Research article Received: 29 April 2014,
Revised: 5 June 2014,
Accepted: 7 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2735
Derivative emission spectrofluorimetry for the simultaneous determination of guaifenesin and phenylephrine hydrochloride in pharmaceutical tablets Hadir M. Maher,a,b* Mona M. Alshehria and Shorog M. Al-taweela ABSTRACT: Rapid, simple and sensitive derivative emission spectrofluorimetric methods have been developed for the simultaneous analysis of binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE). The methods are based upon measurement of the native fluorescence intensity of the two drugs at λex = 275 nm in methanolic solutions, followed by differentiation using first (D1) and second (D2) derivative techniques. The derivative fluorescence intensity–concentration plots were rectilinear over a range of 0.1–2 μg/mL for both GUA and PHE. The limits of detection were 0.027 (D1, GUA), 0.025 (D2, GUA), 0.031 (D1, PHE) and 0.033 (D2, PHE) μg/mL and limits of quantitation were 0.089 (D1, GUA), 0.083 (D2, GUA), 0.095 (D1, PHE) and 0.097 (D2, PHE) μg/mL. The proposed derivative emission spectrofluorimetric methods (D1 and D2) were successfully applied for the determination of the two compounds in binary mixtures and tablets with high precision and accuracy. The proposed methods were fully validated as per ICH guidelines. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: spectrofluorimetry; derivative emission; guaifenesin; phenylephrine; tablets
Introduction Spectroscopic techniques are among the earliest methods used in pharmaceutical analysis; one such technique is fluorescence spectroscopy (1). Although fluorimetry was one of the earliest instrumental techniques available to analytical chemists, recent developments in instrumentation and sample handling have made it possible for the full potential of this method, particularly its very high sensitivity and selectivity, to be realized in routine analysis (1). Moreover, fluorescence spectroscopy is an adaptable analytical technique, which is more sensitive than other detection systems such as classical UV absorption, less expensive than LC-MS/MS detection, and time saving compared with HPLC (2). The technique has not, however, been widely applied to the simultaneous determination of several fluorescent components in mixtures, mainly because the fluorescence spectra of individual substances contain broad bands that may easily overlap. In general, these compounds are determined using a prior separation step, which is time consuming for routine analysis and in some cases requires special expensive instrumentation. For this reason, the development of techniques that allow the direct determination of related compounds through the careful selection of instrumental variables is of great interest. Among these techniques, synchronous and derivative fluorescence spectrometry is the most popular (3–7). The application of derivative techniques to luminescence spectrometry has great advantages (3–7). Differentiation narrows spectral bandwidths and enhances minor spectral features, thus improving the selectivity of multi-component spectra. In this study, first- and second-derivative emission spectrofluorimetry were used to resolve binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE), which show overlapping emission spectra.
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GUA (R,S)3-(2-methoxyphenoxy)-1,2-propanediol (Fig. 1) is reported to increase the volume and reduce the viscosity of tenacious sputum and is used as an expectorant for productive coughs (8). Different methods have been reported for the analysis of GUA either alone or in combination, including HPLC (9,10), GC (11,12), CE (13), X-ray diffraction (14), voltammetry (15) and spectrophotometry (16,17). PHE, a hydrochloride salt of (R)-2-methylamino-1(3-hydroxyphenyl) ethanol (Fig. 1), is a sympathomimetic agent with direct effects on α-adrenoreceptors. It is used mainly as a nasal decongestant either alone or in combination with other agents for the symptomatic relief of cold symptoms (8). PHE has been previously determined in several pharmaceutical forms using HPLC (9,18–20), CE (21), spectrophotometry (20,22), spectrofluorimetry (23,24) and electrochemical analysis (25). Binary combinations of GUA and PHE are available as cough and cold preparations. Products containing GUA and PHE are used to treat symptoms (coughing, runny/stuffy nose, congestion) of the common cold, allergies, asthma, bronchitis, sinusitis and other illnesses that affect breathing (8). A survey of the literature revealed that only one HPLC method is reported for the simultaneous determination of GUA and PHE in binary bulk powders and
* Correspondence to: H. M. Maher, College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, Riyadh 11495, P.O. Box 22452, Saudi Arabia. E-mail: [email protected] a
College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, P.O. Box 22452, Riyadh 11495, P.O. Box 22452, Saudi Arabia
b
Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria 21521, Egypt
Copyright © 2014 John Wiley & Sons, Ltd.
H. M. Maher et al. Materials and reagents
Figure 1. Structures of GUA and PHE.
pharmaceutical dosage forms (9). To our knowledge, no method has been reported for the spectrofluorimetric determination of such a drug combination. Furthermore, no study has been found that uses the intrinsic fluorescence properties of GUA as a tool in its quantitative analysis. The aim of this work was to develop a simple and validated spectrofluorimetric method for the simultaneous determination of GUA and PHE in their tablet dosage form. The method made use of the intrinsic fluorescence properties of both drugs under experimentally optimized conditions. This work was based on the application of a derivative technique (D method) to resolve the overlapping emission spectra of both GUA and PHE in order to obtain highly specific derivative analytical signals. This enabled the determination of each drug at a zero contribution point from the other.
Experimental Instrumentation Fluorescence measurements were performed on a Spectramax (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA). Data acquisition and data analysis were performed with SoftMax Pro v. 5.4® software (Molecular Devices). The following instrumental parameters were selected throughout the measurements: sensitivity, medium; scan speed, normal; and emission step, 1. A microprocessor laboratory pH meter (Mettler-Toledo International Inc., Zürich, Switzerland), calibrated daily using standard buffers at room temperature was used.
GUA and PHE standards were from Sigma Aldrich (St. Louis, MO, USA). All reagents were of analytical grade, namely, methanol (Panreac Co., Barcelona, Spain), disodium hydrogen phosphate, phosphoric acid and sodium hydroxide. The water used was double glass distilled. Because of the unavailability of a marketed tablet formulation for GUA and PHE, simulated powdered tablets were prepared in the laboratory by mixing both drugs in the same amounts as in actual tablets, namely, Norel® EX labeled to contain 400 mg GUA and 40 mg PHE, along with other excipients normally encountered in tablet formulations (9).
Standard solutions and calibration graphs Stock solutions of 100.0 μg/mL GUA and PHE were prepared in methanol. A step dilution was conducted with the same solvent to prepare intermediate stock solutions of 10.0 μg/mL for both drugs. These solutions were found to be stable for at least one week when kept in the refrigerator. Accurately measured volumes of the intermediate stock solutions of each drug were further transferred into a series of 10 mL volumetric flasks and completed to volume with methanol to obtain working standard solutions at suitable concentrations (corresponding to the linearity range in Table 1). A reagent blank consisting of methanol was used throughout the study. For each standard solution of GUA and PHE, the emission spectra were recorded in the range 290–360 nm with excitation at 275 nm and were corrected for the blank signal. First and second derivatives of the emission data were calculated. For firstderivative emission fluorimetry (D1 method), the absolute values of the first derivative were measured at 307 and 317 nm for the determination of GUA and PHE, respectively. For the second derivative (D2 method), values were measured at 317 and 302 nm for the determination of GUA and PHE, respectively. Derivative values, calculated at the selected points, were plotted against the final concentration of each drug for both D1 and D2 methods, to obtain the calibration graphs. Alternatively, the corresponding regression equations were derived.
Table 1. Regression and statistical parameters for the determination of GUA and PHE by the proposed spectrofluorimetric method Parameter
Linearity range (μg/mL) LOD (μg/mL) LOQ (μg/mL) Intercept (a) Slope (b) Correlation coefficient (r) Sa Sb Sy/x F Significance F
First derivative of emission spectra (D1)
Second derivative of emission spectra (D2)
GUA
PHE
GUA
PHE
0.1–2 0.027 0.089 –0.120 16.863 0.9998 0.126 0.218 0.149 5968.48 1.680E-04
0.1–2 0.031 0.095 0.254 –15.997 0.9997 0.167 0.288 0.197 3081.51 3.240E-04
0.1–2 0.025 0.083 0.017 –2.408 0.9998 0.018 0.031 0.021 5950.42 1.680E-04
0.1–2 0.033 0.097 0.073 –4.636 0.9997 0.048 0.083 0.057 3084.77 3.240E-04
LOD, limit of detection; LOQ, limit of quantitation; Sy/x, standard deviation of residuals; Sa, standard deviation of intercept; Sb, standard deviation of slope; F, variance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals).
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Derivative fluorimetric analysis of guaifenesin and phenylephrine Preparation and analysis of laboratory-made mixtures Accurate volumes of GUA and PHE stock solutions were transferred into 10 mL volumetric flasks and diluted to volume with methanol to prepare mixtures within the concentration range of each compound (Table 2). The recommended procedure under the calibration graph was then performed. Derivative values were measured and the corresponding concentrations were derived from the calibration curves or the corresponding regression equations.
Procedure for tablets An accurately weighed quantity of powdered tablets equivalent to 10 mg of GUA and 1 mg PHE was transferred into a 100 mL volumetric flask. Sixty milliliters of methanol were added and the flask was sonicated in an ultrasonic bath for 30 min, diluted to volume with the same solvent, then filtered. Portions of 0.5 mL of the previously prepared solution were diluted with methanol in 50 mL volumetric flasks to prepare tablet solutions containing 1.0 μg/mL GUA and 0.1 μg/mL PHE. The emission spectra of the prepared tablet solutions were recorded in the range 290–360 nm with excitation at 275 nm and corrected for the blank signal. First- and second-derivative values of emission spectra were calculated and signals were measured at the selected wavelengths, as shown under construction of calibration graphs. The nominal contents were calculated using the corresponding regression equation for each of GUA and PHE, using both (D1) and (D2) methods.
Figure 2. Excitation (a, c) and emission (b, d) spectra of GUA (0.5 μg/mL) and PHE (0.5 μg/mL). All spectra were acquired in methanol.
(Fig. 2). For simultaneous determination of GUA and PHE, a single excitation wavelength of 275 nm was selected where a suitable response for both drugs was obtained. Factors affecting fluorescence intensity
Results and discussion
Different experimental parameters affecting the performance of the proposed methods were carefully studied and optimized to obtain the best measurement conditions and maximum fluorescence signals of GUA and PHE. Such factors were changed individually, whereas others were kept constant. These factors included the effect of pH and type of diluting solvent.
Spectral characteristics GUA exhibits one excitation maximum at 274 nm, and one emission maximum at 317 nm (Fig. 2). Also, PHE presents one excitation maximum at 276 nm and one emission maximum at 307 nm
Table 2. Intra- and interday precision and accuracy for the determination of GUA and PHE in laboratory-prepared mixturesa using the proposed spectrofluorimetric methods Taken concentration (μg/mL)
Mean % recovery ± SD
RSD (%)
Er (%)
Taken concentration (μg/mL)
Mean % recovery ± SD
RSD (%)
Er (%)
First Derivative (D1) (a) Intraday precision and accuracy (n = 3) GUA
PHE
0.1 0.5 1.0 0.1 0.5 1.0
99.92 ± 100.55 ± 100.62 ± 99.92 ± 99.93 ± 100.56 ±
(b) Interday precision and accuracy (n = 9) 0.57 0.52 0.75 0.86 0.73 0.55
0.572 –0.08 0.1 99.22±1.16 1.17 0.51 0.55 0.5 98.83 ± 1.13 1.15 0.74 0.62 1.0 101.51 ± 1.93 1.90 0.86 –0.08 0.1 100.81 ± 1.48 1.47 0.74 –0.07 0.5 101.16 ± 1.05 1.04 0.55 0.56 1.0 99.52 ± 1.02 1.03 Second Derivative (D2) (b) Interday precision and accuracy (n = 9)
0.66 0.52 0.90 1.07 0.84 0.63
0.67 0.52 0.89 1.06 0.85 0.63
(a) Intra-day precision and accuracy (n = 3) GUA
PHE
0.1 0.5 1.0 0.1 0.5 1.0
99.51 ± 100.61 ± 101.02 ± 100.26 ± 99.23 ± 100.01 ±
–0.49 0.61 1.02 0.26 –0.77 0.01
0.1 0.5 1.0 0.1 0.5 1.0
98.84 98.89 101.95 101.16 100.55 99.22
± ± ± ± ± ±
1.02 1.19 1.71 1.62 1.11 1.02
1.03 1.20 1.67 1.60 1.10 1.03
–0.78 –1.17 1.51 0.81 1.16 –0.48
–1.16 –1.11 1.95 1.60 0.55 –0.78
Mixture composition: 0.1 μg/mL GUA + 1 μg/mL PHE; 0.5 μg/mL GUA + 0.5 μg/mL PHE; 1 μg/mL GUA + 0.1 μg/mL PHE. RSD (%), percent relative standard deviation Er (%), percent relative error. a
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H. M. Maher et al. Selection of optimum pH The influence of pH on the fluorescence intensity of both GUA and PHE was studied using different buffers covering the whole pH range, e.g. acetate buffer (pH 3.6–5.6), phosphate buffer (pH 5.8–9.0) and borate buffer (pH 9.0–11.0). PHE was previously reported to be fluorescent in acid media (24). It was found that solutions diluted with acetate buffer (pH 3.6–5) showed maximum fluorescence intensity for both drugs (Fig. 3a), and increasing the pH resulted in a decrease in the relative fluorescence intensity of both drugs.
In addition, the effect of SDS surfactant on the fluorescence intensity of both GUA and PHE was studied. This effect was examined by adding 1 mL of 0.5% SDS to a methanolic solution of each drug (final concentration 0.5 μg/mL). It was observed that the use of SDS resulted in a minor decrease in the fluorescence intensity of both drugs. Therefore, no surfactant was used in this work. Again, this matches findings obtained for methocarbamol (26).
Determination of GUA and PHE by derivative emission spectra Effect of diluting solvent. Dilution with different solvents including water, acetate buffer (pH 3.6), 0.1 M sodium hydroxide, methanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) was employed (Fig. 3b). Methanol gave the highest relative fluorescence intensity for both GUA and PHE compared with the other solvents. Thus, diluting solvents consisting of different ratios of methanol and acetate buffer (pH 3.6) were tested for their effect on the relative fluorescence intensity of both drugs (Fig. 3c). An increase in the methanol percentage of the medium results in an increase in the fluorescence intensity of both drugs, markedly for PHE. In combined pharmaceutical preparations, PHE is commonly present in much lower concentration ratios than GUA, mostly 0.1 or less, therefore, its fluorescence is strongly favored to allow their simultaneous determination in a mixture. Thus 100% methanol was chosen as the diluting solvent throughout the study. Also, this matches the results of a previous report on the intrinsic fluorescence of methocarbamol, GUA carbamate, where methanol yielded the maximum fluorescence intensity, compared with other solvents (26).
The emission spectra of both GUA and PHE overlapped greatly (Fig. 4a). This hindered the use of direct measurements for the simultaneous determination of these drugs in binary mixtures. This problem is greater if these compounds are to be determined in their co-formulated preparations. For this reason, it is necessary to use other techniques. As a result, the first (D1) and second (D2) derivatives of the emission spectra were calculated (Fig. 4b,c) and the zero-crossing technique was applied to resolve the overlapping derivative spectra. Instrumental parameters affecting the fluorescence analysis were first optimized. The wavelength increment (Δλ) used to calculate the derivative spectra greatly affects the resolution of the overlapping spectra and their intensity (7). Generally, an increase in Δλ results in a decrease in the noise level and hence simpler spectra. However, values that are too high result in a consequent loss of the fine spectral characteristics and poorer resolution. In this study, a Δλ value of 1 nm was found to be optimal for spectral resolution, selectivity and sensitivity. Moreover, other instrumental parameters were selected throughout the measurements: sensitivity, medium; scan speed, normal; and
Figure 3. Illustration of the factors affecting the relative fluorescence intensity of GUA (0.5 μg/mL) and PHE (0.5 μg/mL), obtained at λex = 275 nm. Effect of (a) pH, (b) diluting solvent and (c) methanol % along with acetate buffer pH 3.6 as the diluting solvent.
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Derivative fluorimetric analysis of guaifenesin and phenylephrine wavelengths, the target drug could be determined without any interference from the other drug.
Validation of the method The validity of the method was checked by testing linearity, specificity, accuracy, and precision according to ICH recommendations (27). Linearity. Under the above-described experimental conditions, a linear relationship was observed by plotting drug concentrations against first- or second-derivative signals calculated for each drug, at the selected points, from emission fluorescence spectra. The corresponding concentration ranges are listed in Table 1. The slopes, intercepts and correlation coefficients obtained by linear least squares regression treatment of the results are also given. High values for the correlation coefficients (r > 0.999) with negligible intercepts indicate the good linearity of the calibration graphs. Standard deviations of the residuals (Sy/x), intercept (Sa) and slope (Sb) are presented for each compound. Sy/x is a measure of the extent to which the found (measured) y-values deviate from the calculated ones. The smaller the value of Sy/x, the closer the points are to the linear regression line. For equal degrees of freedom, an increase in the variance ratio (F-values) means an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the greater the steepness of the regression line. The smaller the mean of squares due to residuals, the less the scatter of the experimental points around the regression line. Consequently, regression lines with high F values (low significance F) are much better than those with lower values. Good regression lines show high values for both r and F (28). Detection and quantitation limits. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated for each compound, using both D1 and D2 methods, as presented in Table 1. LOD and LOQ were calculated according to the ICH Q2B recommendations (27), LOD = 3s/k and LOQ = 10s/k, where s is the standard deviation of replicate determination values under the same conditions as for sample analysis in the absence of the analyte, and k is the sensitivity, namely the slope, of the calibration graph.
Figure 4. Emission fluorescence spectra of (a) GUA (1 μg/mL) and PHE (0.1 μg/mL), and (b) their corresponding first derivative spectra (D1) and (c) second derivative spectra (D2). All measured in methanol at λex = 275 nm.
emission step, 1. This selection was based on a satisfactory signal-to-noise ratio. Using the first derivative of the emission spectra (D1), GUA was determined by measuring the first derivative signals at 307 nm, with zero contribution from PHE. Similarly, PHE was determined by measuring the first derivative signals at 317 nm, with zero contribution from GUA. For the second derivative technique (D2), the points selected were 317 nm for GUA and 302 nm for PHE. At these
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Accuracy. The accuracy of the proposed methods was evaluated by analyzing laboratory-prepared mixtures of GUA and PHE, at different concentrations within the working range of each drug (Table 2). Accuracy was expressed as the percent recovery and relative error (27,28). It can be seen that, for both D1 and D2 methods, the concentration of each drug in the mixture could be determined, at the selected zero-crossing points, without any interference from the other drug, indicating good accuracy (Er% < 2). Precision. To test the precision of the proposed method, three replicate determinations were conducted of laboratory-prepared mixtures, prepared as described above. The assay was repeated three times on the same day (intraday precision) or on three different days (interday precision) for each mixture. The calculated relative standard deviations (RSD) for intra- and interday precision were found to be < 2% for both drugs, indicating that the proposed methods are highly precise (Table 2).
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H. M. Maher et al. Table 3. Assay of GUA and PHE in tablets using the proposed spectrofluorimetric methods Mean % recovery ± RSD GUA
t F
PHE
(D1) method
(D2) method
Reference method (9)
(D1) method
(D2) method
Reference method (9)
100.60 ± 1.06 1.87 1.54
101.02 ± 1.33 1.15 2.45
102.06 ± 0.85
100.27 ± 1.10 1.59 1.67
101.20 ± 1.56 0.33 3.37
101.48 ± 0.85
Results are the average of five determinations. Critical values of t and F are 2.31 and 6.39, respectively, at the 95% confidence limit.
Selectivity. The proposed methods allowed the selective determination of each drug in the presence of the other without interference; Er% was < 2 for the analysis of drug mixtures, proving the methods’ selectivity and ability to resolve a mixture of the two drugs.
Pharmaceutical application The proposed methods were applied to the determination of GUA and PHE in their laboratory-prepared tablets. The analysis results obtained using both D1 and D2 methods were compared with the reported HPLC method (9). The Student’s t-test and variance ratio F-test (28) revealed no significant difference between the performance of the two proposed methods and the reference method, in terms of accuracy and precision (Table 3). The specificity of the method was shown by the lack of interference from common excipients. This was proved by good recovery values obtained during the determination of GUA and PHE in combined tablets (Table 3).
Conclusion The proposed molecular fluorescence techniques (emission spectra) in combination with derivative spectrometry, first (D1) and second (D2) techniques, can be readily applied to the resolution of binary mixtures of GUA and PHE. These methods permit determination of the two drugs with high precision and accuracy in both laboratory-prepared mixtures and pharmaceutical preparations. The methods are specific and there is no interference from any of the sample components. Overall, the proposed methods can be used for the rapid determination of the above-mentioned drug combination and have a great promise in the routine analysis of commercial formulations and quality control of such mixtures. Moreover, this report was the first to study the intrinsic fluorescence of GUA under different experimental conditions, indicating its application for the simple and direct determination of GUA in bulk powders or pharmaceutical preparations. Minimum sample preparation, speed of analysis and low cost are the main advantage of these methods when compared with the alternative HPLC method reported in the literature (9). The proposed methods do not use sophisticated instruments or any separation step. Acknowledgement This research project was supported by a grant from the Research Center of the Center for Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University.
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Revised: 5 June 2014,
Accepted: 7 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2735
Derivative emission spectrofluorimetry for the simultaneous determination of guaifenesin and phenylephrine hydrochloride in pharmaceutical tablets Hadir M. Maher,a,b* Mona M. Alshehria and Shorog M. Al-taweela ABSTRACT: Rapid, simple and sensitive derivative emission spectrofluorimetric methods have been developed for the simultaneous analysis of binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE). The methods are based upon measurement of the native fluorescence intensity of the two drugs at λex = 275 nm in methanolic solutions, followed by differentiation using first (D1) and second (D2) derivative techniques. The derivative fluorescence intensity–concentration plots were rectilinear over a range of 0.1–2 μg/mL for both GUA and PHE. The limits of detection were 0.027 (D1, GUA), 0.025 (D2, GUA), 0.031 (D1, PHE) and 0.033 (D2, PHE) μg/mL and limits of quantitation were 0.089 (D1, GUA), 0.083 (D2, GUA), 0.095 (D1, PHE) and 0.097 (D2, PHE) μg/mL. The proposed derivative emission spectrofluorimetric methods (D1 and D2) were successfully applied for the determination of the two compounds in binary mixtures and tablets with high precision and accuracy. The proposed methods were fully validated as per ICH guidelines. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: spectrofluorimetry; derivative emission; guaifenesin; phenylephrine; tablets
Introduction Spectroscopic techniques are among the earliest methods used in pharmaceutical analysis; one such technique is fluorescence spectroscopy (1). Although fluorimetry was one of the earliest instrumental techniques available to analytical chemists, recent developments in instrumentation and sample handling have made it possible for the full potential of this method, particularly its very high sensitivity and selectivity, to be realized in routine analysis (1). Moreover, fluorescence spectroscopy is an adaptable analytical technique, which is more sensitive than other detection systems such as classical UV absorption, less expensive than LC-MS/MS detection, and time saving compared with HPLC (2). The technique has not, however, been widely applied to the simultaneous determination of several fluorescent components in mixtures, mainly because the fluorescence spectra of individual substances contain broad bands that may easily overlap. In general, these compounds are determined using a prior separation step, which is time consuming for routine analysis and in some cases requires special expensive instrumentation. For this reason, the development of techniques that allow the direct determination of related compounds through the careful selection of instrumental variables is of great interest. Among these techniques, synchronous and derivative fluorescence spectrometry is the most popular (3–7). The application of derivative techniques to luminescence spectrometry has great advantages (3–7). Differentiation narrows spectral bandwidths and enhances minor spectral features, thus improving the selectivity of multi-component spectra. In this study, first- and second-derivative emission spectrofluorimetry were used to resolve binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE), which show overlapping emission spectra.
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GUA (R,S)3-(2-methoxyphenoxy)-1,2-propanediol (Fig. 1) is reported to increase the volume and reduce the viscosity of tenacious sputum and is used as an expectorant for productive coughs (8). Different methods have been reported for the analysis of GUA either alone or in combination, including HPLC (9,10), GC (11,12), CE (13), X-ray diffraction (14), voltammetry (15) and spectrophotometry (16,17). PHE, a hydrochloride salt of (R)-2-methylamino-1(3-hydroxyphenyl) ethanol (Fig. 1), is a sympathomimetic agent with direct effects on α-adrenoreceptors. It is used mainly as a nasal decongestant either alone or in combination with other agents for the symptomatic relief of cold symptoms (8). PHE has been previously determined in several pharmaceutical forms using HPLC (9,18–20), CE (21), spectrophotometry (20,22), spectrofluorimetry (23,24) and electrochemical analysis (25). Binary combinations of GUA and PHE are available as cough and cold preparations. Products containing GUA and PHE are used to treat symptoms (coughing, runny/stuffy nose, congestion) of the common cold, allergies, asthma, bronchitis, sinusitis and other illnesses that affect breathing (8). A survey of the literature revealed that only one HPLC method is reported for the simultaneous determination of GUA and PHE in binary bulk powders and
* Correspondence to: H. M. Maher, College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, Riyadh 11495, P.O. Box 22452, Saudi Arabia. E-mail: [email protected] a
College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, P.O. Box 22452, Riyadh 11495, P.O. Box 22452, Saudi Arabia
b
Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria 21521, Egypt
Copyright © 2014 John Wiley & Sons, Ltd.
H. M. Maher et al. Materials and reagents
Figure 1. Structures of GUA and PHE.
pharmaceutical dosage forms (9). To our knowledge, no method has been reported for the spectrofluorimetric determination of such a drug combination. Furthermore, no study has been found that uses the intrinsic fluorescence properties of GUA as a tool in its quantitative analysis. The aim of this work was to develop a simple and validated spectrofluorimetric method for the simultaneous determination of GUA and PHE in their tablet dosage form. The method made use of the intrinsic fluorescence properties of both drugs under experimentally optimized conditions. This work was based on the application of a derivative technique (D method) to resolve the overlapping emission spectra of both GUA and PHE in order to obtain highly specific derivative analytical signals. This enabled the determination of each drug at a zero contribution point from the other.
Experimental Instrumentation Fluorescence measurements were performed on a Spectramax (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA). Data acquisition and data analysis were performed with SoftMax Pro v. 5.4® software (Molecular Devices). The following instrumental parameters were selected throughout the measurements: sensitivity, medium; scan speed, normal; and emission step, 1. A microprocessor laboratory pH meter (Mettler-Toledo International Inc., Zürich, Switzerland), calibrated daily using standard buffers at room temperature was used.
GUA and PHE standards were from Sigma Aldrich (St. Louis, MO, USA). All reagents were of analytical grade, namely, methanol (Panreac Co., Barcelona, Spain), disodium hydrogen phosphate, phosphoric acid and sodium hydroxide. The water used was double glass distilled. Because of the unavailability of a marketed tablet formulation for GUA and PHE, simulated powdered tablets were prepared in the laboratory by mixing both drugs in the same amounts as in actual tablets, namely, Norel® EX labeled to contain 400 mg GUA and 40 mg PHE, along with other excipients normally encountered in tablet formulations (9).
Standard solutions and calibration graphs Stock solutions of 100.0 μg/mL GUA and PHE were prepared in methanol. A step dilution was conducted with the same solvent to prepare intermediate stock solutions of 10.0 μg/mL for both drugs. These solutions were found to be stable for at least one week when kept in the refrigerator. Accurately measured volumes of the intermediate stock solutions of each drug were further transferred into a series of 10 mL volumetric flasks and completed to volume with methanol to obtain working standard solutions at suitable concentrations (corresponding to the linearity range in Table 1). A reagent blank consisting of methanol was used throughout the study. For each standard solution of GUA and PHE, the emission spectra were recorded in the range 290–360 nm with excitation at 275 nm and were corrected for the blank signal. First and second derivatives of the emission data were calculated. For firstderivative emission fluorimetry (D1 method), the absolute values of the first derivative were measured at 307 and 317 nm for the determination of GUA and PHE, respectively. For the second derivative (D2 method), values were measured at 317 and 302 nm for the determination of GUA and PHE, respectively. Derivative values, calculated at the selected points, were plotted against the final concentration of each drug for both D1 and D2 methods, to obtain the calibration graphs. Alternatively, the corresponding regression equations were derived.
Table 1. Regression and statistical parameters for the determination of GUA and PHE by the proposed spectrofluorimetric method Parameter
Linearity range (μg/mL) LOD (μg/mL) LOQ (μg/mL) Intercept (a) Slope (b) Correlation coefficient (r) Sa Sb Sy/x F Significance F
First derivative of emission spectra (D1)
Second derivative of emission spectra (D2)
GUA
PHE
GUA
PHE
0.1–2 0.027 0.089 –0.120 16.863 0.9998 0.126 0.218 0.149 5968.48 1.680E-04
0.1–2 0.031 0.095 0.254 –15.997 0.9997 0.167 0.288 0.197 3081.51 3.240E-04
0.1–2 0.025 0.083 0.017 –2.408 0.9998 0.018 0.031 0.021 5950.42 1.680E-04
0.1–2 0.033 0.097 0.073 –4.636 0.9997 0.048 0.083 0.057 3084.77 3.240E-04
LOD, limit of detection; LOQ, limit of quantitation; Sy/x, standard deviation of residuals; Sa, standard deviation of intercept; Sb, standard deviation of slope; F, variance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals).
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Derivative fluorimetric analysis of guaifenesin and phenylephrine Preparation and analysis of laboratory-made mixtures Accurate volumes of GUA and PHE stock solutions were transferred into 10 mL volumetric flasks and diluted to volume with methanol to prepare mixtures within the concentration range of each compound (Table 2). The recommended procedure under the calibration graph was then performed. Derivative values were measured and the corresponding concentrations were derived from the calibration curves or the corresponding regression equations.
Procedure for tablets An accurately weighed quantity of powdered tablets equivalent to 10 mg of GUA and 1 mg PHE was transferred into a 100 mL volumetric flask. Sixty milliliters of methanol were added and the flask was sonicated in an ultrasonic bath for 30 min, diluted to volume with the same solvent, then filtered. Portions of 0.5 mL of the previously prepared solution were diluted with methanol in 50 mL volumetric flasks to prepare tablet solutions containing 1.0 μg/mL GUA and 0.1 μg/mL PHE. The emission spectra of the prepared tablet solutions were recorded in the range 290–360 nm with excitation at 275 nm and corrected for the blank signal. First- and second-derivative values of emission spectra were calculated and signals were measured at the selected wavelengths, as shown under construction of calibration graphs. The nominal contents were calculated using the corresponding regression equation for each of GUA and PHE, using both (D1) and (D2) methods.
Figure 2. Excitation (a, c) and emission (b, d) spectra of GUA (0.5 μg/mL) and PHE (0.5 μg/mL). All spectra were acquired in methanol.
(Fig. 2). For simultaneous determination of GUA and PHE, a single excitation wavelength of 275 nm was selected where a suitable response for both drugs was obtained. Factors affecting fluorescence intensity
Results and discussion
Different experimental parameters affecting the performance of the proposed methods were carefully studied and optimized to obtain the best measurement conditions and maximum fluorescence signals of GUA and PHE. Such factors were changed individually, whereas others were kept constant. These factors included the effect of pH and type of diluting solvent.
Spectral characteristics GUA exhibits one excitation maximum at 274 nm, and one emission maximum at 317 nm (Fig. 2). Also, PHE presents one excitation maximum at 276 nm and one emission maximum at 307 nm
Table 2. Intra- and interday precision and accuracy for the determination of GUA and PHE in laboratory-prepared mixturesa using the proposed spectrofluorimetric methods Taken concentration (μg/mL)
Mean % recovery ± SD
RSD (%)
Er (%)
Taken concentration (μg/mL)
Mean % recovery ± SD
RSD (%)
Er (%)
First Derivative (D1) (a) Intraday precision and accuracy (n = 3) GUA
PHE
0.1 0.5 1.0 0.1 0.5 1.0
99.92 ± 100.55 ± 100.62 ± 99.92 ± 99.93 ± 100.56 ±
(b) Interday precision and accuracy (n = 9) 0.57 0.52 0.75 0.86 0.73 0.55
0.572 –0.08 0.1 99.22±1.16 1.17 0.51 0.55 0.5 98.83 ± 1.13 1.15 0.74 0.62 1.0 101.51 ± 1.93 1.90 0.86 –0.08 0.1 100.81 ± 1.48 1.47 0.74 –0.07 0.5 101.16 ± 1.05 1.04 0.55 0.56 1.0 99.52 ± 1.02 1.03 Second Derivative (D2) (b) Interday precision and accuracy (n = 9)
0.66 0.52 0.90 1.07 0.84 0.63
0.67 0.52 0.89 1.06 0.85 0.63
(a) Intra-day precision and accuracy (n = 3) GUA
PHE
0.1 0.5 1.0 0.1 0.5 1.0
99.51 ± 100.61 ± 101.02 ± 100.26 ± 99.23 ± 100.01 ±
–0.49 0.61 1.02 0.26 –0.77 0.01
0.1 0.5 1.0 0.1 0.5 1.0
98.84 98.89 101.95 101.16 100.55 99.22
± ± ± ± ± ±
1.02 1.19 1.71 1.62 1.11 1.02
1.03 1.20 1.67 1.60 1.10 1.03
–0.78 –1.17 1.51 0.81 1.16 –0.48
–1.16 –1.11 1.95 1.60 0.55 –0.78
Mixture composition: 0.1 μg/mL GUA + 1 μg/mL PHE; 0.5 μg/mL GUA + 0.5 μg/mL PHE; 1 μg/mL GUA + 0.1 μg/mL PHE. RSD (%), percent relative standard deviation Er (%), percent relative error. a
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H. M. Maher et al. Selection of optimum pH The influence of pH on the fluorescence intensity of both GUA and PHE was studied using different buffers covering the whole pH range, e.g. acetate buffer (pH 3.6–5.6), phosphate buffer (pH 5.8–9.0) and borate buffer (pH 9.0–11.0). PHE was previously reported to be fluorescent in acid media (24). It was found that solutions diluted with acetate buffer (pH 3.6–5) showed maximum fluorescence intensity for both drugs (Fig. 3a), and increasing the pH resulted in a decrease in the relative fluorescence intensity of both drugs.
In addition, the effect of SDS surfactant on the fluorescence intensity of both GUA and PHE was studied. This effect was examined by adding 1 mL of 0.5% SDS to a methanolic solution of each drug (final concentration 0.5 μg/mL). It was observed that the use of SDS resulted in a minor decrease in the fluorescence intensity of both drugs. Therefore, no surfactant was used in this work. Again, this matches findings obtained for methocarbamol (26).
Determination of GUA and PHE by derivative emission spectra Effect of diluting solvent. Dilution with different solvents including water, acetate buffer (pH 3.6), 0.1 M sodium hydroxide, methanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) was employed (Fig. 3b). Methanol gave the highest relative fluorescence intensity for both GUA and PHE compared with the other solvents. Thus, diluting solvents consisting of different ratios of methanol and acetate buffer (pH 3.6) were tested for their effect on the relative fluorescence intensity of both drugs (Fig. 3c). An increase in the methanol percentage of the medium results in an increase in the fluorescence intensity of both drugs, markedly for PHE. In combined pharmaceutical preparations, PHE is commonly present in much lower concentration ratios than GUA, mostly 0.1 or less, therefore, its fluorescence is strongly favored to allow their simultaneous determination in a mixture. Thus 100% methanol was chosen as the diluting solvent throughout the study. Also, this matches the results of a previous report on the intrinsic fluorescence of methocarbamol, GUA carbamate, where methanol yielded the maximum fluorescence intensity, compared with other solvents (26).
The emission spectra of both GUA and PHE overlapped greatly (Fig. 4a). This hindered the use of direct measurements for the simultaneous determination of these drugs in binary mixtures. This problem is greater if these compounds are to be determined in their co-formulated preparations. For this reason, it is necessary to use other techniques. As a result, the first (D1) and second (D2) derivatives of the emission spectra were calculated (Fig. 4b,c) and the zero-crossing technique was applied to resolve the overlapping derivative spectra. Instrumental parameters affecting the fluorescence analysis were first optimized. The wavelength increment (Δλ) used to calculate the derivative spectra greatly affects the resolution of the overlapping spectra and their intensity (7). Generally, an increase in Δλ results in a decrease in the noise level and hence simpler spectra. However, values that are too high result in a consequent loss of the fine spectral characteristics and poorer resolution. In this study, a Δλ value of 1 nm was found to be optimal for spectral resolution, selectivity and sensitivity. Moreover, other instrumental parameters were selected throughout the measurements: sensitivity, medium; scan speed, normal; and
Figure 3. Illustration of the factors affecting the relative fluorescence intensity of GUA (0.5 μg/mL) and PHE (0.5 μg/mL), obtained at λex = 275 nm. Effect of (a) pH, (b) diluting solvent and (c) methanol % along with acetate buffer pH 3.6 as the diluting solvent.
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Derivative fluorimetric analysis of guaifenesin and phenylephrine wavelengths, the target drug could be determined without any interference from the other drug.
Validation of the method The validity of the method was checked by testing linearity, specificity, accuracy, and precision according to ICH recommendations (27). Linearity. Under the above-described experimental conditions, a linear relationship was observed by plotting drug concentrations against first- or second-derivative signals calculated for each drug, at the selected points, from emission fluorescence spectra. The corresponding concentration ranges are listed in Table 1. The slopes, intercepts and correlation coefficients obtained by linear least squares regression treatment of the results are also given. High values for the correlation coefficients (r > 0.999) with negligible intercepts indicate the good linearity of the calibration graphs. Standard deviations of the residuals (Sy/x), intercept (Sa) and slope (Sb) are presented for each compound. Sy/x is a measure of the extent to which the found (measured) y-values deviate from the calculated ones. The smaller the value of Sy/x, the closer the points are to the linear regression line. For equal degrees of freedom, an increase in the variance ratio (F-values) means an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the greater the steepness of the regression line. The smaller the mean of squares due to residuals, the less the scatter of the experimental points around the regression line. Consequently, regression lines with high F values (low significance F) are much better than those with lower values. Good regression lines show high values for both r and F (28). Detection and quantitation limits. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated for each compound, using both D1 and D2 methods, as presented in Table 1. LOD and LOQ were calculated according to the ICH Q2B recommendations (27), LOD = 3s/k and LOQ = 10s/k, where s is the standard deviation of replicate determination values under the same conditions as for sample analysis in the absence of the analyte, and k is the sensitivity, namely the slope, of the calibration graph.
Figure 4. Emission fluorescence spectra of (a) GUA (1 μg/mL) and PHE (0.1 μg/mL), and (b) their corresponding first derivative spectra (D1) and (c) second derivative spectra (D2). All measured in methanol at λex = 275 nm.
emission step, 1. This selection was based on a satisfactory signal-to-noise ratio. Using the first derivative of the emission spectra (D1), GUA was determined by measuring the first derivative signals at 307 nm, with zero contribution from PHE. Similarly, PHE was determined by measuring the first derivative signals at 317 nm, with zero contribution from GUA. For the second derivative technique (D2), the points selected were 317 nm for GUA and 302 nm for PHE. At these
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Accuracy. The accuracy of the proposed methods was evaluated by analyzing laboratory-prepared mixtures of GUA and PHE, at different concentrations within the working range of each drug (Table 2). Accuracy was expressed as the percent recovery and relative error (27,28). It can be seen that, for both D1 and D2 methods, the concentration of each drug in the mixture could be determined, at the selected zero-crossing points, without any interference from the other drug, indicating good accuracy (Er% < 2). Precision. To test the precision of the proposed method, three replicate determinations were conducted of laboratory-prepared mixtures, prepared as described above. The assay was repeated three times on the same day (intraday precision) or on three different days (interday precision) for each mixture. The calculated relative standard deviations (RSD) for intra- and interday precision were found to be < 2% for both drugs, indicating that the proposed methods are highly precise (Table 2).
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H. M. Maher et al. Table 3. Assay of GUA and PHE in tablets using the proposed spectrofluorimetric methods Mean % recovery ± RSD GUA
t F
PHE
(D1) method
(D2) method
Reference method (9)
(D1) method
(D2) method
Reference method (9)
100.60 ± 1.06 1.87 1.54
101.02 ± 1.33 1.15 2.45
102.06 ± 0.85
100.27 ± 1.10 1.59 1.67
101.20 ± 1.56 0.33 3.37
101.48 ± 0.85
Results are the average of five determinations. Critical values of t and F are 2.31 and 6.39, respectively, at the 95% confidence limit.
Selectivity. The proposed methods allowed the selective determination of each drug in the presence of the other without interference; Er% was < 2 for the analysis of drug mixtures, proving the methods’ selectivity and ability to resolve a mixture of the two drugs.
Pharmaceutical application The proposed methods were applied to the determination of GUA and PHE in their laboratory-prepared tablets. The analysis results obtained using both D1 and D2 methods were compared with the reported HPLC method (9). The Student’s t-test and variance ratio F-test (28) revealed no significant difference between the performance of the two proposed methods and the reference method, in terms of accuracy and precision (Table 3). The specificity of the method was shown by the lack of interference from common excipients. This was proved by good recovery values obtained during the determination of GUA and PHE in combined tablets (Table 3).
Conclusion The proposed molecular fluorescence techniques (emission spectra) in combination with derivative spectrometry, first (D1) and second (D2) techniques, can be readily applied to the resolution of binary mixtures of GUA and PHE. These methods permit determination of the two drugs with high precision and accuracy in both laboratory-prepared mixtures and pharmaceutical preparations. The methods are specific and there is no interference from any of the sample components. Overall, the proposed methods can be used for the rapid determination of the above-mentioned drug combination and have a great promise in the routine analysis of commercial formulations and quality control of such mixtures. Moreover, this report was the first to study the intrinsic fluorescence of GUA under different experimental conditions, indicating its application for the simple and direct determination of GUA in bulk powders or pharmaceutical preparations. Minimum sample preparation, speed of analysis and low cost are the main advantage of these methods when compared with the alternative HPLC method reported in the literature (9). The proposed methods do not use sophisticated instruments or any separation step. Acknowledgement This research project was supported by a grant from the Research Center of the Center for Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University.
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