A Novel Stability Indicating RP-HPLC Assay Method Development and Validation for the Quantification of Cyamemazine Tartrate in bulk and its Pharmaceutical Dosage Form

 

S. Muneer¹*, Hindustan Abdul Ahad², Chandra Sekhar Kothapalli Bonnoth³

¹Research Scholar, Research and Development, Jawaharlal Nehru Technological University, Anantapur 515002, AP, India.

²Professor in Department of Pharmaceutical Sciences, JNTUA- Oil Technological and Pharmaceutical Research Institute, Ananthapuramu 515002, AP, India.

³Professor in Chemistry, Director Foreign Affairs and Alumni Matters, JNTUA University, Ananthapuramu, A.P., India

 

ABSTRACT:

A simple and rapid stability-indicating reverse phase HPLC assay procedure has been developed and validated for Cyamemazine tartrate in bulk and its Pharmaceutical dosage form. Chromatographic separation was achieved on Phenomenex Luna C18 (250 mm x 4.6, 5μ particle size) column using isocratic elution with mobile phase consisting of water, Acetonitrile and Methanol (60:30:10 v/v/v) under ambient temperature at 1.0 mL min−1. The procedure showed a dynamic linear response over 10– 60 μg mL-1 demonstrating correlation coefficient (r²) above 0.999, with the limits of detection and quantitation of 1.72 and 5.23 μg mL-1 respectively. The batch injection repeatability for intra and inter assay precision and accuracy studies were within the acceptance. The analyte was subjected for various stress conditions. The procedure well separated Cyamemazine tartrate and its potential degradation products which prove the stability indicating nature of the method. 

 

 

 

INTRODUCTION:

Cyamemazine tartarate (CMT)¹ is a Phenothiazine derivative from the class of typical antipsychotic and a new drug for the treatment of Schizophrenia² due to its Dopamine D2 receptor antagonistic activity. It is a neuroleptic analyte with anxiolytic properties^3,4 and also exhibits strong cutaneous phototoxicity in humans^5-7. CMT possesses high affinity for 5-HT3- and 5-HT2C-receptor types⁸. It is chemically 10-(3-dimethylamino-2-methyl-propyl)-Phenothiazine-2-carbonitrile. The chemical structure of CMT was shown in Figure 1.

 

 

Fig. 1: Chemical structure of Cyamemazine tartrate

 

The Literature availability of analytical methods for the analysis of Cyamemazine Tartrate and its metabolites in biological samples such as LC-MS/MS tandem⁹, GC-MS tandem mass spectrometry¹⁰. A sensitive HPTLC¹¹ and spectrophotometric method¹² were recently has been developed for the determination of CMT in pharmaceutical dosage forms.

 

The reported methods so far done were in biological samples, there are no significant liquid chromatographic reverse phase HPLC method development of CMT in bulk drug and its formulation and also stability indicating studies. Based on the non availability of reliable methods, in the present study an attempt was made to develop and validate stability indicating RP-HPLC assay method as according to ICH guidelines^13-15 .

 

MATERIALS AND METHODS:

Instrument and Optimum chromatographic conditions

The liquid chromatographic system employed to develop this technique was Shimadzu LC 20 AD series consisting binary pump with PDA detector. Data acquisition was monitored and performed by means of a compatible integrator having a chart speed of 0.25 cm: min using chromatography software (LC solutions). Chromatographic separation was achieved on Phenomenex Luna C18 (250x4.6mm, 5μ particle size) column with mobile phase water: Acetonitrile and methanol (60:30:10 v/v/v) under ambient temperature. The flow rate was 1.0 ml/min and detector wavelength was kept at 270 nm for monitoring the elution. Injection volume was 20 μL and total run time was 8 min.

 

Chemicals and reagents

Standard drug of CMT (99.99 %) was kindly provided by MSN laboratories, Hyderabad, Telangana, India. HPLC grade methanol, acetonitrile and water were purchased from Merck Co., Mumbai, India. All other reagents were of analytical reagent grade. All the solutions were filtered through 0.45 mm silicon membrane filter. All the glass wares used were calibrated for class A type.

 

Preparation of standard solutions

A stock solution of 1.0 mg mL-1 was prepared by dissolving appropriate amount of CMT in the diluent. Working solution of 40µg mL-1 was prepared from stock solution for related substances and assay determination.

 

Sample preparation:

Accurately weighed sample equivalent to 10 mg of CMT was transferred into a 10 ml volumetric flask. The drug was dissolved with few ml of mobile phase, and sonicate to dissolve it completely and made up to the mark with the same mobile phase. From this solution working standards of required concentrations were prepared.

 

Solution stability and mobile phase stability

The stability of CMT in solution was determined by leaving test solutions of the sample and reference standard in tightly capped volumetric flasks at room temperature for 48 h during which they were assayed at 12 h intervals. Stability of mobile phase was determined by analysis of freshly prepared sample solutions at 12 h intervals for 48 h and comparing the results with those obtained from freshly prepared reference standard solutions. The mobile phase was prepared at the beginning of the study period and not changed during the experiment. The % assay of the results was calculated for both the mobile phase and solution-stability- experiments.

 

The stability of CMT and its impurities in solution for the related substance method was determined by leaving spiked sample solution in a tightly capped volumetric flask at room temperature for 48 h and measuring the amounts of the impurities at every 12 h. The stability of mobile phase was also determined by analysis freshly prepared solution of CMT and its impurities at 12 h intervals for 48 h. The mobile phase was not changed during the study period.

 

Preparation of Stock Solution for stress studies

Weighed a quantity of 10 mg of CMT was carefully transferred into a 10 ml volumetric flask, dissolved completely in water (for neutral degradation studies) and the volume was made up to mark to get 1000 μg/ml. The same procedure was used to prepare stock solutions for stress studies viz for acid hydrolysis, base hydrolysis, and oxidation respectively with HCl (0.1N), NaOH (0.1N) and hydrogen peroxide (3.0% v/v). Thermal degradation was carried out for solid State CMT by heating the samples over a period in a hot air oven, at 70 ⁰C. Photo-degradation was carried on solid Sample by exposing to natural sunlight. Blank was analyzed under the same condition to assess the method specificity.

 

RESULTS AND DISCUSSION:

The most important aspect in method development was achievement of chromatographic separation with sufficient resolution and reasonable analysis time. The choice of the method depends on factors such as the nature of the drug, the complexity of the sample and the intended use. In this study, the conditions were influenced by solubility, polarity and UV absorption. Prior to chromatographic method development, the detection wavelength was determined by obtaining the UV spectra of solution of CMT. The absorbance maximum at 270 nm of analyte showed good sensitivity for the determination of all the analytes as well as any other unknown degradation product in the bulk drug. The chromatographic separation of CMT was achieved, using isocratic elution with solvents water, methanol and acetonitrile on different analytical C8 and C18 columns. Finally, a mobile phase consisting of water: acetonitrile and methanol 60:30:10 v/v/v on Phenomenex Luna C18 (250x4.6mm, 5μ) offered a good peak shape of the analyte. Under these conditions, and using a flow rate of 1.0 ml/min and a run time of 10 min, CMT was eluted at about 3.75 ± 0.048 min. Interference from the excipients was also checked, no interference was observed. The optimized chromatogram was shown in fig 2 and system suitability parameters were within the limits and presented in table 1 and 2.

 

 

Fig 2. Optimized chromatogram of Cyamemazine Tartrate (CMT) on C18 column

 

Table 1. System suitability parameters for the developed method (CMT)

 

Validation of the method:

The method was validated with respect to parameters including linearity, precision, accuracy (recovery), limit of quantitation (LOQ), limit of detection (LOD), selectivity and robustness.

 

Solution stability and mobile phase stability

Assay (%) of CMT during solution stability and mobile phase stability experiments was within ±1%. The variability in the estimation of CMT impurities was within ±10% during solution stability and mobile phase experiments. The results from solution stability and mobile phase stability experiments confirmed that standard solutions and solutions in the mobile phase were stable up to 48 for assay analysis.

 

Linearity

The linear calibration plot for the CMT assay method was obtained over the calibration ranges tested, i.e. 10–60 µg/ml and correlation coefficient obtained was greater than 0.999. The result shows that an excellent correlation existed between the peak area and concentration of the analyte shown in fig 3. The correlation coefficient obtained was greater than 0.999 represented in table 2.

 

 

Fig 3. Calibration curve of CMT

 

Precision

The precision of the method was verified by repeatability and by intermediate precision. Repeatability was checked by injecting six individual preparations of CMT real sample.  The intermediate precision of the method was also evaluated performing the analysis on different days. Assay method precision was evaluated by carrying out six independent assays of real sample of CMT at 0.1 mg mL-1 level against qualified reference standard. The %RSD of the assay results obtained was within limit conforming good precision of the method and was shown in table 2.

 

Limits of detection (LOD) and quantification (LOQ)

The LOD and LOQ for CMT were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision study was also carried out at the LOQ level by injecting six individual preparations and calculating the %RSD of the area. The LOD and LOQ were determined as 1.72 and 5.23 µg mL-1 respectively.

 

Accuracy

For the developed method recovery studies was conducted. Known amount of pure drug was spiked in placebo at three different levels, i.e. 50, 100 and 150% and percent recovery was calculated. The % recovery values for CMT are presented in table 2.

 

Robustness

In all the deliberate varied chromatographic conditions (flow rate and column temperature), all analytes were adequately resolved and elution orders remained unchanged. The assay variability of CMT was within ±1%. From the result obtained the method was found to be robust and shown in table 2.

 

Table 2. Summary of the performance parameters of the RP-HPLC procedure for CMT

 

Forced decomposition studies

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. The specificity of the developed LC method for CMT was carried out in bulk drug. Stress studies were performed at an initial concentration 40 µg/ml of CMT in formulation to provide an indication of the stability-indicating property and specificity of the proposed method. Intentional degradation was attempted to stress conditions of photolytic, thermal (70^oC), acid (0.1N HCl), base (0.1N NaOH) and oxidation (3.0% H₂O₂) to determine the ability of the proposed method to separate CMT from degradation products generated during forced decomposition studies. For thermal and photolytic studies, study period was 5 days whereas for acid, base and oxidation it was 48 h. Thermal degradation was carried for solid state of CMT by heating the samples over a period in a hot air oven, at 70 ^oC. Photolytic studies were carried on solid sample by exposing to natural sunlight. Peak purity test was carried out on the stressed samples by using PDA detector. Assay studies were carried out for stress samples against qualified reference standard and the mass balance (% assay+ % degradation products) was calculated. Assay was also calculated for bulk sample by spiking with CMT at the specification level (0.40%). Blank was analyzed under the same condition to assess the method specificity. Representative chromatograms of stress studies of various conditions were shown in fig. 2, 3 and table 3 shows the results of the experiments.

 

 

Figure 3(a): Chromatogram of acid degradation in 0.1M HCl

Figure 3(b): Chromatogram of alkaline degradation in 0.1M NaOH

Figure 3(c): Chromatogram of oxidative degradation in 3% v/v H₂O₂

 

 

 

 

Figure 4(a): Chromatogram of thermal degradation

Figure 4(b): Chromatogram of photolytic degradation

 

Table 3. Summary of forced degradation results

 

 

CONCLUSION:

A stability-indicating reverse phase HPLC assay method was developed for the quantitation of CMT. The method is simple, precise, accurate and robust. The procedure permitted an accurate and quantitative determination of CMT. All the degradation products formed during forced degradation studies were well separated from the analyte peak demonstrates that the developed method was specific and stability-indicating. This method can be used to carry out the analysis of CMT in stability samples.

 

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Received on 03.09.2018       Accepted on 20.09.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(3): 169-173.

DOI: 10.5958/2231-5675.2018.00031.5