Validated UV spectrophotometric and HPLC methods for quantitative determination of zotepine

 

A.S.  Manjula Devi* and T.K.  Ravi

Department of Pharma. Analysis, College of Pharmacy, SRIPMS, Coimbatore, Tamil Nadu, India. PIN 641 044

*Corresponding Author E-mail: hari1509@gmail.com

ABSTRACT:

A high-performance liquid chromatographic method with PDA detection and UV spectrophotometric method were developed for the quantitative determination of zotepine.  Chromatographic separation was achieved on a LiChrospher® RP-18 HPLC column (5 μ particle size and 25 cm × 4.6 mm internal diameter) using 0.5%trifluoroacetic acid:methanol:acetonitrile (5:40:55 v/v/v; pH 2.23)  as mobile phase and aceclofenac as the internal standard. The effluent was monitored at 265 nm. Two sharp peaks were obtained for internal standard and zotepine at 3.4 and 5.1min, respectively. UV spectrophotometric method was performed at 265 nm using methanol as the solvent. Analytical performance parameters such as linearity, specificity, accuracy, precision, limit of detection and limit of quantification were determined according to International Conference on Harmonization (Q₂B) guidelines.  Linear range obtained was 1-10 µg mL^-1 (r²>0.99) for HPLC method and 2.5-25 μg mL ^-1 (r²>0.99) for UV spectrophotometric method. Validation as per ICH guidelines and statistical analysis showed that both the methods were precise, accurate, sensitive, and can be used for the routine quality control of zotepine.

 

 

INTRODUCTION:

Zotepine, 2-((8-chloro dibenzo [b, f] thiepin-10-yl) oxy)-N,N-dimethylethanamine), is an atypical antipsychotic drug indicated for the treatment of  acute and chronic schizophrenia. The drug is administered orally in daily doses of 25, 50 and 100 mg¹. A thorough literature survey has revealed that an on-line liquid chromatographic electrochemistry/electrospray/ tandem mass spectrometry method for the determination of zotepine metabolites in human serum² and strategy for structure elucidation of zotepine metabolites by ESI-MS³ have been reported. A gas-liquid chromatographic method involving time consuming multiple extraction steps⁴ and a GC-MS method using solid phase extraction and evaporation processes⁵ were reported for the quatitation of zotepine. The aim of the present study was to develop simple, sensitive validated RP-HPLC method and UV spectrophotometric method for the quantitative estimation of zotepine.

MATERIALS AND METHODS:

Analytically pure samples of zotepine and aceclofenac (assigned purity >99% w/w) were obtained as gift samples from Symed Labs Limited, Hyderabad, and Sun Pharmaceuticals Ltd, Chennai, India. HPLC grade methanol and acetonitrile were obtained from Merck specialties Pvt. Ltd (Mumbai, India). Other chemicals and reagents were analytical grade.  Water for RP-HPLC was prepared using Millipore purification system (Milli-Q, Bangalore, India) and double distilled water was used to prepare all the solutions for UV spectrophotometric experiments.

 

Instrumentation and analytical conditions

Chromatography was performed under ambient conditions with liquid chromatograph mass spectrometer (Shimadzu LCMS-2010EV) equipment comprising of a binary gradient pump (LC-20AD), degasser (DGU-20A₃), and a variable wavelength programmable PDA detector (SPD-M20A) with auto sampler system (SIL-20AC). The instrumentation was controlled by Shimadzu LCMS Solution Software. Chromatographic separation was achieved on a LiChrospher® RP-18 HPLC column (5 μ particle size and 25 cm × 4.6 mm internal diameter) using the mobile phase composed of 0.5%trifluoroacetic acid:methanol:acetonitrile (5:40:55 v/v/v) at flow rate of 1 ml/min. The pH of the mobile phase was 2.23 (adjusted with 1% triethylamine). The mobile phase was filtered through 0.45 μ nylon filter and degassed by sonication prior to use. Column temperature was maintained at ambient and the run time was set at 10 min. The column was equilibrated for 30-40 min with mobile phase prior to injection of the analyte. The volume of injection was 20µL. Detection was performed at 265 nm since both the components showed reasonable absorbance at this wavelength.  The UV spectrophotometric method was performed using a Jasco V-630 UV/VIS Spectrophotometer with Spectra Manager (Version 2) controlling software at 265 nm in 1 cm quartz cell. The samples were prepared using methanol.

 

Preparation of standard solutions

For HPLC method, 100µg mL^-1 of zotepine and aceclofenac were prepared separately by dissolving 10 mg each in 100 ml methanol. Working standard solutions of zotepine (1 - 10 µg mL^-1) were prepared by transferring 0.1 to 1 ml of zotepine stock solution to serially arranged 10 ml standard flasks and diluting to volume using the mobile phase. A constant volume (0.2 ml) of stock solution containing aceclofenac was added as the internal standard before making the dilutions.

For the UV Spectrophotometric method, stock solution of 100 μg mL^-1 of zotepine was prepared in methanol. The working standard solutions were prepared by dilution of the stock solution with methanol to get a concentration range 2.5 - 25 μg mL^-1.  The absorbance was measured at 265 nm against methanol as blank.

Method Validation

Both the methods were validated by following ICH recommendations for validation of analytical procedures⁶.   

 

Linearity and range

For the HPLC method, stock solution of zotepine was suitably diluted with the mobile phase to get concentrations in the linear range of 1 - 10 μg mL^-1. Then 2  μg mL^-1  of aceclofenac (IS) was added to each dilution and 20 μl was injected into the column, the peak area and retention time were recorded. The calibration curve for zotepine was constructed by plotting the ratio of the peak area of zotepine to the peak area of the internal standard (Y) against concentration (X) and linearity was evaluated by linear regression equation. The slope, intercept and correlation coefficient values were recorded. Each experiment was performed in six replicates. For the UV Spectrophotometric method, calibration graph was prepared with 2.5 - 25 µg mL^-1 of zotepine and absorbance was recorded at 265 nm. The experiment was performed in six replicates.

 

Accuracy

Standard addition method was followed to determine the method accuracy. A known quantity of standard was added at three levels (50, 100 and 150% of the assay concentration) to pre-analyzed sample solution. A constant concentration (2 µg/ml) of IS was added to all the samples and the mixtures were analyzed by the developed method. The experiment was performed in six replicates. %RSD and % recovery were calculated for all the concentration.  Absorbance was measured directly for the UV spectrophotometric method. %Recovery was obtained by comparing the calculated and measured concentrations.

 

Precision

The precision of the method was studied by repeatability (within-day) and intermediate precision (inter-day).The intra-day precision studies were carried out by estimating the response six times on the same day using three different concentrations (2.5, 3 and 3.5 µg mL^-1 for HPLC method and 10, 15 and 20 µg mL^-1 for UV spectrophotometric method) of zotepine and inter-day precision studies were done by repeating the above procedure on three different days. A fixed concentration (2 µg mL^-1) of the internal standard was added to all the samples in HPLC experiment. The results of precision studies were expressed as %RSD.

LOD and LOQ

Sensitivity of HPLC and UV methods were determined from limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were calculated from the calibration curve using the following equations

LOD = 3.3xσ/S and

LOQ = 10 x σ/S, where,  

σ = standard deviation of y intercept of regression line

S = slope of the calibration curve

 

Specificity

Purity of zotepine and IS peaks were assessed in HPLC method by comparing the individual spectrum at three regions i.e. peak start, peak apex and peak end.

 

Robustness

Robustness of HPLC method was studied to evaluate the effect of small but deliberate variations of the chromatographic conditions on the method parameters. Robustness was determined by changing individually the flow rate (1±0.1ml/min), organic solvent (95±1%), pH (0.5±0.1) and evaluating their effects on peak parameters.

 

System suitability tests

The test was carried out by making six replicate injections of a standard solution containing zotepine (5 μg mL^-1) and IS (2 μg mL^-1) and evaluating each analyte for their peak area, theoretical plates (N), resolution(R_s), tailing factor (T),capacity factor (k’), and asymmetric factor (A_s).

 

Stability Studies

A suitably diluted solution of zotepine and IS in methanol was stored under laboratory bench conditions upto 48 hours, and under refrigeration (8±0.5ºC) for 5 days. The solutions were assayed by the proposed method. 

 

RESULTS AND DISCUSSION:

The current study describes a new RP-HPLC method and UV-spectrophotometric method for the determination of the antipsychotic drug, zotepine.

 

HPLC Method: The retention time of IS and zotepine was 3.4 and 5.1 min, respectively. Figure 1 shows the overlay spectrum of IS and zotepine. The typical chromatograms of zotepine with internal standard are shown in Figure 2.

 

The developed method was validated in terms of specificity, linearity and range, accuracy, intra-day and inter-day precision, LOD, LOQ, robustness and system suitability by following the recommendations of ICH guidelines. Specificity of the method was established by the complete separation of the peaks of zotepine and internal standard as well as their peak purity index which was found to be 1. The standard calibration curve was linear over the concentration range 1 to 10 μg mL^-1 with a correlation coefficient (r²) 0.9989±0.00051, the corresponding linear regression equation being y = 0.7946x+0.07438. LOD and LOQ were 0.318 µg mL^-1 and 0.964 µg mL^-1 respectively, indicating the sensitivity of the method. Recovery experiments demonstrated satisfactory accuracy with small relative standard deviations (RSD %). The developed RP-HPLC method was found to be precise since the %RSD values were <2 for both repeatability and intermediate precision studies as recommended by ICH guidelines. RSD of repeatability (intra-day) and intermediate precision (inter-day) ranged from 0.188 to 0.687 and 0.219 to 0.566 respectively. Table 1 illustrates the regression parameters and validation parameters of zotepine by RP-HPLC method.

Table I. Regression parameters and validation parameters of zotepine by RP-HPLC and UV-spectrophotometric method

^*Average of 6 determinations ± SD

 

 

Deliberate changes in experimental conditions did not alter peak symmetry and there was no significant change in the retention time of IS and zotepine during these experiments. The %RSD for each method parameter was calculated and was found to be <2. The percentage amount of zotepine present in the assayed sample was found to be 99.956 ±0.1026. Adequacy of the proposed RP-HPLC method for routine analysis of zotepine was assured by system suitability tests. The capacity factor (k’) of IS and drug were found to be 7.54 and 11.84 respectively, signifying that both the peaks were well resolved with respect to the void volume. The tailing factor of 1.3 for both IS and zotepine peaks reflect good peak symmetry. The resolution (Rs) for the principle peak and internal standard was found to be 8.521, showing good separation. The theoretical plate number (N) was found to be 10620 for drug, thus demonstrating good column efficiency. The result of system suitability tests (table 2) shows that the newly developed method fulfils these requirements within the accepted limits. Solution stability studies demonstrated good stability of standard  solutions during all storage conditions.

 

Table 2. System suitability parameters of the proposed RP-HPLC method

UV Spectrophotometric method: The solutions were scanned in the wavelength range of 200-400 nm after making suitable dilutions from the stock solutions. Zotepine showed absorption maxima at 265 nm. The method was validated as per ICH guidelines. Zotepine in methanol showed linear relationship in the concentration range of 2.5 to 25 µg mL^-1 with a correlation coefficient (r²) 0.9995. The regression equation was found to be y= 0.06334x+0.01091. The recoveries of zotepine estimated by standard addition method for the three concentrations ranged from 99.9 to 100.18% with acceptable RSD values. LOD and and LOQ were found to be 0.417 µg mL^-1 and 1.265µg mL^-1 respectively (table 1). Assay results varied from 99.92% to 100.14% with satisfactory RSD values.

Chromatographic conditions were carefully optimized to get satisfactory resolution between analyte and IS. The final decision on mobile phase composition and flow rate was made on the basis of peak shape (peak area, asymmetry, tailing factor), baseline drift, time required for analysis, and cost of solvents. Several mobile phases were tried to get good separation between analyte and internal standard. Zotepine was not eluted when water:methanol, ammonium formate:methanol and ammonium acetate:methanol in different ratio were tried. No analyte peak was observed in subsequent trials using 0.01% - 0.1% TFA and methanol (5:95 v/v) while the use of 0.5% TFA resulted in a broad asymmetric peak. The peak shape improved when the mobile phase composition was changed to 0.5% TFA:MeOH:ACN (5:75:20 v/v/v) but tailing factor was 2.7. Finally the optimized mobile phase which gave acceptable K value, adequate resolution, short run time and symmetric peak was 0.5%TFA:MeOH:ACN (5:40:55) at pH 2.23.  Internal standard method was preferred in the present study due its advantage of eliminating errors from sample preparation and run to run variations, thus improving the accuracy and precision of the method^{7, 8}. Six drugs, viz. mefenamic acid, paracetamol, diclofenac, ibuprofen,  ketoprofen, and aceclofenac were analyzed to select a suitable internal standard. All the criteria for a compound to be used as an internal standard were met by aceclofenac. It was well resolved from analyte peak (R_s 8.5) with good peak shape (tailing factor 1.3). Its elution time was short (3.4 min) and was stable during the analysis. In addition it was readily available. Peak shape of diclofenac and mefenamic acid was not symmetric while the resolution between the analyte and IS such as ibuprofen, ketoprofen and paracetamol was >15.  Linear regression analysis showed that excellent correlation exists between peak area and ratio of concentration of analyte and IS. High accuracy and precision of the method was apparent from the results shown in table1and their statistical evaluations. Further, the high values of correlation coefficients and small values of intercepts validated the linearity of the calibration plots. Slopes and intercepts of the calibration graphs indicate the high reproducibility of the proposed method. The method was also found to be robust as there was no significant change in the peak area, peak shape, retention time and other system suitability parameters.  The retention time observed at 3.4 and 5.1 min for drug and IS allowed rapid quantification of zotepine which is important in routine analysis. Recovery values met the acceptance criteria of 100±2% at the three different concentration levels evaluated. The current UV spectrophotometric method allowed rapid quantification of zotepine. Methanol was chosen as the solvent because of good solubility and stability. The spectral characteristics were also good in this solvent. Low values of %RSD signify the precision of the method. A good accuracy of the method was documented with mean recoveries close to 100%. Moreover, the method is highly sensitive as evidenced by the LOD and LOQ values.

 

CONCLUSIONS:

The validated HPLC and UV methods for the quantitative estimation of zotepine were found to be accurate, precise, reliable and less time consuming.  UV spectrophotometric method was simpler and sensitive than HPLC method and the same may be used as an alternative method when advanced instruments like HPLC are not available for routine quantification purpose.

 

ACKNOWLEDGEMENTS:

The authors thank Symed Labs Limited, Hyderabad and Sun Pharmaceuticals Ltd, Chennai, India, for providing reference samples of zotepine and aceclofenac. The authors also wish to thank the management, SNR Sons Charitable trust, Coimbatore for providing facilities to carry out the study.

 

REFERENCES:

1.     Thomson Reuters,  Micromedex (R) Healthcare series, 148 (2011).

2.     Nozaki K, Issey O, Hideya K and Ryuichi A. Application of on-line  electrochemistry/electrospray/tandem mass spectrometry to a quantification method for the antipsychotic drug zotepine in human serum. Analytical Sciences. 25 (1); 2009:1197 - 1201.

3.     Tozuka Z, Hayato K, Toshifumi S, Yasuyuki M, Akio K, Akira K and Anri A. Strategy for structure elucidation of drug metabolites derived from protonated molecules and (MS)ⁿ fragmentation of zotepine, tiaramide and their metabolites. Drug Metabolism and Pharmacokinetics. 17 (4); 2002: 316–339.

4.     Urlrich S, Meyer FP and B.Bogerts. J.Pharmaceutical and Biomedical Analysis. A capillary gas-liquid chromatographic method for the assay of the neuroleptic drug zotepine in human serum or plasma. 14 (4 ); 1996: 441-449.

5.     Tanaka O, Kondo T, Kaneko S, Mihara K, Otani K and Tokinaga N. A method for rapid determination of zotepine by gas chromatography-mass spectrometry.  Therapeutic Drug Monitoring. 18; 1996: 294-296.

6.     International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline – Validation of Analytical Procedures:Text and Methodology Q2(R1),  version 4, London 2005.

7.     Snyder LR,  Kirkl JJ and Glajch JL.  Practical HPLC Method Development. 2^nd ed. John Wiley and Sons, New York. 1997.

8.     Willard HL, Merritt LL, Dean JA and Settle FA. Instrumental Methods of Analysis. 7^th ed. Wadsworth Publishing Company,  California. 1988.

 

 

 

Received on 21.12.2011          Modified on 20.01.2012

Accepted on 22.01.2012         © RJPT All right reserved