Validated RP-HPLC Method for Simultaneous Estimation of Perphenazine and Amitriptyline in Bulk and Tablet Dosage form

 

P. Aravinda Reddy¹*, Vommidarapu Srujana¹, Ramya Sri. S²

¹Department of Pharmaceutical Analysis, Samskruti College of Pharmacy,

Affiliated to JNTUH University, Hyderabad 501301, Telangana, India.

²Department of Pharmacy, University College of Technology, Osmania University,

Hyderabad, Telangana, 500007, India.

*Corresponding Author E-mail: aravindareddyp1@gmail.com

A new, simple, precise, rapid, selective and stability reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous quantification of Perphenazine and Amitriptyline in pure form and its pharmaceutical dosage form. The method is based on Phenomenex Gemini C18 (4.6×250mm) 5µ column. The separation is achieved using isocratic elution by Methanol: TEA Buffer in the ratio of 65:35% v/v, pumped at flow rate 1.0mL/min and UV detection at 230nm. The column is maintained at 40°C throughout the analysis. The total run time is about 6min. The method is validated for specificity, accuracy, precision and linearity, robustness and ruggedness, system suitability, limit of detection and limit of quantitation as per International conference of harmonization (ICH) Guidelines. The method is accurate and linear for quantification of Perphenazine, Amitriptyline between 10 - 50µg/mL and 20 - 100µg/mL respectively. Further, satisfactory results are also established in terms of mean percent- age recovery (100.37% for Perphenazine and 100.34% for Amitriptyline, intra-day and inter-day precision (<2%) and robustness. The advantages of this method are good resolution with sharper peaks and sufficient precision. The results indicate that the method is suitable for the routine quality control testing of marketed tablet formulations.

 

 

INTRODUCTION:

In HPLC, separation occurs due to partitioning between a stationary phase contained in a column and a liquid phase, which is pumped under pressure through this column. Each of the components will have a certain affinity for the stationary phase and a certain affinity for the mobile phase. Provided there is sufficient difference between the analytes in their relative affinities for the two phases, then in HPLC system they will separate ¹.

 

The components themselves are first dissolved in a solvent and then required to flow (via the mobile phase) complete a column (stationary phase) in high pressure.

 

The mixture is determined into its components within the column and the amount of resolution is dependent upon the interaction between the solute components and the column stationary phase and liquid phase. The interaction of the solute with the mobile and stationary phases can be worked through different choices of both solvent and stationary phases ².

 

Schizophrenia is a disorder that affects the way a person acts, thinks, and sees the world. People with schizophrenia have an altered perception of reality and may withdraw from the outside world and or act out in confusion and fear ³. Schizophrenia strikes without regard to gender, race, social class or culture ⁴. Pharmacological therapies and psychosocial interventions play a role in the prognosis of schizophrenia as an essential component of a comprehensive schizophrenia treatment ⁵.

 

Fig 1: Chemical Structure of Perphenazine⁶

 

Amitriptyline is a tricyclic antidepressant (TCA). 3(10,11dihydro 5H dibenzo (a,b) cyclohetene-5-cylidene)-N,N-dimethylpropan-1-amine ⁷. It is a white or practically white, odourless or practically odourless, crystalline powder or small crystals ⁸. It acts primarily as a serotonin-norepinephrine reuptake inhibitor, with strong actions on the norepinephrine transporter, and moderate effects on the serotonin transporter ⁹.

 

Fig 2: Chemical Structure of Amitriptyline¹⁰

 

MATERIALS AND METHODS:

Perphenazine from Sura labs, Amitriptyline from Sura labs, Water and Methanol for HPLC from Lichrosolv (Merck), Acetonitrile for HPLC from Merck

 

HPLC METHOD DEVELOPMENT:

Trails:

Preparation of standard solution:

Accurately weigh and transfer 10 mg of Perphenazine and Amitriptyline working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

 

Further pipette 0.3 ml of Perphenazine and 0.6ml of Amitriptyline from the above stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was methanol: Water, Methanol: Phosphate buffer and ACN: Water with varying proportions. Finally, the mobile phase was optimized to TEA buffer (pH 4.0), Methanol in proportion 65:35 v/v respectively. 

 

Optimization of Column:

The method was performed with various C18columns like Symmetry, X terra and ODS column. Phenomenex Gemini C18 (4.6×250mm) 5µ was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard):

Mobile phase ratio: Methanol: TEA Buffer (65:35 v/v)

Column: Phenomenex Gemini C18 (4.6×250mm) 5µ

Column temperature: 40ºC

Wavelength: 230nm

Flow rate: 1ml/min

Injection volume: 10µl

Run time: 6minutes

 

Figure 3-: Optimized Chromatogram (Standard)

 

Optimized Chromatogram (Sample):

 

Figure 4-: Optimized Chromatogram (Sample)

Table1-: Optimized Chromatogram (Standard)

S. No.	Name	RT	Area	Height	USP Tailing	USP Plate Count	Resolution
1	Perphenazine	2.157	526541	78564	1.62	5859
2	Amitriptyline	3.631	1645875	265842	1.48	7965	9.9

 

Table 2: Optimized Chromatogram (Sample)

S. No.	Name	Rt	Area	Height	USP Tailing	USP Plate Count	Resolution
1	Perphenazine	2.142	538954	79658	1.63	5986
2	Amitriptyline	3.649	1658745	275854	1.49	8056	10.1

 

System Suitability:

 

Table 3-: Results of system suitability for Perphenazine

S. No.	Peak Name	RT	Area (µV*sec)	Height (µV)	USP Plate Count	USP Tailing
1	Perphenazine	2.152	526856	78569	1.63	5856
2	Perphenazine	2.157	528794	78545	1.63	5874
3	Perphenazine	2.141	526598	78954	1.62	5869
4	Perphenazine	2.133	524875	78224	1.63	5897
5	Perphenazine	2.166	526584	78965	1.62	5829
Mean			526741.4
Std. Dev.			1392.398
% RSD			0.264342

 

Table 4-: Results of system suitability for Amitriptyline

S. No	Peak Name	RT	Area (µV*sec)	Height (µV)	USP Plate Count	USP Tailing	Resolution
1	Amitriptyline	3.674	1645985	268542	5869	1.48	10.01
2	Amitriptyline	3.631	1648579	267854	5874	1.49	10.01
3	Amitriptyline	3.625	1645739	268598	5864	1.48	9.99
4	Amitriptyline	3.692	1645285	268745	5826	1.49	10.01
5	Amitriptyline	3.629	1648598	268598	5824	1.48	10.02
Mean			1646837
Std. Dev.			1618.325
% RSD			0.098269

 

Assay (Standard):

 

Table 5-: Peak results for assay standard of Perphenazine

S. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	Injection
1	Perphenazine	2.152	526595	78569	1.63	5896	1
2	Perphenazine	2.198	524658	78496	1.63	5879	2
3	Perphenazine	2.179	528476	78459	1.62	5895	3

 

Table 6-: Peak results for assay standard of Amitriptyline

S. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	Injection
1	Amitriptyline	3.646	1648546	265845	1.48	8012	1
2	Amitriptyline	3.604	1648598	265418	1.49	7955	2
3	Amitriptyline	3.610	1648574	265365	1.48	7989	3

 

Assay (Sample):

Table 7-: Peak results for Assay sample of Perphenazine

S. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	Injection
1	Perphenazine	2.152	536598	79856	1.64	5969	1
2	Perphenazine	2.150	536589	79265	1.65	5997	2
3	Perphenazine	2.187	534658	79898	1.65	5986	3

 

Table 8-: Peak results for Assay sample of Amitriptyline

S. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	Injection
1	Amitriptyline	3.646	1658952	278598	1.49	8016	1
2	Amitriptyline	3.651	1658954	276984	1.48	8041	2
3	Amitriptyline	3.601	1653659	275849	1.49	8079	3

 

%ASSAY =

  Sample area        Weight of standard     Dilution of sample     Purity      Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

  Standard area      Dilution of standard    Weight of sample       100          Label claim

 

The % purity of Perphenazine and Amitriptyline in pharmaceutical dosage form was found to be 99.63%

 

Linearity

Chromatographic Data for Linearity Study of Perphenazine:

 

Fig-5: Calibration Curve of Perphenazine

 

 

 

Chromatographic Data for Linearity Study of Amitriptyline:

Concentration mg/ml	Average Peak Area
20	665985
40	1298698
60	1927852
80	2548545
100	3162468

 

Fig 6-: Calibration Curve of Amitriptyline

 

 

Repeatability:

Table 9-: Results of Repeatability for Perphenazine:

S. No.	Peak name	Retention time	Area (µV*sec)	Height (µV)	USP Plate Count	USP  Tailing
1	Perphenazine	2.157	526854	78569	5869	1.62
2	Perphenazine	2.159	523659	78469	5874	1.63
3	Perphenazine	2.186	523856	78525	5896	1.63
4	Perphenazine	2.160	523485	78548	5818	1.62
5	Perphenazine	2.170	523485	78594	5879	1.63
Mean			524267.8
Std.dev			1453.805
%RSD			0.277302

 

Table 10-: Results of repeatability for Amitriptyline:

S. No.	Peak name	Retention time	Area (µV*sec)	Height (µV)	USP Plate Count	USP  Tailing
1	Amitriptyline	3.603	1645879	265845	7985	5869
2	Amitriptyline	3.608	1648578	265487	7964	5849
3	Amitriptyline	3.600	1645985	265982	7915	5879
4	Amitriptyline	3.696	1648759	265478	7928	5874
5	Amitriptyline	3.629	1648572	265422	7964	5829
Mean			1647555
Std.dev			1483.603
%RSD			0.090049

 

Accuracy:

Table 11-: The accuracy results for Perphenazine

% Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	263572	15	15.038	100.253%	100.37%
100%	518870.3	30	30.147	100.490%
150%	772572.3	45	45.162	100.360%

                                                                               

Table 12-: The accuracy results for Amitriptyline

% Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	972935.7	30	30.109	100.363%	100.34%
100%	1919319	60	60.100	100.166%
150%	2877020	90	90.449	100.498%

Limit of Detection:

The detection  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

LOD= 3.3 × σ / s

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Perphenazine:

= 0.9µg/ml

 

Amitriptyline:

= 1.2µg/ml

 

 

 

Quantitation Limit:

The  quantitation  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte  in  a  sample  which  can  be  quantitatively  determined. 

LOQ=10×σ/S

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Perphenazine:

=2.7µg/ml

 

Amitriptyline

Result:

=3.6µg/ml

 

Robustness

 

Table 13-: Results for Robustness

Perphenazine

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.0 mL/min	526541	2.157	5859	1.62
Less Flow rate of 0.9 mL/min	589564	2.210	5635	1.61
More Flow rate of 1.1 mL/min	515246	2.184	5569	1.64
Less organic phase	502659	2.200	5154	1.63
More Organic phase	526485	2.172	5365	1.62

 

Table 14-: Results for Robustness

Amitriptyline

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.0 mL/min	1645875	3.643	7965	1.48
Less Flow rate of 0.9 mL/min	1635985	4.498	7856	1.46
More Flow rate of 1.1 mL/min	1624587	3.505	7425	1.43
Less organic phase	1652834	4.504	7621	1.45
More organic phase	1625548	3.512	7582	1.42

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Perphenazine and Amitriptyline in bulk drug and pharmaceutical dosage forms.

 

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Perphenazine was found to be soluble in methanol and in ethanol (95), soluble in acetic acid (100), sparingly soluble in diethyl ether and practically insoluble in water and also dissolves in dilute hydrochloric acid. Amitriptyline was found to be soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide (DMF) and freely soluble in water, methanol and Acetonitrile and freely soluble in alcohol, chloroform, methyl alcohol and methyl chloride, practically insoluble in ether.

Methanol: TEA Buffer (65:35 v/v)was chosen as the mobile phase. The solvent system used in this method was economical.

 

The %RSD values were within 2 and the method was found to be precise.

 

The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

 

This method can be used for the routine determination of Perphenazine and Amitriptyline in bulk drug and in pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

The Authors are thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

 

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Received on 01.11.2022                    Modified on 08.12.2022

Accepted on 30.12.2022                   ©AJRC All right reserved