TY - JOUR
AU - Moneim, Mona M Abdel
AB - Abstract A validated and selective high-performance thin-layer chromatography (HPTLC) method was developed for the analysis of mixures of tamsulosin hydrochloride (TAM) with either tolterodine tartrate (TOL) or solifenacin succinate (SOL) in bulk drug and in combined dosage forms. The proposed method is based on HPTLC separation of the three drugs followed by densitometric measurements of their spots at 224 nm. Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using ethyl acetate–methanol–ammonia (6:4:0.05, v/v) as mobile phase. The linear regression analysis data were used for the regression line in the range of 0.1–0.7, 0.4–4 and 1–6 μg band–1 for TAM, TOL and SOL, respectively. The proposed method was validated and successfully applied for the analysis of their pharmaceutical formulations and laboratory-prepared mixtures containing the two bicomponent combinations. The method was validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. Introduction Lower urinary tract symptoms (LUTS) are prevalent in men aged above 45 years and have a significant impact on men’s health life. Alpha blockers are widely prescribed to manage LUTS that are usually associated with benign prostatic hyperplasia (BPH) (1). Available data suggest that treatment with antimuscarinics plus the α1-blockers may be more effective in reducing LUTS associated with BPH than α-blockers alone (2). Tamsulosin hydrochloride (TAM), (R)-5-(2-{[2-(2-ethoxyphenoxy)ethyl]amino}propyl)-2-methoxybenzene-1-sulphonamide, exists in two enantiomeric forms, but only R-isomer is the pharmaceutically active component. The chemical structure is shown in Figure 1a. It is a new type of highly selective α-1-blocker for treatment of BPH (3, 4). Figure 1. View largeDownload slide Chemical structures of (a) tamsulosin hydrochloride (TAM), (b) tolterodine tartrate (TOL) and (c) solifenacin succinate (SOL). Figure 1. View largeDownload slide Chemical structures of (a) tamsulosin hydrochloride (TAM), (b) tolterodine tartrate (TOL) and (c) solifenacin succinate (SOL). Tolterodine Tartrate (TOL), (R)-2-[3-(diisopropylamino)-1-phenylpropyl]-4-methylphenol (Figure 1b). It is an antimuscarinic drug that is used to treat urinary incontinence (3). Also, Solifenacin Succinate (SOL), 1-azabicyclo[2.2.2]oct-3-yl (1 R)-1-phenyl-3,4-dihydro-1 H-isoquinoline-2-carboxylate (Figure 1c), is an antimuscarinic prescription drug used for the treatment of overactive bladder in adults with symptoms of urinary incontinence, urinary urgency and urinary frequency (2, 5). It is important to develop new analytical methods to resolve new pharmaceutical mixtures with classical active compounds such as those newly marketed pharmaceutical preparations containing TAM with SOL (Mixture 1) and TAM with TOL (Mixture 2) used in the treatment of BPH/LUTS. TAM is official in the USP (6) where a potentiometric titration method was described for its assay in bulk powder and HPLC method for its determination in capsules. The literature also reveals different methods for the determination of TAM in dosage forms. These include spectrophotometry (7–12), spectrofluorimetry (13, 14), HPLC (15, 16), voltametry (17, 18) and conductimetry (19). Also, HPLC–MS (20–22) and HPLC (23) were described for TAM assay in different biological fluids. Different methods have been also reported for the determination of TAM in presence of its impurities and related compounds as LC–MS (24) and HPLC (25, 26) or its degradation products by HPLC (27, 28) and HPTLC (29–31). On the other hand, for the simultaneous determination of TAM with TOL, a few methods have been reported. These include spectrophotometry (32, 33), HPLC (34–36) and one recently reported HPTLC method (37) for their assay in dosage forms. Similarly, the literature reveals only one HPLC method (38) for the simultaneous determination of TAM and SOL in their combined dosage forms. HPTLC represents an analytical technique having several advantages when compared with HPLC. These include high sample output where several samples could be run simultaneously within a comparatively short time and lower cost per analysis. In addition to the need of minimum sample cleaning compared to HPLC. To date, there have been no published reports about the simultaneous determination of these two binary mixtures containing TAM by high-performance thin-layer chromatography. This study describes for the first time the simultaneous determination of TAM in the selected binary mixtures drugs by HPTLC. The proposed study involves separation of TAM and TOL or SOL by HPTLC followed by densitometric measurements of their spots at 224 nm. The advantage of the proposed method is that all three drugs, TAM, TOL and SOL, were successfully separated from each other. Hence, the two mixtures can be separated in the same run using the same mobile phase, which allows quality control laboratories to analyze the two mixtures in their pharmaceutical preparations in a short time with minimum solvent and cost. Validation of the proposed method according to the United States Pharmacopeia (USP) (6) validation elements and the International Conference on Harmonization (ICH) (39) guidelines is presented. The results were also in good agreement with those obtained with the reference methods (32, 38). Experimental Instrumentation and chromatographic conditions HPTLC plates (20 × 10 cm, aluminum plates with 250-μm thickness precoated with silica gel 60 F254) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100-μL CAMAG microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Linomat IV applicator (CAMAG, Muttenz, Switzerland). The slit dimension was kept at 5.00 × 0.45 mm, and 20 mm s–1 scanning speed was employed. Ascending development of the mobile phase was carried out in a CAMAG 20 cm × 10 cm twin trough glass chamber. The optimized chamber saturation time using 20 mL of the mobile phase was 30 min at room temperature (25 ± 2°C). Densitometric scanning was performed at 224 nm on a CAMAG TLC Scanner 3 operated in the reflectance–absorbance mode and controlled by CAMAG CATS software (V 3.15). The source of radiation utilized was deuterium lamp emitting a continuous ultraviolet (UV) spectrum between 190 and 400 nm. Materials and reagents Pharmaceutical grades of Tamsulosin, Tolterodine and Solifenacin were kindly supplied by Medizen Pharmaceuticals (Borg El Arab, Alexandria, Egypt.) and their purity was 99.99%, 99.95% and 99.90%, respectively. Methanol (S.D. Fine-Chem Limited, India) was of analytical grade. Ethyl acetate and ammonia 30% (El-Nasr Pharmaceutical Co., Abu Zaabal, Egypt). In addition, Whatman No. 1 filter paper 110 mm diameter (Sigma-Aldrich) was used for filtration of sample solutions. The pharmaceutical preparations were purchased from their commercial market. Roliflo-OD® labeled to contain 0.4 and 4 mg of TAM and TOL, respectively, per capsule is manufactured by Ranbaxy Laboratories Ltd, India. Vesomni® labeled to contain 0.4 and 6 mg of TAM and SOL, respectively, per tablet is manufactured by Astellas Pharma Ltd, Surrey, UK. Standard solutions and calibration graphs Standard solutions Standard solutions containing 0.5 mg.mL−1 of TAM and 2 mg.mL–1 of TOL and SOL were prepared separately by dissolving the reference materials in methanol. Regarding the stability of solutions of the drugs, stock solutions were stored at 4°C in amber glass vessels and were found to be stable for at least 10 days. Working solutions for construction of calibration graphs Preparation of working solutions was done by diluting the standard solutions with methanol. For Mixture 1, different volumes to give concentrations in the range of 0.01–0.07 and 0.04–0.4 mg.mL−1 for TAM and TOL, respectively, were diluted with methanol in 10-mL volumetric flasks. For Mixture 2, different volumes to give concentrations in the range of 0.01–0.07 and 0.1–0.6 mg.mL−1 for TAM and SOL, respectively, were diluted with methanol in 10-mL volumetric flasks. From each working standard solution, 10-μL portions were spotted as bands on HPTLC plate to obtain final concentrations of TAM, TOL and SOL as cited in Table I. The bands (5 mm width) were separated by a distance of 4 mm apart and 15 mm from the bottom of the plate. Each solution was applied in triplicate. The plate was then developed using ethyl acetate–methanol–ammonia (6:4:0.05, v/v) as mobile phase and scanned densitometrically at 224 nm for both mixtures. The calibration graphs were obtained upon plotting the obtained peak areas versus the corresponding concentrations for each compound. The concentrations of TAM, TOL and SOL were computed from their corresponding calibration graphs. Table I. Characteristic Parameters for the Regression Equations of the Proposed HPTLC Method for the Determination of TAM, TOL and SOL Parameters  TAM  TOL  SOL  Linearity range (μg.band−1)  0.1–0.7  0.4–4  1–6  LOD (μg.band−1)  0.03  0.12  0.30  LOQ (μg.band−1)  0.10  0.40  1.00  Intercept (a)  496.50  290.87  1003.23  Slope (b)  4405.18  1988.42  1256.68  Correlation coefficient (r)  0.9990  0.9992  0.9996  Sa  44.31  98.78  71.89  Sb  111.46  45.65  19.46  Sy/x  51.32  125.59  87.45  F  1561.92  1897.21  4171.21  Significance F  3.56 × 10−5  2.66 × 10−5  8.18 × 10−6  Parameters  TAM  TOL  SOL  Linearity range (μg.band−1)  0.1–0.7  0.4–4  1–6  LOD (μg.band−1)  0.03  0.12  0.30  LOQ (μg.band−1)  0.10  0.40  1.00  Intercept (a)  496.50  290.87  1003.23  Slope (b)  4405.18  1988.42  1256.68  Correlation coefficient (r)  0.9990  0.9992  0.9996  Sa  44.31  98.78  71.89  Sb  111.46  45.65  19.46  Sy/x  51.32  125.59  87.45  F  1561.92  1897.21  4171.21  Significance F  3.56 × 10−5  2.66 × 10−5  8.18 × 10−6  Sa is standard deviation of intercept, Sb is standard deviation of slope and Sy/x is standard deviation of residuals. Analysis of synthetic mixtures Accurate volumes of standard solutions of TAM and TOL (Mixture 1) or TAM and SOL (Mixture 2) were transferred into two sets each of three 10-mL volumetric flasks then completed to volume with methanol so that the final concentration of each drug was within its linearity range as cited in Table I. The procedure was then completed as under (Constructions of calibration graphs). Then, the peak area values for each drug were measured, and the % found values were calculated. Analysis of pharmaceutical preparations Twenty Roliflo-OD® capsules were opened and emptied and the contents were accurately weighed. A portion of the capsules contents equivalent to 2.5 mg TAM and 25 mg TOL was weighed and accurately transferred into a 25-mL volumetric flask using about 15 mL methanol. On the other hand, twenty Vesomni® tablets were weighed and finely powdered. A portion of the tablet powder equivalent to about 2.5 mg TAM and 37.5 mg SOL was transferred into another 25-mL volumetric flask using about 15 mL methanol. The sample solutions of both flasks were sonicated for 15 min. In both flasks, the volume was completed to 25 mL using methanol and filtered through Whatman No. 1 filter paper. Portions of 1, 2 and 3 mL of each of the prepared Roliflo-OD® capsules sample solution (Mixture 1) and the prepared Vesomni® tablets sample solution (Mixture 2) were transferred into two separate sets of 10-mL volumetric flasks and diluted to volume with methanol. The concentrations achieved after the above dilution were 10, 20 and 40 μg.mL−1 of TAM and 100, 200 and 400 μg.mL−1 of TOL (Mixture 1), and 10, 20 and 40 μg.mL−1 of TAM and 150, 300 and 600 μg.mL−1 of SOL (Mixture 2). Ten-microliter volume of each sample solution was spotted three times to achieve a final concentration of 0.1, 0.2 and 0.4 μg.band−1 of TAM and 1, 2 and 4 μg.band−1 of TOL (Mixture 1), and 0.1, 0.2 and 0.4 μg.band−1 of TAM and 1.5, 3 and 6 μg.band−1 of SOL (Mixture 2). The plate was developed under the previously described chromatographic conditions and the concentration of each of TAM, TOL and SOL was calculated. Results Method development and optimization The experimental conditions for HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible, compact flat bands for the simultaneous determination of TAM with either TOL or SOL. The three pure drugs were spotted on TLC silica plates. Different solvent systems were tried for their separation. Initially, a system using chloroform and methanol in different ratios was tried but TAM and TOL bands were not separated efficiently and the bands were slightly tailed. When ethyl acetate replaced chloroform, the required separation between the three bands without any tailing was achieved. Different ratios of ethyl acetate to methanol were tried to obtain the optimum separation of bands in each mixture. The greatest differences between the Rf values of the three compounds with no tailing for Mixture 1 (0.61 ± 0.02 for TAM and 0.47 ± 0.03 for TOL) and (0.61 ± 0.02 for TAM and 0.20 ± 0.03 for SOL) for Mixture 2 were obtained by using a mobile phase consisting of ethyl acetate–methanol–ammonia (6:4:0.05, v/v). Figure 2 not only shows that the two compounds in both mixtures could be separated with good resolution as sharp and symmetrical peaks but also the three drugs could be simultaneously determined in the same run with very good separation. Well-defined bands for both drugs in both mixtures were obtained when the chamber was saturated with the mobile phase at room temperature for at least 30 min. This was required to eliminate the edge effect and, thus, avoiding unequal solvent evaporation from the developing plate which might cause random behavior for the developing drugs and resulting in lack of reproducibility of Rf values. Different scanning wavelengths were tried, but the best sensitivity of TAM, the minor component in the two mixtures, was obtained at 224 nm (wavelength at which TAM shows maximum absorption). The optimum band width chosen was 5 mm, taking into consideration the range of concentrations applied and number of tracks. Figure 2. View largeDownload slide A typical HPTLC chromatogram of (a) 0.3 and 3 μg.band–1 of TAM and TOL, respectively, in their mixture (Mixture 1), (b) 0.3 and 4.5 μg.band–1 of TAM and SOL, respectively, in their mixture (Mixture 2) and (c) 0.2, 2 and 3 μg.band–1 of TAM, TOL and SOL, respectively, in their mixture, using 10-μL band volume. Figure 2. View largeDownload slide A typical HPTLC chromatogram of (a) 0.3 and 3 μg.band–1 of TAM and TOL, respectively, in their mixture (Mixture 1), (b) 0.3 and 4.5 μg.band–1 of TAM and SOL, respectively, in their mixture (Mixture 2) and (c) 0.2, 2 and 3 μg.band–1 of TAM, TOL and SOL, respectively, in their mixture, using 10-μL band volume. Validation of the proposed method ICH guidelines (39) for method validation were followed for the developed HPTLC method. Linearity and range The linearity of the proposed method was evaluated by analyzing series of at least five different concentrations of each of TAM, TOL and SOL. By plotting peak area of each drug against its corresponding concentration, linear correlation was obtained within the ranges stated in Table I. Statistical parameters of the regression equation including intercepts, slopes and correlation coefficients obtained by the linear least squares regression treatment of the results are also given. Standard deviation of the residuals, Sy/x is an important statistical parameter for representing the random error in the estimated values of y. Small values of the standard error of the residuals indicate closeness of the points to the straight line. In addition, standard deviation of intercept (Sa) and of slope (Sb) are presented for each compound. Good linearity of the calibration graphs was confirmed by the high correlation coefficients and F-values (40, 41). For equal degrees of freedom, an increase in the variance ratio (F-values) describes an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals (41). Limit of detection and limit of quantification The limit of detection (LOD) is considered as the concentration which has a signal-to-noise ratio of 3:1. For the limit of quantification (LOQ), the ratio considered is 10:1 with a % RSD value less than 10% (6). Using the proposed method, LOD and LOQ for each compound were calculated and are presented in Table I. Their values indicate high sensitivity of the proposed method. Accuracy The accuracy of the proposed method was studied by applying the method to pharmaceutical dosage forms (TAM and TOL combination capsules or TAM and SOL combination tablets) after addition of known quantities of TAM and TOL (Mixture 1) or TAM and SOL (Mixture 2) standard powder equivalent to 80%, 100% and 120% of label claim (standard addition method) followed by extraction, filtration and dilution to obtain the final concentrations within each drug linearity range (Table I). Calculation of the percentage recovery was done by comparison of the peak areas of standard solutions of TAM, TOL and SOL relative to the peak areas of standards added in the fortified samples of different concentrations. Five replicates were performed at each level of concentration. The data obtained from the recovery studies are presented in Table II. The obtained percentage recoveries for the added amounts were in the range of 98–102% with small percentage relative standard deviations (RSDs) (less than 2%) indicating accuracy of the presented work for the simultaneous analysis of the two mixtures. Table II. Evaluation of Accuracy of the Proposed Method for the Determination of TAM and TOL (Mixture 1) and TAM and SOL (Mixture 2) Drug, label claim (μg.mL−1)  Amount of label claim added (%)  Total amount (μg.mL−1)  Theoretical conc. (μg band−1)  Mean % recoverya  RSD %b  Er %c  Mixture 1 (TAM and TOL)   TAM, 10  80  18  0.18  100.20  1.20  0.20  100  20  0.20  099.97  1.37  −0.03  120  22  0.22  099.54  0.50  −0.46   TOL, 100  80  180  1.80  100.94  1.65  0.94  100  200  2.00  100.57  1.50  0.57  120  220  2.20  099.22  1.22  −0.78  Mixture 2 (TAM and SOL)   TAM, 10  80  18  0.18  099.90  0.90  −0.10  100  20  0.20  098.95  1.22  −1.05  120  22  0.22  100.01  0.80  0.01   SOL, 150  80  270  2.70  101.90  1.90  1.90  100  300  3.00  100.89  0.77  0.89  120  330  3.30  101.50  1.12  1.50  Drug, label claim (μg.mL−1)  Amount of label claim added (%)  Total amount (μg.mL−1)  Theoretical conc. (μg band−1)  Mean % recoverya  RSD %b  Er %c  Mixture 1 (TAM and TOL)   TAM, 10  80  18  0.18  100.20  1.20  0.20  100  20  0.20  099.97  1.37  −0.03  120  22  0.22  099.54  0.50  −0.46   TOL, 100  80  180  1.80  100.94  1.65  0.94  100  200  2.00  100.57  1.50  0.57  120  220  2.20  099.22  1.22  −0.78  Mixture 2 (TAM and SOL)   TAM, 10  80  18  0.18  099.90  0.90  −0.10  100  20  0.20  098.95  1.22  −1.05  120  22  0.22  100.01  0.80  0.01   SOL, 150  80  270  2.70  101.90  1.90  1.90  100  300  3.00  100.89  0.77  0.89  120  330  3.30  101.50  1.12  1.50  aMean ± standard deviation of five determinations. bPercentage RSD. cPercentage relative error. Precision The precision of the proposed method was studied by the analysis of five replicate determinations of laboratory-made mixtures containing the two drugs in each mixture in concentrations within the linearity range of each drug. For each mixture, the laboratory-made mixtures were analyzed five times on the same day or on five different days for studying the intra-day and inter-day precision, respectively. The percentage relative error (Er%) and percentage relative standard deviation (RSD %) values for intra- and inter-day precisions were found to be less than 2% demonstrating good precision of the proposed method (Table III). Table III. Evaluation of Intra-day and Inter-day Precision of the Proposed Method for the Determination of TAM and TOL (Mixture 1) and TAM and SOL (Mixture 2) in their Synthetic Mixtures Concentration (μg.band−1)  Mean % recovery ± SDa  RSD %b  Er %c  (a) Intra-day precision (repeatability)   TAM  TOL  TAM  TOL  TAM  TOL  TAM  TOL    0.10  1.00  100.53 ± 1.01  99.79 ± 0.68  1.00  0.68  −0.53  −0.21    0.40  0.40  99.52 ± 0.68  100.90 ± 1.07  0.68  1.06  −0.48  −0.90    0.30  1.50  100.27 ± 0.81  100.05 ± 0.06  0.81  0.06  −0.27  0.05    Grand mean  100.11 ± 0.83  100.25 ± 0.60  0.83  0.60  0.1111  0.25   TAM  SOL  TAM  SOL  TAM  SOL  TAM  SOL    0.10  1.50  99.76 ± 1.49  99.46 ± 0.76  1.49  0.76  −0.24  −0.54    0.50  1.00  199.21 ± 1.11  99.68 ± 0.46  1.12  0.46  −0.79  −0.32    0.60  6.00  199.69 ± 0.40  100. 81 ± 0.71  0.40  0.70  −0.31  −0.81    Grand mean  99.55 ± 1.00  99.98 ± 0.64  1.00  0.64  −0.45  −0.02  (b) Inter-day precision   TAM  TOL  TAM  TOL  TAM  TOL  TAM  TOL    0.10  1.00  99.35 ± 0.92  99.71 ± 0.59  0.93  0.59  −0.65  −0.29    0.40  0.40  99.77 ± 1.57  101.01 ± 1.43  1.57  1.42  −0.23  1.01    0.30  1.50  100.28 ± 1.83  99.49 ± 1.06  1.82  1.07  −0.28  −0.51    Grand mean  99.80 ± 1.44  100.07 ± 1.03  1.44  1.03  −0.20  0.07   TAM  SOL  TAM  SOL  TAM  SOL  TAM  SOL    0.10  1.50  99.53 ± 0.76  100.48 ± 1.84  0.76  1.83  −0.47  −0.48    0.50  1.00  100.71 ± 1.47  99.56 ± 2.00  1.46  2.01  0.71  −0.44    0.60  6.00  101.08 ± 1.53  99.39 ± 2.00  1.51  2.01  1.08  −0.61    Grand mean  100.44 ± 1.25  99.81 ± 1.95  1.24  1.95  0.44  −0.19  Concentration (μg.band−1)  Mean % recovery ± SDa  RSD %b  Er %c  (a) Intra-day precision (repeatability)   TAM  TOL  TAM  TOL  TAM  TOL  TAM  TOL    0.10  1.00  100.53 ± 1.01  99.79 ± 0.68  1.00  0.68  −0.53  −0.21    0.40  0.40  99.52 ± 0.68  100.90 ± 1.07  0.68  1.06  −0.48  −0.90    0.30  1.50  100.27 ± 0.81  100.05 ± 0.06  0.81  0.06  −0.27  0.05    Grand mean  100.11 ± 0.83  100.25 ± 0.60  0.83  0.60  0.1111  0.25   TAM  SOL  TAM  SOL  TAM  SOL  TAM  SOL    0.10  1.50  99.76 ± 1.49  99.46 ± 0.76  1.49  0.76  −0.24  −0.54    0.50  1.00  199.21 ± 1.11  99.68 ± 0.46  1.12  0.46  −0.79  −0.32    0.60  6.00  199.69 ± 0.40  100. 81 ± 0.71  0.40  0.70  −0.31  −0.81    Grand mean  99.55 ± 1.00  99.98 ± 0.64  1.00  0.64  −0.45  −0.02  (b) Inter-day precision   TAM  TOL  TAM  TOL  TAM  TOL  TAM  TOL    0.10  1.00  99.35 ± 0.92  99.71 ± 0.59  0.93  0.59  −0.65  −0.29    0.40  0.40  99.77 ± 1.57  101.01 ± 1.43  1.57  1.42  −0.23  1.01    0.30  1.50  100.28 ± 1.83  99.49 ± 1.06  1.82  1.07  −0.28  −0.51    Grand mean  99.80 ± 1.44  100.07 ± 1.03  1.44  1.03  −0.20  0.07   TAM  SOL  TAM  SOL  TAM  SOL  TAM  SOL    0.10  1.50  99.53 ± 0.76  100.48 ± 1.84  0.76  1.83  −0.47  −0.48    0.50  1.00  100.71 ± 1.47  99.56 ± 2.00  1.46  2.01  0.71  −0.44    0.60  6.00  101.08 ± 1.53  99.39 ± 2.00  1.51  2.01  1.08  −0.61    Grand mean  100.44 ± 1.25  99.81 ± 1.95  1.24  1.95  0.44  −0.19  aMean ± standard deviation of five determinations. bPercentage RSD. cPercentage relative error. Robustness The robustness of the proposed method was evaluated by analyzing TAM, TOL and SOL at three concentration levels as cited in Table III. The parameters studied were mobile phase composition, mobile phase volume, duration of saturation and time from chromatography to scan. It was found that variation in the above-mentioned parameters had no significant influence on the determination of any of the three drugs using the proposed method. After introducing small deliberate changes in the method parameters, low values of RSD% along with nearly unchanged Rf values were obtained indicating the robustness of the developed method (Table IV). Table IV. Evaluation of the Robustness of the Proposed HPTLC Method for the Determination of TAM, TOL and SOL Parameters tested  HPTLCa method  TAM  TOL  SOL  RSD % of peak areas  Rf ± SD  RSD % of peak areas  Rf ± SD  RSD % of peak areas  Rf ± SD  1) Mobile phase composition [methanol:ethyl acetate:ammonia (6:4:0.05, 5.9:4.1:0.05, 6.1:3.9:0.05 v/v)]  0.50  0.61 ± 2.00 × 10–2  1.10  0.47 ± 1.16 × 10–2  1.50  0.21 ± 1.15 × 10–2  [methanol:ethyl acetate:ammonia (6:4:0.05, 6:4:0.1, 6:4:0.15 v/v)]  0.90  0.61 ± 5.77 × 10–3  0.87  0.44 ± 3.00 × 10–2  1.00  0.21 ± 1.00 × 10–2  2) Mobile phase volume [15, 20 and 25 mL]  1.00  0.63 ± 1.20 × 10–2  1.35  0.45 ± 1.53 × 10–2  1.55  0.22 ± 1.00 × 10–2  3) Duration of saturation [30, 40 and 50 min]  1.20  0.63 ± 2.08 × 10–2  1.00  0.44 ± 1.73 × 10–2  1.32  0.22 ± 2.89 × 10–3  4) Time from chromatography to scan [10, 20, 30 and 60 min]  1.25  0.62 ± 1.16 × 10–2  1.55  0.43 ± 2.65 × 10–2  0.70  0.21 ± 2.08 × 10–2  Parameters tested  HPTLCa method  TAM  TOL  SOL  RSD % of peak areas  Rf ± SD  RSD % of peak areas  Rf ± SD  RSD % of peak areas  Rf ± SD  1) Mobile phase composition [methanol:ethyl acetate:ammonia (6:4:0.05, 5.9:4.1:0.05, 6.1:3.9:0.05 v/v)]  0.50  0.61 ± 2.00 × 10–2  1.10  0.47 ± 1.16 × 10–2  1.50  0.21 ± 1.15 × 10–2  [methanol:ethyl acetate:ammonia (6:4:0.05, 6:4:0.1, 6:4:0.15 v/v)]  0.90  0.61 ± 5.77 × 10–3  0.87  0.44 ± 3.00 × 10–2  1.00  0.21 ± 1.00 × 10–2  2) Mobile phase volume [15, 20 and 25 mL]  1.00  0.63 ± 1.20 × 10–2  1.35  0.45 ± 1.53 × 10–2  1.55  0.22 ± 1.00 × 10–2  3) Duration of saturation [30, 40 and 50 min]  1.20  0.63 ± 2.08 × 10–2  1.00  0.44 ± 1.73 × 10–2  1.32  0.22 ± 2.89 × 10–3  4) Time from chromatography to scan [10, 20, 30 and 60 min]  1.25  0.62 ± 1.16 × 10–2  1.55  0.43 ± 2.65 × 10–2  0.70  0.21 ± 2.08 × 10–2  aAverage of three concentrations 0.1, 0.3 and 0.7 μg.band–1 of TAM; 0.5, 1 and 3 μg.band–1 of TOL and 1, 3 and 6 μg.band–1 of SOL. Specificity Laboratory-made mixtures containing different ratios of the two drugs in each mixture was prepared for studying the method specificity. Good percentage recoveries were obtained indicating that they did not interfere with each other (Table III). In addition, the selectivity of the proposed method was demonstrated by applying the proposed method for the analysis of the two mixtures in their drug products, without any interference from the excipients (Table V). Table V. Assay Results for the Determination of TAM, TOL and SOL in their Drug Products Using the Proposed HPTLC Method Drug product Mixture 1 (TAM/TOL)b  Mean % recovery ± RSDa  TAM  TOL  Proposed HPTLC method  Comparative spectrophotometric method (32)  Proposed HPTLC method  Comparative spectrophotometric method (32)  99.05 ± 0.94  100.00 ± 1.00  99.05 ± 1.99  99.28 ± 1.07  tc  1.56  0.23  Fc  1.16  3.63  Drug product Mixture 1 (TAM/TOL)b  Mean % recovery ± RSDa  TAM  TOL  Proposed HPTLC method  Comparative spectrophotometric method (32)  Proposed HPTLC method  Comparative spectrophotometric method (32)  99.05 ± 0.94  100.00 ± 1.00  99.05 ± 1.99  99.28 ± 1.07  tc  1.56  0.23  Fc  1.16  3.63  Drug product Mixture 2 (TAM/SOL)d  Mean % recovery ± RSDa  TAM  SOL  Proposed HPTLC method  Comparative HPLC method (38)  Proposed HPTLC method  Comparative HPLC method (38)  99.89 ± 1.20  100.90 ± 1.49  98.97 ± 1.74  100.79 ± 0.80  tc  1.176  2.14  Fc  1.562  4.51  Drug product Mixture 2 (TAM/SOL)d  Mean % recovery ± RSDa  TAM  SOL  Proposed HPTLC method  Comparative HPLC method (38)  Proposed HPTLC method  Comparative HPLC method (38)  99.89 ± 1.20  100.90 ± 1.49  98.97 ± 1.74  100.79 ± 0.80  tc  1.176  2.14  Fc  1.562  4.51  aMean ± standard deviation of nine determinations. bRoliflo OD-4® Capsules labeled to contain 0.4 mg TAM and 4 mg TOL/capsule. cTheoretical values of t and F are 2.31 and 6.39, respectively, at 95% confidence limit. dVesomni® tablets labeled to contain 0.4 mg TAM and 6 mg SOL/tablet. The specificity of the proposed HPTLC method was also ascertained after peak purity assessment of the three drugs. This was done by the analysis of each drug in standard and sample solutions. The spots for the three drugs in their samples were confirmed by comparing the retardation factor values together with the UV spectra of the spots with those of the standards. The peak purity assessment of the three drugs was done by comparing the spectra at the peak start (S), peak apex (M) and peak end (E) positions of the spot. The calculated values of r(S, M) and r(M, E) of the three drugs were not <0.999 which indicates peaks’ homogeneity (42). Furthermore, the specificity was confirmed by the overlaid UV spectra of each drug in its sample, either tablet or capsule, and standard solutions (Figure 3). Figure 3. View largeDownload slide Spectra illustrating peak purity of (a) TAM, (b) TOL and (c) SOL; each is obtained from corresponding standards and capsules or tablets sample solution. Figure 3. View largeDownload slide Spectra illustrating peak purity of (a) TAM, (b) TOL and (c) SOL; each is obtained from corresponding standards and capsules or tablets sample solution. Solution stability The stability of TAM, TOL and SOL in their both standard and pharmaceutical preparations solutions was investigated. This was done by storing solutions of the three drugs at room temperature for different time intervals (1, 3 and 5 h) before being analyzed by the proposed method. No decomposition of the drugs was observed during development which was confirmed by the absence of any additional peaks in the chromatograms obtained throughout the analysis time together with the nearly unchangeable peak area of the drugs during stability studies (RSD % less than 2%) indicating stability of the drugs solutions for at least 5 h, which was enough for completing the analytical procedure. Spot stability The time for which the sample stands on the plate between spotting and chromatographic development can affect its stability and should be assessed for validation (43). Two-dimensional chromatography was applied to study decomposition occurring during spotting and development. Appearance of peak(s) of decomposition product(s) in both the first and second development directions indicates decomposition during development. Using the proposed conditions, no decomposition was observed during spotting and development indicating stability of the three drugs in their solutions. Analysis of pharmaceutical preparations The applicability of the proposed method was verified by analyzing the two binary mixtures in their pharmaceutical formulations (Table V). The content of the drugs in their dosage forms was calculated by triplicate spotting of solutions of each mixture at three concentration levels. Agreement of the obtained recoveries of all drugs with the labeled claims (as indicated by closeness of the recoveries to 100%) together with their agreement with those obtained with the reported methods (32, 38) show good applicability of the proposed method. The results of both the proposed and the reported methods were statistically compared using the Student’s t-test and the variance ratio F-test. As illustrated in Table V, none of the calculated values exceeds its theoretical one, confirming absence of any significant difference in performance of the compared methods regarding accuracy and precision. Figure 4 illustrates the chromatograms of Roliflo-OD® capsules and Vesomni® tablets where no interfering peaks from common tablet or capsule excipients were observed. Figure 4. View largeDownload slide A typical HPTLC chromatogram of (a) Roliflo OD-4® capsules (0.3 and 3 μg.band–1 of TAM and TOL, respectively) and (b) Vesomni® tablets (0.3 and 4.5 μg.band–1 of TAM and SOL, respectively) using 10-μL band volume. Figure 4. View largeDownload slide A typical HPTLC chromatogram of (a) Roliflo OD-4® capsules (0.3 and 3 μg.band–1 of TAM and TOL, respectively) and (b) Vesomni® tablets (0.3 and 4.5 μg.band–1 of TAM and SOL, respectively) using 10-μL band volume. Discussion Optimization of method parameters is important for the simultaneous determination of the three drugs in their mixtures. Based on minimum tailing and maximum separation of the drugs, different solvent systems were tried. The optimum separation with minimum tailing for the two drugs in each mixture was achieved by using a mobile phase consisting of ethyl acetate–methanol–ammonia (6:4:0.05, v/v). The presence of ammonia in the mobile phase was important to decrease tailing of the spots and to obtain symmetrical peaks for the three drugs. Also, for the selection of optimum scanning wavelength, different wavelengths were tried considering the drugs ratios in their pharmaceutical preparations. The optimum wavelength for detection for all drugs was 224 nm. These optimized chromatographic conditions resulted in good separation of the two drugs in both mixtures with sharp and symmetrical peaks. The developed HPTLC method could be regarded as a beneficial substitute to the other reported HPLC techniques as it involves the use of relatively cheaper instrumentation, lower solvent consumption that ensure higher environmental protection and easier applicability by running several samples simultaneously in the same run which leads to less time and cost of analysis. There are few other previously reported spectrophotometric methods for TAM and TOL simultaneous determination involving the use of long simultaneous equations and calculations to resolve the components of the mixture. On the contrary the proposed HPTLC method offers direct determination of the drugs from their standard calibration curves after the densitometric measurements of the spots without any tedious calculations or previous preparations. Meanwhile, at the end of our study, one HPTLC method for the simultaneous determination of TAM and TOL mixture has been published (37). Although the published HPTLC method was applied for simultaneous determination of TAM and TOL in its combined pharmaceutical preparation, the results were not mentioned (recoveries or SDs) and the results were not compared to a reported method to ensure the selectivity of the proposed method and its real applicability for determination of TAM and TOL in their capsules dosage forms. The published method did not even mention the name of the marketed pharmaceutical preparation of TAM and TOL analyzed nor its source. There is also a huge lack in the results of validation of the published HPTLC method unlike the proposed HPTLC method that was also validated as per ICH guidelines (39). Overall, the proposed work is applicable in the simultaneous quantitation of TAM in its two binary mixtures with TOL and SOL, not only TOL, using the same solvent. The advantage of the proposed method is that all three drugs, TAM, TOL and SOL, were successfully separated from each other. Hence, the two mixtures can be separated in the same run, which allows quality control labs to analyze the two mixtures in their pharmaceutical preparations in a short time with minimal solvent and cost. Conclusion The present method provides simple, rapid, selective, precise, accurate and robust quantitative analysis for the simultaneous determination of TAM and TOL and TAM and SOL in commercial pharmaceutical preparations and content uniformity tests. The major advantage of HPTLC is that several samples can run simultaneously using a small amount of mobile phase, which reduces the time and cost per analysis. Also this method has the advantage of analysing the two mixtures in the same run using the same mobile phase making the method suitable in quality control laboratories especially in developing countries due to its low cost compared to other chromatographic methods (HPLC and GC). 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Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
TI - Novel Validated HPTLC Method for the Analysis of Two Binary Mixtures Containing Tamsulosin Hydrochloride with Antimuscarinic Agents
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmx081
DA - 2018-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/novel-validated-hptlc-method-for-the-analysis-of-two-binary-mixtures-LUR90weMHo
SP - 81
EP - 91
VL - 56
IS - 1
DP - DeepDyve
ER -