Table 6).

The percentage difference in the surface areas of peaks of Clz for measuring points of t0-t48 cannot be more than 5 %. T[ he surface area of peaks corresponding to the incubated samples cannot be greater than surface area of t0 sample.On the basis of UV-Vis spectrum, two wavelengths of 240 and 294 nm were chosen for the analysis of Clz. The obtained chromatograms indicated the higher absorption and bigger peak area at 240 nm. Thus, the method was set at the wavelength of 240 nm.

A C8 column was used for the separation and short analytical time and better resolution were obtained when compared to a C18 column. In order to get the most appropriate mobile phase, different ratio of acetonitrile-water, methanol-water and different concentration of ammonia acetate or sodium dihydrophosphate in water were studied. Finally, a mixture of ammonia acetate (0.03 g/mL, pH 5.5) - acetonitrile (60: 40, v/v) was chosen as the mobile phase to achieve the best resolution factors.

The affection of flow rate and temperature was studied. Temperature does not bring significant changes either in peak area or retention time. Slowing the flow rate prolongs the retention time and has the impact on peak areas.

The proposed method had good analytical performance: the R2 value of the proposed method was 0,9996. The repeatability and intermediate precision were less than 5 % at the lowest concentration of linearity range. The recovery at all concentration levels did not exceed 1.00 %.

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Thus, developed HPLC method for identification and quantitative determination of clozapine in tablets and biomaterial is specific, accurate and reproducible and can be used in the practice of chemical and toxicological analysis.

REFERENCES

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  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
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Year: 2014
City: Shymkent
Category: Medicine