Simultaneous Estimation of Amitriptyline and Perphenazine by RP HPLC

Introduction

Amitriptyline is a tricyclic antidepressant that works by inhibiting the reuptake of norepinephrine and serotonin in the central nervous system. It is primarily used in the treatment of depression, anxiety disorders, and certain types of neuropathic pain. Perphenazine is a typical antipsychotic belonging to the phenothiazine class. It exerts its therapeutic effect by blocking dopamine receptors in the brain and is used in the treatment of schizophrenia and other psychotic disorders.

The combination of amitriptyline and perphenazine is sometimes prescribed for patients with depression who also exhibit psychotic features, as they can provide complementary therapeutic effects. This combination may also be used in certain cases of treatment-resistant depression where augmentation strategies are needed. Therefore, a reliable analytical method for the simultaneous estimation of both drugs in pharmaceutical formulations is essential for quality control purposes and therapeutic drug monitoring.

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is the most widely used technique for simultaneous drug estimation due to its high resolution, sensitivity, reproducibility, and speed of analysis. This paper describes a validated RP-HPLC method for the simultaneous determination of amitriptyline and perphenazine in combined dosage forms.

Experimental

Chemicals and Reagents

Amitriptyline hydrochloride and perphenazine reference standards were obtained from the pharmaceutical industry. HPLC grade acetonitrile and methanol were purchased from Merck, India. Analytical grade potassium dihydrogen phosphate and orthophosphoric acid were obtained from SD Fine Chemicals, India. HPLC grade water was obtained using a Milli-Q water purification system.

Instrumentation

The HPLC system used consisted of a quaternary pump, auto sampler, column oven, and UV detector with data acquisition and processing software. A C18 column (250 4.6 mm, 5 m particle size) was used for the separation.

Chromatographic Conditions

Isocratic elution was performed using a mobile phase consisting of phosphate buffer (pH 3.0):acetonitrile (60:40 v/v). The phosphate buffer was prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 1000 mL of HPLC grade water, and the pH was adjusted to 3.0 using orthophosphoric acid. The mobile phase was filtered through a 0.45 m membrane filter and degassed before use. The flow rate was maintained at 1.0 mL/min, and the detection was performed at 254 nm. The column temperature was maintained at 30C, and the injection volume was 20 L.

Preparation of Standard Solutions

Stock solutions of amitriptyline hydrochloride and perphenazine (1000 g/mL each) were prepared separately by dissolving accurately weighed amounts in methanol. Working standard solutions were prepared by appropriate dilution of the stock solutions with the mobile phase.

Sample Preparation

For the estimation of both drugs in marketed tablet formulations, twenty tablets were weighed and finely powdered. An amount equivalent to one tablet was weighed and transferred into a 100 mL volumetric flask, to which 70 mL of methanol was added. The flask was sonicated for 30 minutes with occasional shaking, and the volume was made up to the mark with methanol. This solution was filtered through Whatman filter paper No. 41. Further dilutions were made with the mobile phase to get concentrations within the linearity range.

Method Validation

Specificity

The specificity of the method was established by analyzing standard drug solutions, sample solutions, and blank solutions. The peaks for amitriptyline and perphenazine were well-resolved from each other and from any excipient peaks present in the sample solutions. The retention times were consistent, and there was no interference from the placebo formulation, indicating the method's specificity.

Linearity and Range

Linearity was determined by preparing standard solutions at six different concentration levels ranging from 10-50 g/mL for amitriptyline and 2-10 g/mL for perphenazine. Each concentration was injected in triplicate, and the peak areas were plotted against the concentrations. The calibration curves showed excellent linearity, with correlation coefficients (r) of 0.9995 for amitriptyline and 0.9998 for perphenazine.

Precision

The precision of the method was evaluated by performing repeatability (intra-day precision) and intermediate precision (inter-day precision) studies. Six replicate injections of standard solutions at three different concentration levels (low, medium, and high) were made on the same day and on three consecutive days. The percentage relative standard deviation (% RSD) values for both drugs were found to be less than 2%, indicating good precision.

Accuracy

Accuracy was determined by performing recovery studies using the standard addition method. Known amounts of standard drugs were added to pre-analyzed sample solutions at three levels (80%, 100%, and 120% of the label claim). The mean percentage recovery for amitriptyline was found to be 99.8-100.3%, and for perphenazine, it was 99.5-100.2%, indicating the method's accuracy.

Limit of Detection and Limit of Quantification

The limits of detection (LOD) and quantification (LOQ) were determined based on the standard deviation of the response and the slope of the calibration curve. The LOD and LOQ for amitriptyline were found to be 0.5 g/mL and 1.5 g/mL, respectively, while for perphenazine, they were 0.2 g/mL and 0.6 g/mL, respectively, indicating high sensitivity.

Robustness

Method robustness was evaluated by intentionally making slight modifications to the chromatographic parameters, including flow rate (0.1 mL/min), mobile phase composition (2%), pH (0.2), and column temperature (5C). The method was found to be robust as all the variations produced no significant changes in the retention times, peak symmetry, or resolution between the analytes.

System Suitability

System suitability parameters were evaluated to ensure that the analytical system was working correctly. The parameters studied included theoretical plates, tailing factor, resolution, and retention time. The results met the acceptance criteria, with theoretical plates greater than 2000, tailing factor less than 2.0, and resolution greater than 2.0 between the two analytes.

Results and Discussion

The developed RP-HPLC method provided good separation between amitriptyline and perphenazine, with retention times of 5.8 minutes for amitriptyline and 7.2 minutes for perphenazine. The resolution between the two peaks was found to be greater than 4.0, indicating excellent separation.

Linear regression analysis gave the following equations:

• For amitriptyline: y = 24580x + 1256 (r = 0.9995)
• For perphenazine: y = 31250x + 892 (r = 0.9998)

where y is the peak area and x is the concentration in g/mL.

Table 1: Summary of validation parameters

Parameter	Amitriptyline	Perphenazine
Linearity range (g/mL)	10-50	2-10
Correlation coefficient (r)	0.9995	0.9998
LOD (g/mL)	0.5	0.2
LOQ (g/mL)	1.5	0.6
Recovery (%)	99.8-100.3	99.5-100.2
Precision (% RSD)	0.5-1.2	0.4-1.0
Theoretical plates	4520	5130
Tailing factor	1.2	1.3

The method was successfully applied to the analysis of marketed tablet formulations containing both drugs. The mean percentage assay for amitriptyline was found to be 98.5-101.2%, and for perphenazine, it was 97.8-100.9%, demonstrating the method's applicability for quality control purposes.

Several methods have been reported for the estimation of amitriptyline and perphenazine individually, but only a few for their simultaneous estimation. Most reported methods have longer run times, use more complex mobile phase systems, or lack thorough validation. The present method overcomes these limitations by providing a simple, rapid, and validated method for the simultaneous estimation of both drugs. The use of a simple binary mobile phase system and isocratic elution makes the method convenient for routine analysis.

Conclusion

A simple, precise, accurate, and rapid RP-HPLC method has been developed and validated for the simultaneous estimation of amitriptyline and perphenazine in pharmaceutical formulations. The method was validated according to ICH guidelines and found to be suitable for routine quality control analysis of these drugs in combined dosage forms. The described method offers advantages over existing methods in terms of simplicity, reduced analysis time, and cost-effectiveness. The use of readily available reagents and simple mobile phase composition makes the method environmentally friendly and economical for routine pharmaceutical analysis.

The method provides good separation with resolution greater than 4.0, and the linearity range covers the expected concentrations in dosage forms. The validation results demonstrate that the method is specific, sensitive, precise, accurate, and robust. Therefore, this method can be recommended for the simultaneous estimation of amitriptyline and perphenazine in combined dosage forms in pharmaceutical quality control laboratories.

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