1. INTRODUCTION:

Many methods have been introduced for the analysis of binary mixtures among which the spectrophotometric based methods were the most simple, fast and applicable in almost all laboratories. Several manipulations were performed on the raw overlapping spectral data to enable mixture resolution for example, using different order derivatives [1-11].The aim of the present work was to develop a new simple, rapid, selective method for the simultaneous determination of components having overlapping spectra in binary mixtures, having the advantages of minimal data processing and a wider range of applications over the previously mentioned methods. To prove the ability of the newly described method in resolving the overlapping spectral data and simultaneous determination of each component, it was applied for the analysis of a mixture of propranolol hydrochloride (PRO) and hydrochlorothiazide (HCT) formulated together in the form of tablets widely used for the treatment of heart related problems accompanying several hypertension.

Propranolol hydrochloride is chemically (2 RS)-1-[(1- methyl ethyl) amino]-3-(naphthalene-l- yloxy)propan-2-ol hydrochloride[12]. Propranolol hydrochloride isa beta-adrenergic blocking agent that is used for treating high blood pressure, heart pain, abnormal rhythms of the heart, and some neurologic conditions, also used to Angina pectoris and coronary artery disease. Propranolol is useful in slowing and regulating Tachycardia[13]. Hydrochlorothiazide is chemically 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide[14]. Hydrochlorothiazide is Diuretic and Antihypertensive used to treat excessive fluid accumulation and swelling (edema) of the body caused by heart failure, cirrhosis, chronic kidney failure, corticosteroid medications, and nephrotic syndrome. Combination of propranolol hydrochloride and hydrochlorothiazide is used to treat hypertension and heart related dieses[15].

Fig.1 (A) is Structure of propranolol hydrochloride and (B) is structure of Hydrochlorothiazide.

1.1. Theory

We can find out concentration of both the drug from combination mixture using the simultaneous equation method. In this method using the absorbance of both the drug and mixture at their wavelength and put this value in following equation and we can find out the concentration of drugs present in combination.

Cx = (A₂ × Ay₁) – (A₁ × Ay₂)

--------------------------------- ------- (1)

(Ay₁ × Ax₂) – (Ay₂ × Ax₁)

Cy = (A₁ × Ax₂) – (A₂ × Ax₁)

--------------------------------- ------- (2)

(Ax₂× Ay₁) – (Ax₁ × Ay₂)

Where,

Cx = Concentration of drug X

Cy = Concentration of drug

A1 = Absorbanceof mixture at wavelength 1

A2 = Absorbance of mixture at wavelength 2

Ax1 = Absorptivity of drug A at wavelength 1

Ax2 = Absorptivity of drug A at wavelength 2

Ay1 = Absorptivity of drug B at wavelength 1

Ay2 =Absorptivity of drug B at wavelength 2

2. MATERIAL AND METHOD:

2.1. Apparatus

A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2nm, 1 cm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software.

2.2. Reference samples

PRO and HCT reference standard are kindly supply by Cipla LTD, Mumbai and CTX Life Science, Surat as a gift sample respectively.

2.3. Pharmaceutical formulation

Cipler-H tablet, labelled to contain 40 mg PRO and 20 mg HCT, manufactured by Cipla Ltd.

2.4. Materials and reagents

Methanol AR grade(RANKEM)

2.5. Standard solutions

2.5.1. Standard solution of Propranolol HCl (PRO)

Accurately weighed quantity of PRO10mg was transferred to 100ml volumetric flask, dissolved and diluted up to mark with Methanol to give a stock solution having strength 100µg/ml.

2.5.2. Standard solution of Hydrochlorothiazide (HCT)

Accurately weighed quantity of HCT 10mg was transferred into 100ml volumetric flask, dissolved and diluted up to mark with Methanol to give a stock solution having strength 100µg/ml.

2.5.3. Preparation of standard mixture

Pipette out accurately 1 ml of PRO stock solution (100µg/ml), 0.5 ml of HCT stock solution (100µg/ml) in 10 ml volumetric flask and make up the volume up to the mark with Methanol. It gives solution containing PRO 10µg/ml, HCT 0.5µg/ml.

2.5.4. Test Sample Preparation

Dissolve tablet sample in 100 ml volumetric flask containing 100 ml methanol. Take 1 ml tablet sample solution in 10 ml volumetric flask and make up volume up to mark with methanol.

2.6. Procedures

2.6.1. Construction of calibration curves (linearity)

This series consisted of five concentrations of standard PRO solution ranging from 10-50μg/ml. The solutions were prepared by pipetting out standard PRO stock solution (1ml, 2ml, 3ml, 4ml, 5ml) was transferred into a series of 10ml volumetric flasks and volume was adjusted up to mark with Methanol. A zero order spectra of the resulting solutions were recorded, measured the absorbance at 289.0nm against are agent blank solution(Methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of PRO.

This series consisted of five concentrations of standard HCT solution ranging from 5-25 μg/ml. The solutions were prepared by pipetting out Standard HCT stock solution (0.5ml, 12ml, 1.5ml, 2ml and 2.5ml) was transferred into a series of 10ml volumetric flasks and volume was adjusted up to mark with Methanol. A zero order spectra of the resulting solutions were recorded and measured the absorbance at 270 nm against a reagent blank solution (Methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of HCT.

2.6.2. Analysis of laboratory-prepared mixtures.

Laboratory-prepared mixtures containing different ratios of PRO and HCT were prepared. By applying the procedure under linearity, absorbance at 289.0 nm was recorded for PRO and 270.0 nm were recorded for HCT. The concentration of each drug in each mixture was calculated from its corresponding Cx and Cy equation. Validity of the method was assessed by spiking the pharmaceutical formulation by known amounts of standard drug powders (standard addition technique). The recovery of the added standards was then calculated after applying the proposed method.

2.6.3. Application of the proposed method for the simultaneous determination of PRO and HCT in Cipler – H tablet

Take one tablet and dissolved into a 100-ml beaker and sonicated in 100 ml methanol for 15 min, filtered into 100- ml volumetric flask. The residue was washed three times each using 10 ml methanol and completed to the mark with the same solvent. Transfer accurately 1 ml of the extracted solution into a 10-ml measuring flask. One millilitres of PRO working solution (20 µg/ml) equivalent to 40 µg PRO was added and completed to the mark with methanol. The general procedure under linearity was followed.

3. RESULTS AND DISCUSSION:

The absorbance wavelength for PRO and HCT found to be 289 and 270 nm, respectively, which is different and hence non-overlapping. Thus simultaneous determination of PRO and HCT in bulk mixture-I and tablet solution-I was found to be successful by simultaneous equation.

3.1. Specificity

The specificity of the method was investigated by observing any interference of one drug with other two drugs in bulk mixture and tablet solution. Similarly the interference of excipients of tablet with drugs was investigated.

3.2. Linearity and range

The linearity of method is its ability within a given range to obtain test results which are directly or through a mathematical transformation, proportional to the concentration of analyte. Linearity of the method was determined at five concentration levels for PRO and HCT independently.

3.3. Accuracy

The accuracy of an analytical method is the closeness of the test results to the true value. It was tested by spiking standard PRO solution in different concentration 80, 100 and 120% to a tablet solution. The tablet solution was analyzed at 289 nm for estimation of PRO. Similarly, the accuracy for HCT was determined at 270 nm, respectively.

3.4. Precision

The intra-day precision (repeatability) of method was determined by measuring the absorbance of tablet solution-I at 289 and 270 nm for PRO and HCT, respectively. Within a laboratory over a short period of time. The inter-day precision (intermediate precision) was determined by measuring the absorbance of tablet solution-I at 289 and 270 nm for PRO and HCT, respectively. Within a laboratory on three consecutive days, by different analysts. The %RSD was calculated for intra and inter-day precision.

3.5. LOD and LOQ

The LOD of an analytical method is the lowest amount of analyte in a sample which can be detected but not necessarily quantified. The detection limit (DL) of method was determined by equation, DL = (3.3 σ)/S, where, σ– standard deviation of blank response, S– slope of the calibration curve. The quantitation limit (QL) of analyte was determined by equation DL = (10 σ)/S, where, σ– standard deviation of blank response, S– slope of the calibration curve

3.6. Robustness and ruggedness

Robustness and ruggedness of the method has been evaluated at two different levels i.e. change in stock solution and changing the instrument.

4. Analysis of commercial tablets

The proposed method was successfully applied to the analysis of both mixtures in their pharmaceutical preparations. Results obtained were precise and in good agreement with the labelled claim as concluded from the satisfactory values of % recovery and RSD(%) gathered in table 2 and 3. Proposed method is precise and accurate, and give same result in same day and between the day and this data is gathered in table 1. When we change in standard stock concentration or changed the instrument that time also this proposed method give good result this data also gathered in table 4 and 5.

Table 1: Inter day and intraday precision data for propranolol and hydrochlorothiazide in three different concentration ranges.

Analyte	Nominal Value (µg/Ml)	Within Day		Between Day
Analyte	Nominal Value (µg/Ml)	Found ± SD(µg/Ml)	%RSD	Found ± SD(µg/Ml)	%RSD
PRO	10	9.82 ± 0.01	0.1133	10.24 ± 0.0662	0.6465
	20	19.32 ± 0.07	0.4041	20.29 ± 0.0724	0.3569
	30	29.58 ± 0.087	0.3156	30.54 ± 0.0566	0.1746
HCT	5	4.96 ± 0.04	0.8064	5.15 ± 0.04836	0.9106
	10	9.9690 ± 0.049	0.51	10.12 ± 0.006	0.0680
	15	15.09 ± 0.082	0.5463	15.42 ± 0.058	0.3558

Table 2: Accuracy data for propranolol hydrochloride with % recovery and % RSD

Concentration of PRO from formulation (µg/ml)	Amount of PRO spiked (µg/ml)	Total amount (µg/ml)	Amount found (µg/ml)	% recovery	S.D	% RSD
40	8	48	48.2234	100.464	0.001	0.000955
40	10	50	50.9428	98.15	0.00577	0.000567
40	12	52	51.1872	101.16	0.00095	0.009885

Table 3: Accuracy data for hydrochlorothiazide with % recovery and % RSD

Concentration of HCT from formulation (µg/ml)	Amount of HCT spiked (µg/ml)	Total amount (µg/ml)	Amount found (µg/ml)	% recovery	S.D	% RSD
20	4	24	25.223	100.892	0.005292	0.005245
20	5	25	24.5388	98.1552	0.00500	0.005095
20	6	26	26.3041	101.17	0.00095	0.009885

Table 4: Robustness and ruggedness data into that change in stock concentration in that concentration of stock – 1 is 100 µg/ml and concentration of stock – 2 is 500 µg/ml.

PRO (Stock 100 µg/ml )		HCT (Stock 100 µg/ml )		PRO (Stock 500 µg/ml )		HCT (Stock 500 µg/ml )
Abs ± S.D	% RSD	Abs ± S.D	% RSD	Abs ± S.D	% RSD	Abs ± S.D	% RSD
0.484 ± 0.0020	0.4295	0.875 ± 0.0017	0.1979	0.4953 ± 0.001525	0.3083	0.776 ± 0.00173	0.2234
0.988 ± 0.002	0.20240	1.804 ± 0.0015	0.0846	0.9066 ± 0.002082	0.2295	1.605 ± 0.00202	0.1296
1.285 ± 0.00202	0.1619	2.358 ± 0.0017	0.0734	1.414 ± 0.0015	0.1079	2.950 ± 0.02569	0.8709

Table 5: Robustness and ruggedness data into that change in instrument used, in that instrument 1 and 2 are used.

PRO (Instrument - 1 )		HCT (Instrument - 1 )		PRO (Instrument - 2 )		HCT (Instrument - 2 )
Abs ± S.D	% RSD	Abs ± S.D	% RSD	Abs ± S.D	% RSD	Abs ± S.D	% RSD
0.484 ± 0.0020	0.4295	0.875 ± 0.0017	0.1979	0.585 ± 0.0015	0.26096	0.864 ± 0.0020	0.2408
0.988 ± 0.002	0.20240	1.804 ± 0.0015	0.0846	0.9866 ± 0.00208	0.21098	1.61 ± 0.010	0.6211
1.285 ± 0.00202	0.1619	2.358 ± 0.0017	0.0734	1.6163 ± 0.001528	0.09450	2.415 ± 0.001	0.04140

Table 6: Result of all validation and development parameters for this proposed method for propranolol and hydrochlorothiazide.

Sr. No	Parameter	Propranolol	Hydrochlorothiazide
1	Range (µg/ml)	10-50	5-25
2	Correlation coefficient (r²)	0.9951	0.9971
3	Regression equation	y = 0.0152x + 0.2829	y = 0.0688x - 0.002
4	Inter day precision	0.1133-0.4041	0.51-8064
5	Intraday precision	0.17468-0.64653	0.068-0.91069
6	Accuracy (%recovery ± SD)	100.2624 ± 0.00486	100.0697 ± 0.006764
7	LOD (µg/ml)	0.090	0.050
8	LOQ (µg/ml)	0.0089	0.0036
9	Robustness and ruggedness	0.10798 - 0.30838	0.03824 – 0.5848

5. CONCLUSION:

A novel, simple, rapid and sensitive method is proposed for the analysis of two binary mixtures with overlapping spectra. The method involves the generation of absorbance spectra followed by measurement of the absorbance. The proposed method does not require any sophisticated mathematical treatment for the absorption data, and it exhibits several advantages over other spectrophotometric methods for resolution of binary mixtures. The applicability of the developed method was evaluated through the determination of both drug combinations in several laboratory-prepared mixtures and in pharmaceutical tablets with good accuracy and precision. Therefore, the presented methodology is adequate for the routine quality control analysis of these fixed-dose combinations.

6. CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

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Received on 25.02.2015 Accepted on 18.03.2015

Asian J. Pharm. Ana. 5(1): Jan.- March 2014; Page 36-40

DOI: 10.5958/2231-5675.2015.00007.1