Synchronous fluorescence spectrofluorimetric method for the simultaneous determination of metoprolol and felodipine in combined pharmaceutical preparation
© Walash et al 2011
Received: 30 July 2011
Accepted: 7 November 2011
Published: 7 November 2011
A rapid, simple and sensitive synchronous specrtofluorimetric method has been developed for the simultaneous analysis of binary mixture of metoprolol (MTP) and felodipine (FDP). The method is based upon measurement of the synchronous fluorescence intensity of the two drugs at Δλ of 70 nm in aqueous solution. The different experimental parameters affecting the synchronous fluorescence intensities of the two drugs were carefully studied and optimized. The fluorescence intensity-concentration plots were rectilinear over the ranges of 0.5-10 μg/mL and 0.2-2 μg/mL for MTP and FDP, respectively. The limits of detection were 0.11 and 0.02 μg/mL and quantification limits were 0.32 and 0.06 μg/mL for MTP and FDP, respectively. The proposed method was successfully applied for the determination of the two compounds in their commercial tablets and the results obtained were favorably compared to those obtained with a comparison method.
Both drugs are official in the United States Pharmacopoeia (USP, 2). The USP  recommended HPLC methods for the determination of each of FDP and MTP in pure form and in different dosage forms. Since the combined therapy of FDP and MTP has been clinically proven to be significantly efficient in the treatment of hypertension, it became important to develop and validate a reliable method for the separation and determination of the two drugs in their commercially available formulations. Only few methods have been reported in the literature for the assay of such mixture, using chemometric assisted spectrophotometric methods [3, 4] and HPLC methods [3–5].
Only one spectrofluorimetric method has been developed for the analysis of MTP in human plasma using trilinear decomposition-based techniques . Regarding FDP, a convential fluorimetric method was reported for its determination in presence of ramipril in their combined tablets Triacor® .
To the best of our knowledge, neither conventional nor synchronous spectrofluorimetry has been reported for the analysis of MTP and FDP in their binary mixtures.
The normal excitation fluorescence spectra of MTP and FDP are greatly overlapped. This observation led us to utilize synchronous fluorescence spectroscopy (SFS) to solve such problem by measuring Synchronous Fluorescence Intensities (SFI) at 260 and 375 nm for MTP and FDP respectively.
Based on chemical features of both drugs, and high therapeutic effect of MTP and FDP combined in treatment of hypertension, the (SFS) technique was developed for the simultaneous determination of both drugs in their combined tablets.
Synchronous fluorescence spectroscopy (SFS) has several advantages over conventional fluorescence spectroscopy, including simple spectra, high selectivity and low interference . Because of its sharp, narrow spectrum, SFS serves as a very simple, effective method for achieving data for quantitative determination in a single run .
Metoprolol tartrate pure sample was kindly provided by Sigma company.
Felodipine pure sample was kindly provided by Minapharm.
Logimax® tablets labeled to contain 50 mg of MTP and 5 mg of FDP (Batch # 90015) were obtained from commercial source in the local market.
All reagents and solvents used were of Analytical Reagent Grade.
Methanol (Merck, Darmstadt, Germany).
Acetic acid, Sodium acetate and Boric acid (BDH, UK).
Acetate buffer 0.2 M (pH 4.5) was prepared by mixing appropriate volume of 0.2 M acetic acid with 0.2 M sodium acetate. Borate buffers (pH 8.5) were prepared by mixing appropriate volumes of 0.02 M boric acid with 0.2 M sodium hydroxide.
Sodium hydroxide ((BDH, UK), 0.1 M aqueous solution was freshly prepared.
Fluorescence spectra and measurements were recorded using a Perkin-Elmer UK model LS 45 luminescence spectrometer, equipped with a 150 Watt Xenon arc lamp, gratting excitation and emission monochromators for all measurements and a Perkin-Elmer recorder. Slit widths for both monochromators were set at 10 nm. A 1 cm quartz cell was used. The SF spectra were estimated at 260 nm and 375 nm for MTP and FDP, respectively.
A Consort NV P901 digital pH Meter (Belgium) calibrated with standard buffers was used for checking the pH of the buffer solutions used.
Stock solutions of MTP and FDP were prepared by dissolving 10.0 mg of the studied compounds in 100 mL of methanol in a calibrated flask and were further diluted with water to obtain standard solutions containing 10 μg/mL of each drug. FDP standard solution was protected from light due to its photosensitivity. The standard solutions were stable for 10 days when kept in the refrigerator.
Performance data of the determination of MTP and FLD in pure form by the proposed method
Concentration range (μg/mL)
Limit of detection (LOD) (μg/mL)
Limit of Quantification (LOQ) (μg/mL)
Procedure for the synthetic mixture
Aliquots of MTP and FDP standard solutions in the ratio of 10:1 were transferred into a series of 10 mL volumetric flasks. Then the solution was diluted to the volume with distilled water, and mixed well. The recommended procedure under Calibration Curve was then performed. The relative SF intensities were measured and the corresponding concentrations were derived from the calibration curves or the corresponding regression equations.
Procedure for commercial tablets
The films of ten coated (individually weighed) tablets were gently removed with water. The tablets were then dried, weighed, powdered and mixed well. A weighed quantity of the powder equivalent to 10.0 mg MTP and 1 mg of FDP (in their ratio of 10:1) was transferred into a small conical flask and extracted with 50 mL of methanol by ultrasonication for 30 min. The extract was filtered with acrodisc GHP (Gelman Hydrophilic Polypropy lene membrane) into a 100 mL volumetric flask. The conical flask was washed with few mLs of methanol. The washings were passed into the same volumetric flask and completed to the mark with the same solvent.
Aliquots covering the working concentration range were transferred into 10 mL volumetric flasks. The recommended procedure under "Calibration Curve" was performed. The nominal content of the Tablets were determined either from a previously plotted calibration curve or using the corresponding regression equation.
Results and discussion
Synchronous fluorescence spectra of MTP and FDP
Optimization of experimental conditions
Different experimental parameters affecting the performance of the proposed method were carefully studied and optimized. Such factors were changed individually while others were kept constant. These factors included: Δλ, λ max, pH and type of the diluting solvent.
Selection of optimum Δλ
Selection of appropriate λ max
Metoprolol SF spectra using Δλ of 70 have two values of λ max, one at 233 and the other at 260 nm. The one at 260 nm didn't afford the higher sensitivity, yet it was used because it enabled the determination of FDP & MTP simultaneously at the ratio of 1:10.
Selection of optimum pH
The influence of pH on the fluorescence intensity of the two drugs was studied using different buffers covering the whole pH range, e.g. acetate buffer (pH 3.6-5.6) and borate buffer pH (6-9.5). It was found that, using any of these two buffers either does not affect the synchronous fluorescence intensity or even decrease it. Therefore, for simplicity of the method no buffer was used throughout the study.
Effect of diluting solvent
Validation of the Method
The validity of the method was tested regarding; linearity & range, accuracy, repeatability, precision and specificity according to ICH Q2B recommendations .
Linearity and Range
The regression plots showed a linear dependence of RSFI values on drug concentration over the range cited in Table 1.
The validity of the methods was proved by statistical evaluation of the regression lines, using the standard deviation of the residuals (Sy/x), the standard deviation of the intercept (Sa) and standard deviation of the slope (Sb). The results are abridged in Table 1. The small values of the figures point out to the low scattering of the points around the calibration curves and high precision.
Limit of quantification (LOQ) and limit of detection (LOD)
The limit of quantification (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B recommendations , below which the calibration graph is non linear. The limit of detection (LOD) was determined by evaluating the lowest concentration of the analyte that can be readily detected. The results of LOD and LOQ of MTP and FDP by SDSFS method are abridged in Table 1.
LOQ and LOD were calculated according to ICH Q2B recommendations :
LOQ = 10 σ/S
LOD = 3.3 σ/S
Where: S is the slope and σ is the standard deviation of the intercept of regression line of the calibration curve.
Accuracy and precision
Application of the synchronous fluorimetry to the determination of the studied drugs in the pure form
Concentration taken (μg/mL)
Concentration found (μg/mL)
Comparison method (4)
Application of the proposed method for determination of the studied drugs in their synthetic mixtures.
Concentration taken (μg/mL)
MTP and FDP mixture
The method was assessed regarding precision by evaluating repeatability and intermediate precision.
Repeatability: The repeatability was performed by applying the proposed methods for the determination of three concentrations of MTP and FDP in pure forms on three successive times, and the results are listed in Table 4.
Validation of the proposed method for determination of MTP and FDP raw materials using SSF mode
Concentration added (μg/ml)
99.96 ± 1.73
100.09 ± 1.75
100.02 ± 0.22
99.66 ± 1.64
100.09 ± 1.00
100.05 ± 0.68
100.10 ± 1.57
100.07 ± 1.52
100.23 ± 1.45
99.79 ± 1.31
100.36 ± 0.91
100.06 ± 1.62
Intermediate precision: Intermediate precision was evaluated through repeated analysis of MTP and FDP in pure form applying the proposed method, using the concentrations showed in Table 4 for a period of 3 successive days.
The proposed SFS method allowed the selective determination of each drug in presence of the other without any interference proving its selectivity and ability to resolve a mixture of the two drugs.
The proposed method was found to be specific for the two studied drugs in their combined tablets without interference from common tablet excipients such as Titanium oxide, Iron oxide, anhydrous lactose, propyl gallate, colloidal silicon dioxide, paraffin, hypromellose, cellulose, hydroxyl propyl cellulose sodium aluminum silicate, macrogel, sodium stearyl fumarate and poloxyl 40-hydrogenated caster oil. These matrix components did not interfere with the proposed method.
Application of the proposed method for determination of the studied drugs in their co-formulated preparation
Concentration taken (μg/mL)
Concentration found (μg/mL)
Comparison method (4)
Logimax ® Tablets a
(MTP 50 mg + FDP 5 mg/Tablet)
Batch # 604346
A new simple and sensitive method was explored for the simultaneous determination of MTP and FDP in binary mixture. The synchronous spectrofluorometric method, by virtue of its high sensitivity, could be applied to the analysis of both drugs in their co-formulated dosage forms. It was possible to measure low concentrations as 0.32 and 0.06 μg/mL for MTP and FDP respectively with good accuracy. Moreover, synchronous spectrofluorimetric technique enables the determination of MTP in the presence of FDP and vice versa. Moreover, the proposed method is time saving.
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