A simple, sensitive, and precise high-performance thin layer chromatographic method has been developed for the estimation of propranolol hydrochloride and Flunarizine dihydrochloride in combined dosage form. The method employed HPTLC aluminum plates precoated with silica gel 60F as the stationary phase while the solvent system was toluene:methanol: ethyl acetate: acetic acid (7 : 1.5 : 1.5 : 0.1 v/v/v/v). The Rf value was observed to be
Propranolol hydrochloride (PRO) is chemically, (RS)-2-(4-(2-methylpropyl) phenyl) 2-Propanol, 1-[(1-methylethyl) amino]-3-(1-naphthalenyloxy), hydrochloride. The empirical formula of PRO is C16H21NO2
Structure of Propranolol hydrochloride.
Structure of flunarizine dihydrochloride.
Propranolol hydrochloride is official in Indian Pharmacopoeia and British Pharmacopoeia. A literature survey regarding quantitative analysis of these drugs propranolol hydrochloride and flunarizine dihydrochloride revealed that attempts have been made to develop analytical methods for the estimation of alone and in combination with other drugs by liquid chromatographic (LC) [
There is no method reported for the estimation of PRO and FLU in combined dosage form. Present study involves development and validation of HPTLC method for the estimation of PRO and FLU in combined dosage form.
The samples were applied in the form of a bands of width 6 mm with a Camag 10
Analytically pure PRO and FLU were obtained as gift samples from CADILA Pharmaceutical, Ahmedabad. Analytical grade methanol, ethyl acetate, toluene, and oxalic acid were obtained from E. Merck Ltd., Mumbai, India. Tablet formulation (BETACAP, Sun pharma, baroda, India) containing labeled amount of 50 mg of Propranolol hydrochloride and 4 mg of Flunarizine dihydrocholride was used for the study.
Plates were developed using a mobile phase consisting of toluene:methanol: ethyl acetate: acetic acid (7 : 1.5 : 1.5 : 0.1 v/v/v/v). Linear ascending development was carried out in a twin-trough glass chamber equilibrated with the mobile phase vapors for 30 min at
Standards and formulation samples of PRO and FLU were applied on the HPTLC plates in the form of narrow bands of 6 mm length, 10 mm from the bottom and left edge, and with 9 mm distance between two bands. Samples were applied under a continuous drying stream of nitrogen gas.
Plates were developed using a mobile phase consisting of toluene:methanol: ethyl acetate: acetic acid (7 : 1.5 : 1.5 : 0.1 v/v/v/v). Linear ascending development was carried out in a twin-trough glass chamber equilibrated with the mobile phase vapors for 30 min at
Densitometric scanning was performed in the absorbance mode under control by winCATS planar chromatography software. The source of radiation was the deuterium lamp, and bands were scanned at 240 nm. The slit dimensions were 5 mm length and 0.45 mm width, with a scanning rate of 20 mm/s. Concentrations of the compound chromatographed were determined from the intensity of diffusely reflected light and evaluated as peak areas against concentrations using a linear regression equation.
PRO and FLU were weighed (25 mg each), and transferred to two separate 25 mL volumetric flasks, and dissolved in few mL of mobile phase. Volumes were made up to the mark with mobile phase to yield a solution containing 1000
Validation of the developed HPTLC method was carried out according to International Conference on Harmonisation (ICH) guidelines Q2 (R1) for specificity, sensitivity, accuracy, precision, repeatability, and robustness [
Linearity of the method was evaluated by constructing calibration curves at six concentration levels over a range of 400–2400 ng/band and 50–300 ng/band of IBU and FAM, respectively. The calibration curves were developed by plotting peak area versus concentration (
The accuracy of the method was determined by calculating recoveries of PRO and FLU by method of standard additions. Known amount of PRO (0, 200, 400, and 600 ng/band) and FLU (0, 25, 50, and 75 ng/band) were added to a prequantified sample, and the amounts of PRO and FLU were estimated by measuring the peak area and by fitting these values to the straight-line equation of calibration curve.
Precision was evaluated in terms of intraday and interday precisions. Intraday precision was determined by analyzing sample solutions of PRO(400, 1600, and 2400 ng/band) and FLU (50, 200, and 300 ng/band) at three levels covering low, medium, and high concentrations of the calibration curve three times on the same day (
Repeatability of measurement of peak area was determined by analyzing PRO and FLU samples (1600 and 200 ng/band) seven times without changing the position of plate.
The specificity of the method was ascertained by analyzing PRO and FLU in presence of excipients commonly used for tablet formulations. The bands of PRO and FLU were confirmed by comparing Rf values and respective spectra of sample with those of standards. The peak purity of PRO and FLU was assured by comparing the spectra at three different levels, that is, peak start, peak apex, and peak end positions.
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can reliably be differentiated from background levels. Limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy. LOD and LOQ were calculated using following equation as per ICH guidelines:
LOD = 3.3 ×
Small changes in the chamber saturation time and solvent migration distance were introduced, and the effects on the results were examined. Robustness of the method was determined in triplicate at a concentration level of 1600 ng/band and 200 ng/band of PRO and FLU, respectively. The mean and RSD of peak areas were calculated.
Stability of sample solutions were studied at
Twenty tablets were weighed accurately and finely powdered. Tablet powder equivalent to 50 mg PRO and 4 mg of FLU was taken in 100 mL volumetric flask. Methanol (50 mL) was added to this flask, and the flask was sonicated for 15 minutes. The solution was filtered using Whatman filter paper No.1, and volume was made up to the mark with the mobile phase.
Appropriate volume of the aliquot was transferred to a 10 mL volumetric flask, and the volume was made up to the mark with the mobile phase to obtain a solution containing 400 ng/band of PRO and 50 ng/band of FLU were applied to HPTLC plates and analyzed for PRO and FLU content using the proposed method as described earlier. The possibility of interference from other components of the tablet formulation in the analysis was studied. From the developed chromatogram spot area and Rf values were determined.
To develop the HPTLC method of analysis of PRO and FLU for routine analysis, selection of the mobile phase was carried out on the basis of polarity. A mobile phase that would give a dense and compact band with an appropriate Rf value for PRO and FLU was desired. Various mobile phases such as methanol, hexane, methanol-ethyl acetate, hexane-ethyl acetate, methanol- toluene, methanol-n-butanol and methanol-ethyl acetate- toluene were evaluated in different proportions. A mobile phase consisting of toluene: methanol: ethyl acetate: acetic acid (7 : 1.5 : 1.5 : 0.1, v/v/v/v) gave good separation of PRO and FLU from its matrix. It was also observed that chamber saturation time and solvent migration distance were crucial in the chromatographic separation, as chamber saturation time of less than 30 min and solvent migration distances greater than 80 mm resulted in diffusion of the analyte band. Therefore, toluene: methanol : ethyl acetate: acetic acid (7 : 1.5 : 1.5 : 0.5 v/v/v/v) mobile phase with a chamber saturation time of 30 min at 25°C and solvent migration distance of 80 mm was used. These chromatographic conditions produced a well-defined compact band of PRO and FLU with optimum migration at Rf
Densitogram of PRO, and FLU using mobile phase toluene:methanol: ethyl acetate: oxalic acid (7 : 1.5 : 1.5 : 0.1, v/v/v/v)
Chromatogram of PROand FLU (400 and 50 ng/band) using mobile phase toluene:methanol: ethyl acetate: oxalic acid (7 : 1.5 : 1.5 : 0.1, v/v/v/v).
Linearity of an analytical method is its ability, within a given range, to obtain test results that are directly, or through a mathematical transformation, proportional to the concentration of the analyte. The method was found to be linear in a concentration range of 400–2400 ng/band and 50–300 ng/band of PRO and FLU, respectively, (
Regression analysis of calibration curve.
Parameter | PRO | FLU |
---|---|---|
Linearity ( |
400–2400 | 50–300 |
Correlation coefficient ( |
0.974 | 0.989 |
Slope of regression | 0.94 | 4.013 |
Standard deviation of slope | 0.060 | 0.2372 |
Intercept of regression | 5356.4 | 1373.4 |
Standard deviation of intercept | 79.128 | 55.456 |
Three dimensional overlay of HPTLC densitograms of calibration bands of PRO and FLU.
Accuracy of an analytical method is the closeness of test results to the true value. It was determined by the application of analytical procedure to recovery studies, where a known amount of standard is spiked into preanalyzed samples solutions. Results of the accuracy studies from excipient matrix are shown in Table
Summary of validation parameters.
Parameters | PRO | FLU |
---|---|---|
Rf |
|
|
Detection limit ( |
118.4 | 13.75 |
Quantitation limit ( |
355.2 | 45.4 |
Accuracy (%) | 99.71–100.92 % | 98.8–101.27 |
Precision (RSDa,%) | ||
Intraday precision ( |
1.06–1.33 | 1.28–1.48 |
Interday precision ( |
0.237–0.65 | 0.66–0.88 |
Instrument precision (RSDa) | 0.09 | 0.24 |
aRSD is relative standard deviation and “
The precision of an analytical method expresses the degree of scatter among a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. Intraday precision refers to the use of an analytical procedure within a laboratory over a short period of time by the same operator with the same equipment, whereas interday precision involves estimation of variations in analysis when a method is used within a laboratory on different days. The results obtained are shown in Table
Under the experimental conditions used, the lowest amount of drug that could be detected (LOD) for PRO and FLU was found to be 118.4 and 13.75 ng/band, respectively. The limit of quantification (LOQ) for PRO and FLU was found to be 355.2 and 45.4 ng/band, respectively, with an
The low values of RSD (Table
Robustness studies.
Method parameter/condition | Deliberate changes |
% RSD of peak area ( |
|
---|---|---|---|
PRO | FLU | ||
Chamber saturation time(a) | 20 min | 0.93 | 0.67 |
40 min | 1.13 | 0.98 | |
Development distance from spot application(b) | 75 mm | 0.39 | 0.86 |
85 mm | 0.98 | 1.16 |
(a)±20% change in set time.
(b)±10% change in set distance.
Specificity is the ability of an analytical method to determine the analyte unequivocally in the presence of sample matrix. Specificity of the method for PRO and FLU was proved from the spectral scan (Figure
Peak purity correlation results of PRO and FLU in given formulations.
Sample | Peak purity |
---|---|
PRO | 0.998 |
PRO formulation | 0.995 |
FLU | 0.997 |
FLU formulation | 0.994 |
Spectra comparison of PRO and FLU.
Marketed formulation was analyzed using proposed method which gave percentage recovery for PRO and FLU were
Analysis of marketed formulation.
Formulations | Labelled amount (mg) | % Recoveryb | ||
---|---|---|---|---|
PRO | FLU | PRO | FLU | |
A | 40 | 5 |
|
|
bMean value
A simple accurate and precise HPTLC method has been developed for the identification and quantification of RPO and FLU. The method was successfully validated in accordance with ICH guidelines. It can be conveniently used for routine QC analysis of PRO and FLU as a bulk drug and in marketed tablets without any interference from excipients.
The authors are thankful to CADILA pharmaceutical, Ahmedabad, India for providing gift sample of PRO and FLU, respectively. They are very thankful to Principal, Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar for providing necessary facilities to carry out research work.