A validated RP-HPLC method for simultaneous determination of propranolol and valsartan in bulk drug and gel formulation.

OBJECTIVE: A simple, precise, and stability indicating high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of propranolol hydrochloride and valsartan in pharmaceutical dosage form.
MATERIALS AND METHODS: The method involves the use of easily available inexpensive laboratory reagents. The separation was achieved on Hypersil ODS C-18 column (250*4.6 mm, i.d., 5 μm particle size) with isocratic flow with UV detector. The mobile phase at a flow rate of 1.0 mL/min consisted of acetonitrile, methanol, and 0.01 M disodium hydrogen phosphate (pH 3.5) in the ratio of 50:35:15 v/v.
RESULTS: A linear response was observed over the concentration range 5-50 μg/mL of propranolol and the concentration range 4-32 μg/mL of valsartan. Limit of detection and limit of quantitation for propranolol were 0.27 μg/mL and 0.85 μg/mL, and for valsartan were 0.45 μg/mL and 1.39 μg/mL, respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for linearity, accuracy, precision, specificity, robustness.
CONCLUSION: The analysis concluded that the method was selective for simultaneous estimation of propranolol and valsartan can be potentially used for the estimation of these drugs in combined dosage form.

Liquid chromatography is the most widely used analytical tool in the pharmaceutical industry and reversed-phase is the most frequently used mode. During the drug development process, liquid chromatographic methods are used to determine the quality of the drug substance (active pharmaceutical ingredient) and drug product. Valsartan (VALS) [Figure 1] is a tetrazol–byphenil–valinic derivative of losartan, structurally characterized by the presence of a sole heterocyclic structure.[1] VALS has shown to be effective in decreasing blood pressure values (in monotherapy or in combination with other anti-hypertensive drugs) and treating heart failure.[23] VALS is rapidly absorbed following oral administration, with a rather poor bioavailability of about 25%. Peak plasma concentrations of VALS occur 2 to 4 hours after ingestion. Methods such as high performance liquid chromatography (HPLC),[4-6] and simultaneous UV spectrophotometric methods[78] are reported for estimation of VALS alone or in combination with other agents. However, no method is available for simultaneous determination of VALS with beta blocker.

Figure 1

Chemical structure of valsartan

Propranolol hydrochloride (PROP), [Figure 2] chemically 1-[(1-methylethyl) amino]-3-(1-naphthylenyloxy)-2-propranolol, is a non-selective β-adrenergic antagonist with no intrinsic sympathomimetic activity.[910] It is used in the management of hypertension, phaeochromocytoma, thyrotoxicosis, angina pectoris, myocardial infarction, and cardiac arrhythmias. Several methods are presently available to measure PROP like LC-MS,[11] and spectrometric methods.[12] Indian Pharmacopoeia describes a spectrometric method,[13] whereas USP describes a HPLC method[14] for assaying the drug. HPLC methods for determination of beta-blockers such as PROP, either alone or in combination with other drugs for both in-vivo studies[1516] and in-vitro studies[17] are abundant in literature. According to the information extracted from literature, to date, there is not even a single method reported for the simultaneous determination of PROP and VALS in pharmaceutical formulations. The objective of this work was to develop a simple, precise, and rapid column liquid chromatography (LC) procedure that would serve as an assay method for combination drug product of VALS and PROP.

Figure 2

Chemical structure of propranolol

Experimental

Chemicals and reagents

Propranolol hydrochloride was received as gratis sample from Dr. Reddy's Lab, India, and Valsartan was received as gift sample from Ranbaxy Research Laboratories Ltd, Gurgaon, India. Acetonitrile (HPLC grade), methanol (HPLC grade) disodium hydrogen phosphate, and acetic acid glacial (analytical reagent grade) were purchased from E-Merck Ltd. (Mumbai, India). Ultra-purified HPLC grade water was obtained from Milli-Q® system (Millipore, Milford, MA, USA) water purification unit. Mobile phase was filtered using 0.45 μ nylon filters made by Millipore (USA) and was sonicated using sonicator.

HPLC instrumentation and chromatographic conditions

HPLC system (Agilent Technologies, USA) equipped with a LC-LC-20AT pump, an SPD-20A variable wavelength detector (set at 250 nm), a CBM-20A interface module with Agilent LC Ezchrome software and a Rheodyne injection valve with a 20 μL loop was used for development and evaluation of this method. A Hypersil ODS C-18 column (250*4.6 mm, i.d., 5 μm particle size) was used. The mobile phase was composed of acetonitrile, methanol, and 0.01 M disodium hydrogen phosphate (pH 3.5) in the ratio of 50:35:15 v/v. Flow rate of the mobile phase was selected as 1 ml/min. Peak identity was confirmed by spectrum, and retention time comparison and the HPLC system was operated at room temperature (25 ± 2°C). The mobile phase was prepared daily and degassed by ultrasonication before use.

Preparation of standard solution

The standard solution was prepared by taking 50 mg of PROP and 20 mg of VALS and transferred into a 100 ml volumetric flask and diluted with mobile phase. The solution was sonicated for 15 minutes to completely dissolve both the components. It was then cooled, and the volume was made up to the mark with mobile phase. The above solution was further diluted with mobile phase to obtain a final concentration of 10 μg/ml for PROP and 16 μg/ml for VALS. The solution was filtered through 0.45 μ nylon filters before analysis.

Calibration curve

Calibration curves were prepared by taking appropriate aliquots of standard PROP and VALS stock solutions in different 10 ml volumetric flask and diluted up to the mark with mobile phase to obtain final concentrations of 4, 8, 12, 16, 20, 24, 28, 32 μg/ml of VALS and 5, 10, 20, 30, 40, 50 μg/ml of PROP. Standard solutions were injected through 20 μl loop system, and chromatograms were obtained using 1.0 ml/min mobile phase flow rate. The effluent was monitored at 250 nm. Calibration curve was constructed by plotting average peak area against concentration, and regression equation was computed.

Preparation of sample solution (gel)

Accurately-weighed gel equivalent to 10 mg of PROP and 40 mg of VALS in 50 ml transferred to volumetric flask, methanol was added and sonicated for 15 minutes to extract all the drugs from the gel formulation. The solution was cooled and then filtered by using 0.45 μ nylon filters. The filtrate was transferred to a 25 ml volumetric flask and diluted to the mark with methanol. The final concentration was achieved 16μg/ml for VALS and 10 μg/ml for PROP. The mean values of peak areas of 5 such determinations were calculated, and the drug content in the gel was quantified using the calibration curve.

Method validation

The proposed method was validated[18] by parameters viz. linearity range, precision, accuracy, specificity, robustness, selectivity, limit of quantitation (LOQ), and limit of detection (LOD).

Linearity

Stock solutions of PROP and VALS were prepared as described earlier. Serial dilution of the stock solution by removal of suitable aliquots were undertaken to yield calibration curves over the concentration range of 4-32 μg/ml for VALS and 5-50 μg/ml for PROP, respectively. 5 replicate analyzes of each of the concentrations were used to establish the calibration curve.

Precision

The precision study was assessed by evaluating the chromatographic responses of repeated injections (n = 5) of known concentrations of both PROP and VALS over the concentration ranges studied. The intra-day precision refers to the use of analytical procedure within a laboratory over a short period of time using the same operator with the same equipment. The inter-day precision involves estimation of variations in analysis when a method is used within a laboratory on different days, by same analysts.

Accuracy

The accuracy study was determined by standard addition methodby injection of solution (n = 3) of known concentrations of both drugs that had been prepared from new stock solutions.

Specifity

To check the specifity of the proposed method, a synthetic mixture of VALS and PROP was prepared with commonly occurring ingredients that are present in most gel formulations. The comparison of its chromatograms with the chromatograms of the standard solution was done along with the percentage recovery of both the analytes.

Limit of quantitation and limit of detection

The parameters LOD and LOQ were determined on the basis of signal to noise ratio, LOD and LOQ was calculated by the method, which was based on the standard deviation (S.D.) of the response and the slope (S) of the calibration curve at levels approximating the LOD and LOQ. LOD and LOQ were determined as follows.

LOD = 3.3 × Standard deviation of y intercept/Slope of calibration curve

LOQ = 10 × Standard deviation of y intercept/Slope of calibration curve

Robustness

As defined by the ICH, the robustness of an analytical procedure refers to its capability to remain unaffected by small and deliberate variations in method parameters.[19] The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The conditions studied were flow rate (altered by ± 0.2 ml/min), mobile phase composition (buffer ± 5%), buffer pH (altered by ± 0.2), and use of LC columns from different batches.

Results and Discussion

In this work, a simple, sensitive, and validated HPLC method has been developed for simultaneous estimation of PROP and VALS using liquid chromatography with ultraviolet detection. A number of mobile phases were initially attempted to elute both components simultaneously and to achieve sharp and gaussian-shaped peaks. The best mobile phase composition was then found to be acetonitrile, methanol, and 0.01 M disodium hydrogen phosphate pH (3.5), in the ratio of 50:35:15 (v/v). Under the mentioned chromatographic conditions, sharp peaks belonging to PROP and VALS were obtained at retention times of 6.62 and 9.76 minutes, respectively. The proposed chromatographic method was validated using ICH guidelines by parameters viz performing linearity, selectivity, robustness, accuracy, repeatability, limits of detection, and quantitation.

Linearity

The linearity of an analytical method is its ability of an analytical method to show a directly-proportional relationship of a quantitative response to a specific concentration of an analyte within a given specified range of concentrations. The range of a method may be defined as the interval between the upper and lower limits of quantitation to which the method produces test results that are proportional to the analyte concentration or to which a linear calibration model may be applied within a known confidence interval. The results of the validation procedure for linearity reveal that the above assay was linear over the concentration range studied and yielded regression coefficients of R2 = 0.9988 for PROP and R2 = 0.9966 for VALS. The relevant equations for these are Y = 18559.3x + 131.7 and Y = 15039x + 633.8 for PROP and VALS, respectively, shown in Table 1. The test for linearity of the proposed analytical method yielded R2 values that were greater than 0.990 for both HPLC systems used during validation. Therefore, the linearity of the method is suitable for the quantitation of PROP and VALS in pharmaceutical dosage forms.

Table 1

Analytical parameters of propranolol, and valsartan

Accuracy (Recovery studies)

Accuracy can be expressed in terms of the % bias, which represents the percentage error difference between a measured value and a reference value. The excellent recoveries of standard addition method [Table 2] suggested the good accuracy of the proposed methods. It is clearly evident that the method can be considered accurate as the % RSD was <2% for all determinations.

Table 2

Accuracy of the proposed HPLC method

Precision

Intra- and inter- day constructions of calibration curves showed the intermediate precision of the method. It is expressed as % R.S.D. for a statistically-significant number of samples. The % R.S.D. values in the regression lines prepared on the same day, and different days were within acceptable the limits [Table 3].

Table 3

Statistical evaluation of precision of developed method

Specificity

The specificity of the HPLC method developed for the analysis of PROP and VALS can be demonstrated from the chromatograms as shown in [Figures 3 and 4]. Specificity and selectivity were studied for the examination of the presence of interfering endogenous components, working solution containing PROP, and VALS were prepared with methanol and mobile phase. The results indicate that there is not much variation in the retention time of standard and test formulation. None of the impurities were interfering in the assay. As is evident in figure with the peaks of interest, viz., PROP, VALS are free from interference from formulation excipients, the solvent front and from each other. This indicates the method is selected and specific for determination VALS and PROP simultaneously.

Figure 3

Chromatograms of propranolol and valsartan reference samples

Figure 4

Chromatograms of propranolol and valsartan test samples

Limit of detection and limit of quantification

The LOD and LOQ of VALS were found to be 0.45 and 1.39 μg/ml, respectively, while those of PROP were found 0.27 and 0.85 μg/ml, respectively. RSD (%) of 6 replicate injections of VALS at LOD (0.45 μg/ml) and LOQ (1.39 μg/ml) were 11.28 and 3.71, respectively. Similarly, % RSD of 6 replicate injections of PROP at LOD (0.27 μg/ml) and LOQ (0.85 μg/ml) were 14.33 and 3.53, respectively. These values [Table 1] indicated that the method was very sensitive to quantify both the drugs.

Robustness of method

In the developed RP-HPLC method, small deliberate variations in the optimized method parameters were done. The effects of change in flow rate, mobile phase ratio, and buffer pH on the retention time and % recovery were studied. The results showed that the slight variations on the chromatographic conditions have negligible effect on the chromatographic parameters, showing the method is highly robust for its intended use. The results are given in Table 4.

Table 4

Robustness study of developed method

Assay of gel

The application of the method was assessed by quantifying the propranolol and valsartan in laboratory developed gel formulation. The results are given in Table 5, which show high percentage recoveries and low percent RSD values.

Table 5

Results of analysis propranolol and valsartan in gel formulation

Conclusion

The proposed method is simple, sensitive, and reproducible and hence can be used for simultaneous determination of propranolol and valsartan in bulk as well as in pharmaceutical preparations. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The RSD for all parameters was found to be less than 2, which indicates the validity of method and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative simultaneous estimation of propranolol and valsartan in pharmaceutical preparations.