INTRODUCTION:

Flupenthixol HCl^1-7is a white to off white powder, amorphous in nature, sensitive to light and moisture. Chemically it is 2-[4-[3-[2-(trifluoromethyl)-9-ylidene]propyl]piperazin-1-yl] Ethanol. It is a thioxanthene drug and acts by antagonism of D1 and D2 Dopamine receptors (as well as serotonin).it effectively relives Hallucination disorders associated with schizophrenia. It is official in B.P, Martindale-the extra Pharmacoeipia. Melitracen HCl^8,9is white powder. Chemically it is 3-(10,10-dimethyl anthracen-9-ylidine)-N,N-dimethyl propan-1-amine.it is a tricylclic antidepressant and work by inhibiting the re-uptake of the neurotransmitters norepinephrine and serotonin by neurons. Tricyclics may also possess an affinity for muscarinic and histamine H1 receptors to varying degrees. Although the pharmacologic effect occurs immediately, often the patient's symptoms do not respond for 2 to 4 weeks it is used in treatment of trigeminal neuralgia and severe depression state. The method reported for simultaneous estimation of Flupenthixol HCl and Melitracen HCl is validation of a sensitive LC/MS/MS method for simultaneous quantitation of Flupenthixol and Melitracen in Human Plasma. Therefore, an attempt has been made to develop a more accurate, precise, reproducible and economic spectrophotometric method^1,2,4 over the available method.

METHOD AND MATERIALS:

All the Chemicals and reagents were of Analytical Grade (AR). The instrument used in the present study was Double beam UV-visible Spectrophotometer with 10mm matched quartz cell.

Standard Solutions:

Flupenthixol HCl Stock standard solution:

An accurately weighed quantity ~ 50.0 mg of Flupenthixol HCl was dissolved in solvent i.e., methanol: in 50.0 ml volumetric flask and volume was made up to the mark with the same solvent.

Melitracen HCl Stock standard solution:

An accurately weighed quantity of Melitracen Hydrochloride ~ 50.0mg was dissolved in solvent i.e., methanol in 50.0 ml volumetric flask and volume was made up to mark with the same solvent.

Study of Spectra and selection of wavelength:

The aliquot portions of stock standard solutions of Flupenthixol HCl and Melitracen HCl were diluted approximately with solvent to obtain concentration of 5 mcg/ml of each drug. The solutions were scanned in the range of 400-200 nm in 1cm cell against blank.

From the overlain spectrum the wavelength selected for estimation of drugs were 283.60nm as Isoabsorptive Point and 229.40nm as λmax of Flupenthixol HCl.

λmax of Flupenthixol HCl = 229.40nm

Isoabsorptive Point = 283.60nm

Fig. 1: Overlain spectra of Flupenthixol HCl and Melitracen HCl

E (1%, 1cm) of individual drugs at selected wavelength:

Aliquot portions of Flupenthixol HCl and Melitracen HCl were diluted with solvent i.e., methanol to obtain different concentrations of each drug. The absorbance of each solution was measured at 283.60nm and 229.40nm.A (1%1cm) the values were calculated using following formula.

Text Box: Absorbance
Concentration (g/100ml)

E (1%, 1cm) =

Study of Beer Lambert Law^10,11:

The aliquot portions of stock standard solutions of Flupenthixol HCl and Melitracen HCl were diluted appropriately with solvent to get a series of concentration between 2-10(mcg/ml).the absorbance of each solution was measured at 229.40nm and 258.40nm in 1cm cell against blank.

Absorbance Additivity Study:

The data obtained in study of Beer-Lambert law was further used to study additivity of absorbance of Flupenthixol HCl and Melitracen HCl at 283.60nm and 229.40nm.Mixture of drugs shows additivity of absorbance at selected wavelength.

Analysis of Laboratory mixture by proposed method:

In order to see feasibility of proposed method for simultaneous estimation of Flupenthixol HCl and Melitracen HCl in Pharmaceutical formulation, the method was first tried for estimation of drugs in standard laboratory mixture.

Accurately weighed quantities ~50.0 mg of Flupenthixol HCl and Melitracen HCl ~ 50.0 mg were taken in 100ml volumetric flask and dissolved in solvent i.e., Methanol: by vigorous shaking. The volume was made upto mark with the same solvent to get final concentration of about 5µg/ml Flupenthixol and 5µg/ml Melitracen, the absorbance at 283.60nm and 229.40nm in 1cm cell against blank.

Amount of each drug was calculated using following formulae.

Cx =

Cy =

Where,

Qm = Absorbance ratio of mixture at 283.60 nm and 229.40nm

Qy = Ratio of Absorptivity of FLU at 283.60nm and 229.40nm

Qx = Ratio of Absorptivity of MEL at 283.60nm and 229.40nm

A = Absorbance ratio of mixture at 283.60nm (i.e. isoabsorptive point)

ax₁and ay₁ = Absorptivity of FLU and MEL at respectively at isoabsorptive wavelength = 283.60 nm

Cx and Cy = Concentration in g/100ml of FLU and MEL respectively in final solution.

The drug can be estimated by the formula

Amount of drug estimated (mg)

Weight of drug taken (mg)

%Drug estimated = x DF

Where, C= concentration in mg/ml of FLU and MEL

D = Dilution factor = 2000

V = Volume of stock = 50.0ml

Estimation of Drugs in Marketed Formulation:

Twenty tablets were accurately weighed and contents removed. Average weight of contents per tablets was calculated. The contents of tablets were reduced to fine powder and mixed thoroughly. a quantity of tablet powder Flupenthixol HCl and Melitracen HCl was transferred to 50.0 ml volumetric flask and 0.0475g of flupenthixol (pure drug was added) and volume was made to 50.0 ml with same solvent. the solution was filtered through whatman filter paper no.41.the aliquot portion of filtrate was further diluted to get final concentration of about 5µg/ml of Flupenthixol HCl and 5µg/ml of Melitracen HCl.

The absorbance of sample solution was measured at 283.60nm and 229.40nm in 1cm cell against the blank. The content of flupenthixol HCl and Melitracen HCl in tablet was calculated using formula

% labeled claim of FLU = Cx x d x Avg. Wt. of tablet

Wm x L

Cx x d x Avg. Wt. of tablet

Wm x L

% labeled claim of MEL =

Where,

Cx and Cy = concentration g/100ml of FLU and MEL in final solution respectively

D = Dilution factor = 2000

Wm = Weight of tablet powder taken in g

L = Labeled claim of drug in g/tablet

TABLES-1(A): ABSORPTIVITY VALUES FOR FLU

Sr No.	Conc. Of FLU (g/100ml)	Absorbance at		E(1%,1cm) at
Sr No.	Conc. Of FLU (g/100ml)	283.60nm	229.40nm	283.60nm	229.40nm
1	0.000515	0.0461	0.2309	92.2	461.8
2	0.000.513	0.0463	0.2306	92.6	461.20
3	0.000516	0.0468	0.230	93.6	460.00
4	0.000513	0.0471	0.2310	94.2	464.00
5	0.000512	0.0476	0.2320	95.4	460.00
			Mean	93.6	461
			+ S.D.	1.28	1.64
			C.V.	1.36	0.35

TABLES-1(B): ABSORPTIVITY VALUES FOR MEL

Sr no	Conc. of MEL (g/100ml)	Absorbance at	E(1%,1cm) at
Sr no	Conc. of MEL (g/100ml)	283.60nm 229.40nm	283.60nm 229.40nm
1	0.000525	0.0395 0.1766	79 353.2
2	0.000520	0.0399 0.1764	79.8 352.8
3	0.000523	0.0397 0.1768	79.4 351.6
4	0.000521	0.0395 0.1769	79.6 353.8
5	0.000526	0.0401 0.1762	80.2 352.4
		Mean	79.6 352.6
		+ S.D.	0.44 0.81

TABLES-2: ESTIMATION OF DRUG IN LABORATORY MIXTURE

Sr No	Wt. of FLU	Wt. of MEL	Absorbance of Mixture	% Drug Estimated
Sr No	Wt. of FLU	Wt. of MEL	283.60nm 229.40nm	283.60nm 229.40nm
1	0.0103	0.0105	0.0863 0.4069	101.18 99.89
2	0.0105	0.0106	0.0865 0.4067	100.33 99.93
3	0.0102	0.0104	0.0867 0.4065	99.86 100.18
4	0.0104	0.0102	0.0860 0.4070	98.22 100.07
5	0.0106	0.0106	0.0873 0.4071	99.89 99.63
			Mean	99.99 99.63
			+ S.D.	1.078 0.2038
			C.V.	1.078 0.2039

TABLE-3 ESTIMATION OF DRUGS IN TABLET

Sr No	Wt .Sample(g)	Absorbance of Mixture	% Drug Estimated
Sr No	Wt .Sample(g)	283.60nm 229.40nm	283.60nm 229.40nm
1	0.1565	0.0871 0.4072	99.26 99.84
2	0.1568	0.0873 0.4074	100.01 99.94
3	0.1570	0.0875 0.4077	99.93 99.82
4	0.1571	0.0877 0.4079	100.20 99.85
5	0.1573	0.0880 0.4081	100.41 100.08
		Mean	99.96 99.90
		+ S.D.	0.4340 0.1076
		C.V.	0.4342 0.1077

TABLE-4: RESULTS OF RECOVERY STUDY

Sr No	Wt. of Tablet Powder	Amount added mcg/mL FLU MEL	Absorbance of Mixture	% Drug Recovery
Sr No	Wt. of Tablet Powder	Amount added mcg/mL FLU MEL	283.60nm 229.40nm	283.60nm 229.40nm
1	0.1568	5 5	0.1745 0.8144	99.26 99.84
2	0.1560	5 5	0.1747 0.8147	100.01 99.95
3	0.1564	5 5	0.1743 0.8149	100.19 99.82
4	0.1565	5 5	0.1742 0.8151	100.20 99.87
5	0.1563	5 5	0.1747 0.8153	100.18 100.08
			Mean	99.98 99.91
			+ S.D.	0.4340 0.1076
			C.V.	0.4342 0.1077

Recovery studies:

Recovery study was done by standard addition method. Sample solution was prepared and final 5mcg/ml concentration solutions were prepared. thereafter sample solution were prepared by preanalysing tablet powder equivalent to 1mg of flupenthixol was taken in 100.0 ml volumetric flask; to it standard solutions of flupenthixol and

melitracen were added in different proportions. then the volume was adjusted upto the mark with the solvent.

Solution was filtered through whatman filter paper no.41.the aliquot portions of filtrate were further added and absorbances of solutions were scanned at 229.40nm and 258.40nm respectively.

% Recovery was then calculated by using formula

% Recovery =

Where, T= total drug estimated

C= drug contributed by preanalysed powder

P= weight of pure drug added

Validation of Proposed Method:

The proposed method was validated by considering the following parameters:

Figure 2: The plot of linearity and range for FLU and MEL

Figure 3: Absorbance of isobestic point plot for Flupenthixol HCl and Melitracen HCl

Accuracy:

Accuracy of analytical method is the closeness of test results obtained by the method to the true value. It was ascertained on the basis of recovery studies performed by standard addition method.

Precision:

Precision of an analytical method is the degree of agreement among individual results when the method is applied repeatedly to multiple readings of a homogenous sample. It is expressed as S.D. or R.S.D. of series of measurements. It was ascertained by replicate estimation of drugs by proposed method.

Specificity:

Accurately weighed quantities of the tablet powder equivalent to about 50.0 mg of MEL were taken in a dry 50.0ml volumetric flask. Each sample was then store for 24hrs under the following different conditions.

1. at room temperature (Normal)

1. At 50⁰C after addition of 1ml of 0.1 M NaOH (Alkali)

2. At 50⁰C after addition of 1ml of 0.1 M HCl (Acid)

3. At 50⁰C after addition of 1 ml of 3% H₂O₂

4. At 60⁰C (Heat)

After 24 hrs each treated sample was dissolved in methanol with vigorous shaking and volume was made upto the mark with Methanol. The solutions were then filtered and aliquots of filtrate were diluted to get final diluted sample solution of FLU and MEL and absorbance were read at 229.40.8nm and 258.40nm against blank. The percent of labeled claim were calculated using same formula given under estimation of FLU and MEL by proposed method.

Ruggedness:

The studies of ruggedness were carried out under two different conditions:

· Days and

· Analyst

Intraday:

It was performed by using procedure as under marketed formulation analysis and absorbance recorded at 3 hrs. Interval within a day. The % label claim was calculated using same formula as for marketed formulation analysis.

TABLE-6: SPECIFICITY STUDY

Sr. No.	Conditions	% Labeled Claim FLU MEL
1	Normal	100.38 101.45
2	Acid	99.98 102.39
3	Alkali	98.36 99.87
4	Oxide	100.12 101.00
5	Heat	100.12 101.00
6	UV	100.04 99.75

Interday:

Same procedure was performed as under marketed formulation analysis and absorbance of same sample were recorded on different days. The % label claim was calculated using same formula as for marketed formulation analysis.

TABLE-7: RESULTS OF RUGGEDNESS STUDY

A) DIFFERENT ANALYST

Sr No	Wt. of Samples(g)	Absorbance at (nm) 283.60nm 229.40nm	% Labeled Claim FLU MEL
1	0.1560	0.1760 0.8146	99.58 99.89
2	0.1562	0.1754 0.8148	99.71 99.83
3	0.1563	0.1762 0.8149	100.15 99.89
		Mean	99.83 99.88
		+ S.D.	0.2987 0.03464
		C.V.	0.2993 0.03469

B) DIFFERENT DAYS

Sr No	Wt. of Samples(g)	Absorbance at (nm) 283.60nm 229.40nm	% Labeled Claim FLU MEL
1	0.1558	0.1763 0.8148	99.67 99.98
2	0.1560	0.1765 0.8150	99.62 100.09
3	0.1562	0.1767 0.8151	100.12 99.87
		Mean	99.80 99.87
		+ S.D.	0.2754 0.1100
		C.V.	0.2760 0.1100

TABLE-8: C) DIFFERENT LABORATORIES

Sr. No.	Wt. of Samples(g)	Absorbance at (nm) 283.60nm 229.40nm	% Labeled Claim FLU MEL
1	0.1555	0.1768 0.8152	99.94 99.85
2	0.1562	0.1771 0.8154	100.18 100.03
		Mean	100.06 99.94
		+ S.D.	0.1697 0.1273
		C.V	0.1696 0.1274

Different analyst:

The sample solutions were prepared by two different analysts and same procedure was followed as described earlier. The % label claim was calculated as done in marketed formulation estimation.

Linearity and range:

Accurately weighed quantities of tablet powder equivalent to 80, 90, 100, 110, 120 % of label claim were taken and dilutions were done appropriately to obtain a concentration in the range of 80-120 % of the test concentration and absorbance were recorded at 229.40nm and 258.40nm. FLU and MEL were found to be linear in 80%-120% of test concentration.

RESULTS AND DISCUSSION:

The attempt was made to develop and alternative and economical method for estimation of Flupenthixol HCl and Melitracen HCl by isobestric point method.

Overlain spectra of FLU and MEL had revealed that both the drug have same absorbance (isobestric point) in methanol at 283.60nm. Flupenthixol HCl shows it’s absorption maxima at 229.40nm in same solvents. Hence it was predicted that simultaneous estimation of both drugs should be possible by solving a pair of equation for isobestric point method, by measuring the absorption of sample solution at 283.60nm and 229.40nm.

Both the drug FLU and MEL and its mixtures follow beer - Lambert’s law in the range of 2-10 μg/ml at all the selected

wave length. Mixture of drugs showed Additivity of absorbance at selective wavelengths. Indicating that there is no interacting between the drug in methanol. Specific absorptivity values determines for both the drugs were 93.6+ 1.28 and 79.6 + 0.44 for FLU and 214.45 + 2.79 and

897.61 + 12.63 for MEL at 283.60nm and 229.40nm respectively (Table 1).

Figure 4: Isobestic point plot of mixture at 283.60nm

The percent estimation of mixed drug in laboratory mixture was done to be 99.99 + 1.078and 99.63 + 0.2038 for FLU and MEL respectively (Table 2). The percent drug estimation in marketed formulation was found to be 99.96 + 0.4340 and 99.90 + 0.1076 (Table 3). The method was validated according to ICH guidelines. The accuracy of method was validated by percent recovery both the drugs. The average percent recovery was found to be 99.98 + 0.4340 and 99.91 + 0.1076 (Table 4). The results of method lies with in the prescribe limit of 98-102% shows that method is free from interference from Excipients. The next parameters studied were precision of the experimental method. The Replicate estimation of both FLU and MEL in same batch of tablet was analyzed by the proposed method and showed quite concurrent results indicating reliability of the method. The values + SD of RSD and co-efficient and correlation. are with in the prescribed limit of 2% showing high precision of the method.

The other parameters studied were specificity, linearity and ruggedness. This studied was carried out to check degradation and stability of the drug (Fig 2, Table 5). In the specificity study the samples were exposed to different stress conditions like acid, alkali, oxide, heat and UV-visible lights. The FLU shows slight degradation of drug under alkali and acidic condition. But MEL shows degradation in alkali and UV-visible light (Table 6). Overall this method is in capable of finding exact degradation of drugs. The linearity and range study were also carried out over the range of 80-120% of labeled claim for both the drug in formulation the percent lable claim verses absorbance plots shows a linear relationship with correlation coefficients very closer to 1. Ruggedness studies were carried out under four different conditions i.e., different analyst interday variation, intraday variation, and different instrument shows that the results of estimation by proposed method are very much reproducible under variety of conditions (Table 7).

With regards to obtained results, proposed method revealed that the experimental study signifies simple, accurate, fast, precise and reproducible isobestric point method for simultaneous estimation of FLU and MEL in their combine dosage form and can be used for routine analysis of both the drug in commercially available marketed formulation.

ACKNOWLEDGMENTS:

The authors thank Biocon Laborateries Bangalore for the sample of Flupenthixol Hydrochloride and Melitracen Hydrochloride. The author also pays his indebtness for Principal J L Chaturvedi College of Pharmacy Nagpur for providing Laboratory Facilities.

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Imran A Sheikh*, Manoj Charde and Avinash V Kasture