Stability Indicating RP-HPLC Method Development and Validation for The Simultaneous Estimation of Flupentixol and Melitracen in API from and Marketed Tablet Dosage form

 

S. Janet Beula¹*, T. Ramamohan Reddy², M. Viswaja³, Y. Ramulu⁴

¹Teegala Ram Reddy College of Pharmacy, Meerpet, RR District, Telangana, India.

²CMR College of Pharmacy, Medchal, RR District, Telangana, India.

³Vijaya College of Pharmacy, Munuganoor, RR District, Telangana, India.

⁴Sree Dattha Institute of Pharmacy, Ibrahimpatanam, RR District, Telangana, India.

 

ABSTRACT:

The present work describes a reverse phase high performance liquid chromatographic method                  (RP-HPLC) for the simultaneous estimation of Flupentixol and Melitracen in bulk and in tablet dosage form. Chromatographic separation was performed on Hypersil (C18) (250mm x 4.6mm,    5µm) Column, with a mobile phase comprising of a mixture of methanol and Acetonitrile in the ratio of 34:66v/v. The flow rate was 1.0ml/min with detection at 257nm. Retention times of Flupentixol and Melitracen were found to be 1.791min and 3.465min respectively. As per International Conference on Harmonization (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of Flupentixol was found to be in the range of 60-140µg/mL. and that for Melitracen was found to be 30-70µg/mL. The Precision (Repeatability, Intra-day and Inter-day) of the Flupentixol and Melitracen was found to be within the limits. The correlation coefficients were 0.999 and 0.999 for Flupentixol and Melitracen respectively. The mean recoveries obtained for Flupentixol and Melitracen were 100.28% and 99.79%. This demonstrates that the developed method is simple, precise, accurate, reproducible and rapid for simultaneous estimation of these drugs in bulk and in tablet dosage forms.

 

 

 

INTRODUCTION:

Combination therapy or poly therapy is therapy that uses more than one medication^1-3. Flupentixol is an antipsychotic neuroleptic drug. It is a thioxanthene, and therefore closely related to the phenothiazines.

 

Its primary use is as a long-acting injection given two or three weeklies to people with schizophrenia who have a poor compliance with medication and suffer frequent relapses of illness. Melitracen is a tricyclic antidepressant (TCA), for the treatment of depression and anxiety. In addition to single drug preparations, it is also available as Deanxit, marketed by Lundbeck, a combination product containing both Melitracen and Flupentixol. This method was validated according to ICH guidelines for specificity, LOD, LOQ, Precision, Robustness, system suitability, accuracy, and              Linearity^4-7. The method showed good reproducibility and recovery with %RSD less than 2. Hence, we had made an attempt to develop a simple accurate and precise RP HPLC method for the simultaneous estimation of Flupentixol and Melitracen in bulk and in tablet dosage form^8-11.

 

Drug Profile:^12-15

 

Name: Flupentixol:

Structure:

IUPAC Name:

2-(4-{3-[(9Z)-2-(trifluoromethyl)-9H- thioxanthen-9-ylidene] propyl} piperazin-1-yl) ethan-1-ol.

 

Indication:

Flupentixol belongs to the family of medications known as thioxanthene. This medication is used in the treatment of schizophrenia. It is thought to work by affecting nerve pathways in certain areas of the brain to help correct certain chemical imbalances that cause the symptoms of schizophrenia.

 

Name: Melitracen:

Structure:

 

IUPAC Name:

[3-(10, 10-dimethyl-9, 10-dihydroanthracen-9-ylidene) propyl] diethylamine.

 

Indication:

Melitracen is prescribed to treat depression and anxiety. Which is indicated for different types of depression (psychogenic, masked, menopausal, in alcoholics), depressive neuroses, psychosomatic affections accompanied by anxiety and apathy, and dysphoria in alcoholics. It is also given as a maintenance dose in psychogenic depressions and other psychoses.

 

MATERIALS AND METHODS:^16-20

Drug samples:

Pharmaceutically pure sample of Flupentixol and Melitracen drug was obtained from Sd fine-Chem ltd; Mumbai. Commercial tablet of Flupentixol and Melitracen (100mg) was procured from the local drug market. Acetonitrile and water, methanol, orthophosphoric acid, KH₂PO_4, K₂HPO₄ were HPLC from merck KGaA, 64271 Darmstadt, Germany.

 

Instruments:

Analytical HPLC –auto sampler-UV detector separation model 2695, UV detector 2487 Empower- software version-2, UV double beam spectrometer UV 3000+, Digital weighing balance (sensitivity 5mg), PH meter, Sonicator.

 

Preparation of the Flupentixol and Melitracen standard solution:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Flupentixol and Melitracen 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 1ml of the above Flupentixol and 0.5ml of the Melitracen stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Initialization of the instrument:

The HPLC instrument was switched on. First the column was washed with the HPLC grade water for 45 minutes. After washing the column that the column is saturated with the mobile phase in 45 minutes. The mobile phase was run to find the peaks or identification of peaks. After 20 minutes the standard drug solution was prepared and injected in HPLC system. 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. Initially the mobile phase tried was Methanol: Water and Water: Acetonitrile and Methanol: Phosphate Buffer: ACN with varying proportions. Finally, the mobile phase was optimized to Methanol: Acetonitrile in proportion 34:66 v/v respectively. The method was performed with various columns like C18 column, Symmetry and Zodiac column. Hypersil (C18) (250mm x 4.6mm, 5µm) Column was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

Optimized Chromatographic conditions:

Mobile phase	Methanol: Acetonitrile (34:66v/v)
Wavelength	257 nm
Flow rate	1ml/min
Auto Sampler Temperature	Ambient
Injection Volume	10µl
Run time	8 min.
Column	Hypersil (C18) (250mm x 4.6mm, 5µm) Column
Column Temperature	Ambient

 

Chromatographic trial for simultaneous estimation of Flupentixol and Melitracen by RP-HPLC:

 

Fig. No.1: The chromatogram obtained after optimized condition Flupentixol (1.791 min) and Melitracen (3.465min)

 

RESULTS AND DISCUSSION:

Method Development:

Determination of wavelength of maximum absorbance for of Flupentixol:

Standard of Flupentixol solution (1ml) was transferred to separate 10ml volumetric flask. The final volume was adjusted to 10ml with the same mobile phase. The absorbance of the final resulted solution was scanned in the range 400 to 220nm against mobile phase as blank.

 

Determination of Maximum wavelength for Melitracen:

First of all, take 1ml of standard Melitracen solution from the above standard solution (1ml) was transferred to separate clean and dry of 10ml volumetric flask. The final volume was adjusted to 10ml with same mobile phase (Solvent). The absorbance of the final resulted solution was scanned in the range 400  to 220nm against solvent mixture as blank.

 

Fig. No. 2: UV Spectrum of Flupentixol (292 nm)

 

Fig. No. 3: UV Spectrum of Melitracen (342 nm)

 

Accuracy:

Table No. 1: Showing Accuracy Results for Flupentixol:

Sample ID	Concentration (mg/ml)			% Recovery of Pure drug	Statistical Analysis
	Conc. Found	Conc. Recovered	Peak Area
S1 : 50 %	8	8.039	348673	100.487	Mean= 100.633% S.D. = 0.182066 % R.S.D.= 0.180921
S2 : 50 %	8	8.046	348945	100.575
S3 : 50 %	8	8.067	349745	100.837
S1 : 100 %	10	9.862	419823	98.62	Mean= 99.95% S.D. = 1.340112% R.S.D.= 1.340782
S2 : 100 %	10	9.993	424941	99.93
S3 : 100 %	10	10.130	430295	101.3
S7 : 150 %	12	12.115	507788	100.958	Mean= 100.9717% S.D. = 0.512637 % R.S.D.= 0.507703
S8 : 150 %	12	12.179	510262	101.491
S9 : 150 %	12	12.056	505468	100.466

 

Table No. 2: showing Accuracy Results for Melitracen:

Sample ID	Concentration (mg/ml)			% Recovery of Pure drug	Statistical Analysis
	Conc. Found	Conc. Recovered	Peak Area
S1 : 50 %	16	15.991	989572	99.943	Mean= 100.1577% S.D. = 0.654939 % R.S.D.= 0.653908
S2 : 50 %	16	16.143	998756	100.893
S3 : 50 %	16	15.942	986589	99.637
S4 : 100 %	20	19.995	1231734	99.975	Mean= 100.795% S.D. = 0.822511% R.S.D.= 0.816024
S5 : 100 %	20	20.158	1241569	100.79
S6 : 100 %	20	20.325	1251694	101.62
S7 : 150 %	24	24.335	1494218	101.395	Mean= 100.805% S.D. = 0.613739 % R.S.D.= 0.608837
S8 : 150 %	24	24.204	1486312	100.85
S9 : 150 %	24	24.041	1476398	100.170

 

Table No. 3: Showing % RSD Results for Flupentixol and Melitracen:

Concentration of Flupentixol and Melitracen in ppm	Rt of Flupentixol	Peak area of Flupentixol	Rt of Melitracen	Peak area of Melitracen
10 +10	2.264	3303800	3.132	951802
10 +10	2.246	3349883	3.132	958267
10 +10	2.264	3353514	3.129	954481
10 +10	2.246	3384162	3.113	952151
10 +10	2.280	3390496	3.113	952308
AVG	2.26	3356371	3.1238	953801.8
S.D.	0.014353	34463.10324	0.009935	2709.017
% RSD		1.026796598		0.284023

 

 

The limit for mean % recovery is 98-102% and as both the values are within the limit, hence it can be said that the proposed method was accurate.

 

Precision:

Repeatability: The precision of each method was achieved separately from the peak areas obtained by actual estimation of 5 injections of fixed homogenous sample concentrations of Flupentixol and Melitracen. The % relative standard deviation for the Flupentixol and Melitracen was calculated.

 

The repeatability study which was conducted on the solution having the concentration of about10mg/ml for Flupentixol and 10 mg/ml for Melitracen (n =5) showed a %RSD of 1.026796598 for Flupentixol and 0.284023 for Melitracen. It was concluded that the analytical technique showed good repeatability.

 

Linearity and Range:

Linearity range was found to be 60-140µg/ml for Flupentixol and 30-70µg/ml for Melitracen. The correlation coefficients were found to be 0.999 and 0.999, the slopes were found to be 4711 and 17979 and intercept were found to be 4588 and 47869 for Flupentixol and Melitracen respectively.

 

Fig. No. 4: Standard curve for Flupentixol

 

Table No. 4: Showing Standard curve for Flupentixol:

Cоncentrаtiоn µg/ml	Аverаge Peak Аreа
60	289658
80	387568
100	478562
120	568546
140	658825

 

Fig. No. 5: Standard curve for Melitracen

 

Table No. 5: Showing Standard curve for Melitracen:

Cоncentrаtiоn µg/ml	Аverаge Peаk Аreа
30	5495852
40	7258456
50	9058654
60	10854253
70	12567853

 

Limit of detection (LOD) and Limit of quantification (LOQ):

The detection limit (LOD) and quantization limit (LOQ) may be expressed as:

 

L.O.D. = 3.3 (SD/S).

L.O.Q. = 10 (SD/S)

 

Where,

SD = Standard deviation of the response

S = Slope of the calibration curve

 

The Minimum concentration level at which an analyse can be reliable detected (LOD) and quantified (LOQ) were found to be 0.09 and 0.29 µg/ml respectively for Letrozole.

 

The LOD was found to be 0.1 mg/ml and LOQ was found to be 0.3 mg/ml for Palbociclib which represents that sensitivity of the method is high.

 

Assay:

                AT      WS       DT        P

Assay % = –––– × –––– × –––– × –––– × Average weight = mg/tab

                    AS       DS       WT     100

 

Where;

AT  = Test Preparation Peak Area

AS  = Standard preparation Peak Area

WS = Working standard weight taken in mg

WT = Sample weight taken in mg

DS  = Standard solution dilution

DT  = Sample solution dilution

P     = Working standard percentage purity

 

The assay of Franxit tablets containing Flupentixol was found to be 9.78 (±0.08) and Melitracen was found to be 49.22 (±0.05) and the % purity of the Flupentixol and Melitracen was found to be 99.78(±0.48) /99.77(±0.12). (Table-6).

 

Robustness:

The robustness was performed for the flоw rate vаriаtiоns from 0.9 ml/min to 1.1ml/min and mobile phase ratio vаriаtiоn from more оrgаnic phase to less оrgаnic phase ratio for Flupentixol and Melitracen. The method is robust only in less flow condition and the method is robust even by change in the Mobile phase ±5%. The standard and samples of Flupentixol and Melitracen were injected by changing the conditions of chrоmаtоgrаphy. There was no significant change in the parameters like resolution, tаiling factor, asymmetric factor, and plate count.

 

Table No. 6: Showing Assay of Flupentixol and Melitracen Tablets:

Brand name of tablets	Labelled amount of Drug (mg) Flupentixol and Melitracen	Mean (±SD) amount (mg) found by the proposed method (n=6)	Mean (± SD) Assay (n = 6)
Franxit Tablet	10/50	9.78 (±0.08) /49.22 (±0.05)	99.78(±0.48) /99.77(±0.12)

 

Table No. 7: Showing Results for Robustness -Flupentixol

Parameter used for sample аnаlysis	Peаk Аreа	Retention Time	Theoretical plates	Tаiling factor
Аctuаl Flow rate of 0.9mL/min	485685	1.791	6527	1.35
Less Flow rate of 0.8mL/min	546523	1.867	7256	1.43
More Flow rate of 1.0mL/min	498652	1.744	6248	1.34
Less оrgаnic phase (аbоut 5 % decrease in оrgаnic phase)	478524	1.831	6423	1.32
More оrgаnic phase (аbоut 5 % Increase in оrgаnic phase)	465382	1.874	6355	1.31

 

Table No. 8: Showing Results for Robustness-Melitracen

Parameter used for sample аnаlysis	Peаk Аreа	Retention Time	Theoretical plates	Tаiling factor
Аctuаl Flow rate of 0.9mL/min	8654527	3.465	7698	1.46
Less Flow rate of 0.8mL/min	9152684	3.721	8254	1.42
More Flow rate of 1.0mL/min	8526472	3.097	7326	1.43
Less оrgаnic phase (аbоut 5 % decrease in оrgаnic phase)	8245635	6.242	7298	1.45
More оrgаnic phase (аbоut 5 % Increase in оrgаnic phase)	8365824	2.402	7199	1.40

 

АCCEPTАNCE CRITERIA:

The Tаiling factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

 

SUMMARY AND CONCLUSION:

The developed method was successfully applied for simultaneous estimation of Flupentixol and Melitracen in compound tablet formulation. The proposed RP-HPLC method has excellent sensitivity, precision and reproducibility. A sensitive and selective stability indicting RP-HPLC method has been developed and validated for the analysis of Flupentixol and Melitracen API.

 

ACKNOWLEDGEMENT:

We take pleasure to express our sincere thanks to our beloved Dr. D. Vikshapathi, Principal, Teegala Ram Reddy College of Pharmacy for his undivided attention, valuable suggestion, and endeavor throughout the during project work. Special thanks to all faculty members of Teegala Ram Reddy College of Pharmacy for their help, support and encouragement during project work.

 

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Received on 11.02.2022       Modified on 12.03.2022

Accepted on 22.03.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2022; 12(2):115-120.