TY - JOUR
AU1 - Elonsy, Sohila M
AU2 - El Yazbi, Fawzy A
AU3 - Shaalan, Rasha A
AU4 - Ahmed, Hytham M
AU5 - Belal, Tarek S
AB - Abstract Objective Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). Methods Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 μm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 μm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. Results Method I showed good linearity over the range 10–100 μg/mL for both dugs. Method II’s linear ranges were 0.001–0.1 and 0.02–1 µg/mL for BIS and AML, respectively. Conclusion The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. Highlights To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol. Introduction Hypertension is currently one of the greatest global health care challenges. Although many effective drugs are available, combinations of two or more medications are often required to meet clinical targets. Combination therapy has several advantages over monotherapy: lower doses of each drug can be used to achieve therapeutic goals; lower doses may lead to less side effects, facilitating patient compliance; and using multiple drugs with different modes of action is definitely more effective in treating multifactorial diseases, including hypertension (1). Among combinations of hypertension medications, a cardio-selective β-blocker such as bisoprolol (BIS) with a calcium channel blocker such as amlodipine (AML) is an effective combination therapy for hypertension, with distinct and complimentary modes of action (1, 2). Chemical structures of both drugs are given in Figure 1. Figure 1. Open in new tabDownload slide Chemical structures of amlodipine besylate (AML), bisoprolol fumarate (BIS), and valsartan (VAL). Figure 1. Open in new tabDownload slide Chemical structures of amlodipine besylate (AML), bisoprolol fumarate (BIS), and valsartan (VAL). Simultaneous determination of AML and BIS was addressed in a few reports describing different liquid chromatographic procedures, such as RP-HPLC with UV detection (3, 4), UHPLC for impurity profiling purposes (5), and hydrophilic interaction liquid chromatographic (HILIC) with UV detection (6). In addition, liquid chromatography–tandem mass spectrometry (LC-MS/MS) was implemented in pharmacokinetic studies (7–9). Moreover, UPLC-MS/MS methods were suggested for quantification of AML and BIS along with other antihypertensive drugs for therapeutic drug monitoring purposes or analysis of Chinese patent medicines and health foods containing illegally added chemical hypotensors (10, 11). Alternatively, analysis of AML/BIS combined tablet dosage form was carried out using HPTLC (12) and spectrophotometric methods (13). Generally, if separation using traditional capillary electrophoresis (CE) was not successful by simple variation of buffer pH, micellar electrokinetic capillary chromatography (MEKC) can be considered. In this technique, a suitably charged surfactant, such as sodium dodecyl sulphate (SDS), is added to the buffer in concentrations above its critical micellar concentration (CMC) and micelles will be formed. These act as a pseudo-stationary phase and promote chromatographic separation mechanism to CE (14). On the other hand, both AML and BIS show native fluorescence which was utilized for their quantitation either alone or in mixtures with other drugs in several reports (15–18). To the best of our knowledge, literature review revealed no published article describing electro-chromatographic or liquid chromatographic with fluorescence detection methods for simultaneous determination of AML and BIS. This encouraged us to take the lead and suggest two efficient separation methods: MEKC with diode-array detection (DAD) and UPLC with fluorescence detection for analysis of this fixed dose combination. Experimental Instrumentation MEKC work was done using Agilent CE instrument 7100 series (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) equipped with DAD and a data handling system comprising a computer loaded with Agilent ChemStation Software. The UPLC system is Agilent 1290 series (Agilent Technologies, Santa Clara, CA, USA) consisting of quaternary pump G4204A, autosampler G4226A, thermostatted column compartment TCC G1316C, diode array detector G4212A, and fluorescence detector FLD G7121A connected to a computer loaded with Agilent OpenLAB CDS ChemStation Edition Software. Materials and Chemicals Pure samples of AML and BIS were kindly obtained from Pfizer Egypt S.A.E., Cairo, Egypt and Global Napi Pharmaceuticals, 6th October city, Egypt, respectively. Valsartan (VAL) used as an internal standard (IS) was kindly supplied by Novartis Pharma S.A.E., Cairo, Egypt. Acetonitrile (HPLC grade), Methanol (HPLC grade), and Trifluoroacetic acid were obtained from Sigma-Aldrich Chemie GmbH, Buchs, Switzerland. Analytical grade of boric acid and SDS (Oxford Lab Chem, Mumbai, India), sodium hydroxide (El-Nasr Chemical Ind. Co., Egypt), and high-purity distilled water were used. Pharmaceutical formulation involved in the study is laboratory-prepared tablets (containing 5 mg AML and 5 mg BIS per tablet as in the brand Concor-AM, in addition to magnesium stearate, lactose, starch, and AEROSIL® as tablet excipients). General Procedures (a) Method I: MEKC.—(1) Separation conditions —A deactivated fused silica capillary (Agilent Technologies, Waldbronn, Germany) was used and it had the following dimensions: 33.5 cm total length, 25 cm effective length, and 50 μm internal diameter. Detection was performed using DAD at 238 nm for AML, 224 nm for BIS, and 210 nm for VAL (IS). (2) Preparation of running buffer —A borate buffer (0.01 M, pH 9.2) was prepared by weighing and dissolving 0.062 g of boric acid and 0.02 g of sodium hydroxide in 100 mL of distilled water then pH was checked and adjusted. To prepare 0.025 M of SDS in buffer, 0.722 g of SDS was added to 100 mL of the previously prepared buffer, and then sonicated for 10 min until complete dissolution of SDS powder. The used background electrolyte (BGE) consists of the prepared 0.01 M borate buffer (pH 9.2) containing 0.025 M SDS and HPLC-grade methanol (80:20, v/v). (3) Capillary conditioning —At the beginning of each working day, the capillary was flushed with 0.5 M NaOH for 15 min then with water for 15 min; it then was flushed with 0.1 M NaOH for 5 min, waiting for 2.5 min to make sure of complete activation of the capillary inner wall, washed with water for 5 min, and finally equilibrated with BGE for 10 min. The capillary was rinsed between successive runs with BGE for 1 min. Injections were performed using hydrodynamic mode under pressure of 50 mbar for 13 s. The applied voltage was 30 kV. (4) Preparation of standard solutions and construction of calibration graphs —Stock standard solutions containing 1000 μg/mL of AML, BIS, and VAL (used as IS) were separately prepared in HPLC-grade methanol. Working standard solutions were prepared by transferring proper aliquots of the stock solutions into a set of 10 mL volumetric flasks to reach concentration ranges of 10–100 μg/mL for both BIS and AML. A constant volume of 200 μL of VAL was added giving constant concentration of 20 μg/mL VAL. The flasks were diluted to volume with distilled water, triplicate injections were performed for each solution, and peak area ratios were plotted against the corresponding concentrations to construct calibration graphs. (b) Method II: UPLC-FD.—(1) Chromatographic conditions —The column used was Zorbax SB-C8 (2.1 × 100 mm, 1.8 μm particle size). The mobile phase consisted of 0.1% trifluoroacetic acid (TFA), acetonitrile, and methanol in a ratio of 55:35:10 v/v. The separation was achieved with isocratic elution at a flow rate of 0.6 mL/min. Injection volume was 15 µL. The eluate was monitored by fluorescence detector with excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. All determinations were performed at 25°C. (2) Preparation of standard solutions for construction of calibration graphs —Primary stock solutions containing 100 μg/mL BIS and AML were separately prepared in HPLC-grade methanol. Secondary stock solutions were prepared by dilution of the primary stock solutions with methanol to obtain final concentrations of 1 μg/mL BIS and 10 μg/mL AML. Working standard solutions were prepared by transferring proper aliquots of the secondary stock solutions into a set of 10 mL volumetric flasks, then diluted with the mobile phase to reach concentration ranges of 1–100 and 20–1000 ng/mL for BIS and AML, respectively. Triplicate injections were made for each concentration. The peak areas were plotted against the corresponding concentrations to construct the calibration graphs. Assay of Tablets Dosage Form Twenty laboratory-prepared tablets were weighed, finely powdered, and homogenized. A portion of 15 mL methanol was added to an accurate weight of the powder equivalent to 25 mg BIS and 25 mg AML. The solution was vortexed for 5 min and then filtered into a 25 mL calibrated flask. The residue was washed with 2 × 3 mL methanol, washings were added to the filtrate, and the solution was diluted to volume with methanol. For method I, aliquots of the tablets’ solution were spiked with 200 µL of IS stock, then diluted with distilled water to obtain final concentrations within the specified ranges and finally treated as previously described. For method II, aliquots of the tablets’ solution were diluted with the mobile phase to obtain final concentrations within the specified ranges, and then treated as previously described. In both methods, the recovery values were calculated from the corresponding similarly treated external standards. For standard addition assays, aliquots of BIS and AML standard solutions were added to sample solutions to obtain total concentrations within the previously specified ranges then treated as previously described under General Procedures. Recovery values were obtained by comparing the analyte response with the increment response detected after addition of the standard. Results and Discussion Optimization of Method I (MEKC) Different trials were made to find suitable conditions for the separation of BIS and AML in CE. First, buffers of different pH and concentrations were tested. This included acetate buffer pH 4.7, phosphate buffer pH 7.4, and borate buffer pH 9.2. Then, trying MEKC mode was done by adding SDS above its CMC to the buffer solution. Unfortunately, all the previous trials could not separate BIS and AML peaks as they appeared at the same migration time. This was confirmed by the aid of DAD where both drugs appeared to be co-eluting. Third, modification of MEKC mode by the addition of organic solvent to the BGE was done. Organic modifiers have been found to produce several effects in MEKC. The addition of organic solvent leads to a significant decrease in the BGE velocity due to changes that occur in viscosity and dielectric constant of the separation electrolyte. As the addition of an organic modifier to the MEKC electrolyte improves the wetting of the capillary inner wall, the silica surface is modified, with resulting changes in zeta-potential and, consequently, in electro-osmotic flow (EOF) (19). Introduction of an organic solvent (methanol) to modify MEKC resulted in a good separation between BIS and AML peaks. The separation is affected by several parameters which were tested and optimized. (a)Buffer type and pH.—The effect of pH was tested using different buffers on either CE or MEKC with the addition of organic modifier. It was discovered that pH did not dramatically affect resolution or elution order of the drugs. By comparing a 0.01 M phosphate buffer pH 7.4 and 0.01 M borate buffer pH 9.2, the only observed difference is that the borate buffer showed better baseline shape, peak shape, reproducible, and shorter migration time. Therefore, the selected buffer was the borate buffer with pH 9.2. (b)Buffer concentration.—The effect of buffer concentration was examined by using borate buffer pH 9.2 with different concentrations 0.01, 0.02, and 0.05 M. The study showed that increasing buffer concentration significantly affects migration times of the analyzed drugs, making separation time longer and more tedious. Finally, 0.01 M buffer concentration was selected to reduce analysis time with good resolution and peak shape. (c)SDS concentration.—The effect of SDS concentration on separation was studied by the addition of 0.015, 0.025, or 0.050 M SDS in the presence of 20% methanol in the BGE. It was found that as SDS concentration increases, migration times and resolution increase as well. However, 0.015 M SDS gave significantly tailed peaks, while 0.05 M of SDS resulted in excessively long migration times. Therefore, 0.025 M of SDS was selected for separation as it was the best compromise between peak shape and analysis time. (d) Organic modifier.—The type and amount of organic modifier have great impact on the separation of the studied drugs. Using acetonitrile resulted in deformation in the peak shape of both dugs, therefore it was excluded. Both drugs were co-eluted in one peak in a low amount of methanol ≤ 10% (v/v). On the other hand, upon adding ≥ 25% (v/v), irreproducible results were obtained, as the high content of organic modifier can inhibit micelle formation from monomers. It is generally accepted that micelles are not stable in mixtures of water and organic solvents containing more than 20–30% of organic solvents (19). Therefore, 20% (v/v) of methanol was found optimum. (e)Capillary length.—It is be expected that a shorter capillary would produce faster elution and therefore shorter migration times. By comparing a capillary of 25 cm effective length with that of 50 cm, the shorter capillary led to a significant decrease of run time from 14 min to only 4 min with acceptable resolution of 2.33 between the two peaks. Also, this led to enhanced peak shape especially for BIS as its USP tailing factor improved from 2.79 to 1.32. (f)Applied voltage.—The following values of the applied voltage (15, 20, 25, and 30 kV) were tested using the optimized BGE. The results revealed that by decreasing voltage, migration times increased as expected due to the decrease of the EOF. Despite different migration times upon changing voltage, resolution was unaffected and its value remained around 2.3. Therefore, a voltage of 30 kV was selected to achieve a time-effective separation. (g)Selection of internal standard (IS).—Adding an IS in CE methods is useful to decrease errors that could be due to fluctuation in injection volumes and migration times and to improve quantitative determination (20). VAL was selected as IS (Figure 1). In the selected BGE, it was found well separated from the tested drugs at 1.97 min. (h)Selection of injection time.—In hydrodynamic injection, injection time affects both peak width and height. To determine the optimum injection time, sample solutions were injected at 50 mbar with varying injection times from 3 to 17 s. By increasing injection time, peak height increased; however, further increase of injection time led to peak shape deformation. The optimum injection time was 13 s as it resulted in good peak symmetry and reproducible measurements. (i)Selection of detection wavelengths.—DAD has the ability of developing electropherograms at more than one wavelength for the same run, and each compound could be measured at its maximum absorption wavelength, thus improving sensitivity of the proposed method. Also, DAD confirms peak purity. Therefore, electropherograms were recorded at 238, 224, and 210 nm which corresponded to the maxima of AML, BIS, and VAL (IS), respectively. The proposed method permitted the separation of both studied drugs and IS to be completed in less than 4 min, as shown in the MEKC electropherogram in Figure 2. Migration times were 1.97, 3.14, and 3.43 min for VAL (IS), BIS, and AML, respectively (Table 1). The proposed method proved to have acceptable resolution values not less than 2.33; other system suitability parameters were calculated and found acceptable (Table 1). Figure 2. Open in new tabDownload slide MEKC electropherograms of a standard mixture containing 50 µg/mL of BIS, 50 µg/mL of AML, and 20 µg/mL of VAL (IS) at 224 nm (A) and 238 nm (B). Figure 2. Open in new tabDownload slide MEKC electropherograms of a standard mixture containing 50 µg/mL of BIS, 50 µg/mL of AML, and 20 µg/mL of VAL (IS) at 224 nm (A) and 238 nm (B). Table 1. System suitability parameters for analysis of the BIS and AML mixture using the proposed methods Parameter . Method I: MECK . Method II: UPLC . VAL (IS) . BIS . AML . BIS . AML . tR ± SD, min 1.97 ± 0.02 3.14 ± 0.05 3.43 ± 0.07 0.87 ± 0.05 2.18 ± 0.07 Retention factors, k' 0.81 1.80 2.20 1.16 4.40 Theoretical plates, N 9115 9975 12376 637 2977 USP tailing factor 0.955 1.32 1.09 1.23 1.18 Selectivity, ɑ — 2.22 1.22 — 3.79 Resolution, Rs — 11.23 2.33 — 8.64 Parameter . Method I: MECK . Method II: UPLC . VAL (IS) . BIS . AML . BIS . AML . tR ± SD, min 1.97 ± 0.02 3.14 ± 0.05 3.43 ± 0.07 0.87 ± 0.05 2.18 ± 0.07 Retention factors, k' 0.81 1.80 2.20 1.16 4.40 Theoretical plates, N 9115 9975 12376 637 2977 USP tailing factor 0.955 1.32 1.09 1.23 1.18 Selectivity, ɑ — 2.22 1.22 — 3.79 Resolution, Rs — 11.23 2.33 — 8.64 Open in new tab Table 1. System suitability parameters for analysis of the BIS and AML mixture using the proposed methods Parameter . Method I: MECK . Method II: UPLC . VAL (IS) . BIS . AML . BIS . AML . tR ± SD, min 1.97 ± 0.02 3.14 ± 0.05 3.43 ± 0.07 0.87 ± 0.05 2.18 ± 0.07 Retention factors, k' 0.81 1.80 2.20 1.16 4.40 Theoretical plates, N 9115 9975 12376 637 2977 USP tailing factor 0.955 1.32 1.09 1.23 1.18 Selectivity, ɑ — 2.22 1.22 — 3.79 Resolution, Rs — 11.23 2.33 — 8.64 Parameter . Method I: MECK . Method II: UPLC . VAL (IS) . BIS . AML . BIS . AML . tR ± SD, min 1.97 ± 0.02 3.14 ± 0.05 3.43 ± 0.07 0.87 ± 0.05 2.18 ± 0.07 Retention factors, k' 0.81 1.80 2.20 1.16 4.40 Theoretical plates, N 9115 9975 12376 637 2977 USP tailing factor 0.955 1.32 1.09 1.23 1.18 Selectivity, ɑ — 2.22 1.22 — 3.79 Resolution, Rs — 11.23 2.33 — 8.64 Open in new tab Optimization of Method II (UPLC-FD) A simple isocratic UPLC method coupled with fluorescence detection was developed to be a used for the routine analysis of BIS and AML in their binary mixture. An LC method development aims to achieving acceptable resolution between analytes in reasonable analysis time and with good peak shape. Method development includes several experiments for the optimization of both the stationary and mobile phases. For optimization of the stationary phase, available reversed-phase UPLC columns Zorbax SB-C8 (2.1 × 50 mm, 1.8 μm) and Zorbax SB-C8 (2.1 × 100 mm, 1.8 μm) were tested. Both columns managed to resolve BIS and AML peaks. However, Zorbax SB-C8 (2.1 × 100 mm) became the column of choice for this study as it managed to give sharper peaks with higher column efficiency (N) values of both drugs. Several mobile phases were evaluated using different aqueous phases and organic modifiers. Trials were started by using only acetonitrile or methanol as an organic modifier with 0.1% TFA. Unfortunately, negative results were obtained ranging from peak co-elution or insufficient resolution especially in high percentages of the organic solvent to excessive retention of AML peak to the column with subsequent delayed elution in low percentages. Occasionally, peak deformation and broadening were also observed. Therefore, it was concluded that adding both methanol and acetonitrile as organic modifiers in the mobile phase may be a good choice. On the other hand, a phosphoric acid 0.1% solution was tried as substitution for TFA. This trial resulted in peak broadening especially for AML. A ratio of 55:35:10% (v/v) for TFA 0.1% solution, acetonitrile, and methanol provided the best separation and peak shape with a resolution value of 8.64 between AML and BIS peaks. Flow rate was kept constant at 0.6 mL/min and column temperature was adjusted at 25°C. By optimization of the excitation and emission wavelengths for the fluorescence detector, BIS showed maximum response using 230 nm for excitation and 305 nm for emission, while AML was measured at 370 and 450 nm as excitation and emission wavelengths respectively. The detector offers time programing for λex and λem within the runtime, which is important in the case of analytes with different absorption and fluorescence characteristics. The run was programmed to start with 230 and 305 nm as λex and λem, respectively, for measurement of BIS. At 1.5 min, wavelengths were switched to 370 and 450 for λex and λem, respectively, for the detection of AML. The described chromatographic conditions showed well-defined BIS and AML peaks at about 0.87 and 2.18 min, respectively. Figure 3 shows typical chromatogram for separation of this binary mixture at the selected wavelengths. Resolution and other system suitability parameters were calculated and found acceptable (Table 1). Figure 3. Open in new tabDownload slide UPLC chromatogram of a standard mixture containing 50 ng/mL BIS (measured at λex 230 nm and λem 305 nm) and 500 ng/mL AML (measured at λex 370 nm and λem 450 nm). Figure 3. Open in new tabDownload slide UPLC chromatogram of a standard mixture containing 50 ng/mL BIS (measured at λex 230 nm and λem 305 nm) and 500 ng/mL AML (measured at λex 370 nm and λem 450 nm). Validation of the Developed Methods Validation of the proposed methods was carried out in compliance with the International Conference on Harmonization (ICH) guidelines (21). (a)Linearity and concentration ranges.—Linearity of the described MEKC procedure was verified by analyzing a set of serial concentrations for each compound in the range of 10–100 µg/mL and recording peak area ratios of each drug against the IS. Similarly, linearity of the UPLC procedure was tested by analysis of a set of serial dilutions in ranges of 1–100 and 20–1000 ng/mL for BIS and AML, respectively. The regression equations were generated by least squares treatment of the calibration data. Under the optimized conditions described previously, the measured responses were found to be proportional to concentrations. Table 2 shows linearity data and other statistical parameters including regression equations, concentration ranges, correlation coefficients, standard deviations of the intercept (Sa), slope (Sb), and standard deviations of residuals (Sy/x). Regression calculations show good linearity as evidenced by the correlation coefficient values ≥ 0.9995. In addition, linearity can be tested by the values of RSD, % of the slope (Sb%), which were less than 1%. Analysis of variance (ANOVA) test showed high F values (low significance F) and that reflects the increase in the mean of squares due to regression and the decrease in the mean of squares due to residuals. Table 2. Analytical parameters for determination of the BIS and AML mixture using the proposed methods. Parameter . Method I: MECK . Method II: UPLC . BIS . AML . BIS . AML . Wavelength, nm 224 238 230 / 305 370 / 450 Concentration range 10–100 μg/mL 10–100 μg/mL 1–100 ng/mL 20–1000 ng/mL Intercept, a 0.024 0.046 30.199 −75.452 Saa 0.012 0.011 23.317 18.891 Slope, b 0.025 0.031 103.829 3.321 Sbb 1.97 × 10−4 1.77 × 10−4 0.412 0.033 RSD% of the slope, Sb% 0.788 0.571 0.397 0.994 Correlation coefficient, r 0.9998 0.9999 0.9999 0.9995 Sy/xc 0.018 0.016 46.493 37.534 Fd 16641 31576 63662 9923 Significance F 1.46 × 10−14 1.13 × 10−15 2.35 × 10−20 2.55 × 10−16 LOD 0.937 μg/mL 0.822 μg/mL 0.107 ng/mL 3.587 ng/mL LOQ 3.123 μg/mL 2.740 μg/mL 0.357 ng/mL 11.957 ng/mL Parameter . Method I: MECK . Method II: UPLC . BIS . AML . BIS . AML . Wavelength, nm 224 238 230 / 305 370 / 450 Concentration range 10–100 μg/mL 10–100 μg/mL 1–100 ng/mL 20–1000 ng/mL Intercept, a 0.024 0.046 30.199 −75.452 Saa 0.012 0.011 23.317 18.891 Slope, b 0.025 0.031 103.829 3.321 Sbb 1.97 × 10−4 1.77 × 10−4 0.412 0.033 RSD% of the slope, Sb% 0.788 0.571 0.397 0.994 Correlation coefficient, r 0.9998 0.9999 0.9999 0.9995 Sy/xc 0.018 0.016 46.493 37.534 Fd 16641 31576 63662 9923 Significance F 1.46 × 10−14 1.13 × 10−15 2.35 × 10−20 2.55 × 10−16 LOD 0.937 μg/mL 0.822 μg/mL 0.107 ng/mL 3.587 ng/mL LOQ 3.123 μg/mL 2.740 μg/mL 0.357 ng/mL 11.957 ng/mL a Standard deviation of the intercept. b Standard deviation of the slope. c Standard deviation of residuals. d Variance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Open in new tab Table 2. Analytical parameters for determination of the BIS and AML mixture using the proposed methods. Parameter . Method I: MECK . Method II: UPLC . BIS . AML . BIS . AML . Wavelength, nm 224 238 230 / 305 370 / 450 Concentration range 10–100 μg/mL 10–100 μg/mL 1–100 ng/mL 20–1000 ng/mL Intercept, a 0.024 0.046 30.199 −75.452 Saa 0.012 0.011 23.317 18.891 Slope, b 0.025 0.031 103.829 3.321 Sbb 1.97 × 10−4 1.77 × 10−4 0.412 0.033 RSD% of the slope, Sb% 0.788 0.571 0.397 0.994 Correlation coefficient, r 0.9998 0.9999 0.9999 0.9995 Sy/xc 0.018 0.016 46.493 37.534 Fd 16641 31576 63662 9923 Significance F 1.46 × 10−14 1.13 × 10−15 2.35 × 10−20 2.55 × 10−16 LOD 0.937 μg/mL 0.822 μg/mL 0.107 ng/mL 3.587 ng/mL LOQ 3.123 μg/mL 2.740 μg/mL 0.357 ng/mL 11.957 ng/mL Parameter . Method I: MECK . Method II: UPLC . BIS . AML . BIS . AML . Wavelength, nm 224 238 230 / 305 370 / 450 Concentration range 10–100 μg/mL 10–100 μg/mL 1–100 ng/mL 20–1000 ng/mL Intercept, a 0.024 0.046 30.199 −75.452 Saa 0.012 0.011 23.317 18.891 Slope, b 0.025 0.031 103.829 3.321 Sbb 1.97 × 10−4 1.77 × 10−4 0.412 0.033 RSD% of the slope, Sb% 0.788 0.571 0.397 0.994 Correlation coefficient, r 0.9998 0.9999 0.9999 0.9995 Sy/xc 0.018 0.016 46.493 37.534 Fd 16641 31576 63662 9923 Significance F 1.46 × 10−14 1.13 × 10−15 2.35 × 10−20 2.55 × 10−16 LOD 0.937 μg/mL 0.822 μg/mL 0.107 ng/mL 3.587 ng/mL LOQ 3.123 μg/mL 2.740 μg/mL 0.357 ng/mL 11.957 ng/mL a Standard deviation of the intercept. b Standard deviation of the slope. c Standard deviation of residuals. d Variance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Open in new tab (c)LOD and LOQ.—In accordance to ICH recommendations, LOD is defined as the concentration that has a signal-to-noise ratio of 3:1, while for LOQ the applicable ratio is 10:1. Limit values for both methods were determined and given in Table 1. Indeed, fluorescence detection provides much better sensitivity than traditional UV or DAD detectors, therefore method II permitted measurement of the drugs in the ng/mL range. (d)Precision and accuracy.—The within-day (intra-day) precision and accuracy for the proposed methods were studied at three concentration levels for each compound using three replicate determinations for each concentration within one day. Similarly, the between-days (inter-day) precision was tested by analyzing the same three concentrations for each compound using three replicate determinations repeated on 3 days. The resulted peak areas were recorded and the corresponding concentrations were calculated using regression equations. Within-day and between-day quantification of BIS and AML using the proposed methods showed RSD% values less than 2% (Table 3). These low RSD% values prove acceptable precision of the methods. Similarly, Er% values were less than 2% (Table 3) which confirmed optimum accuracy and performance of the described methods. Table 3. Precision and accuracy for determination of BIS and AML in bulk form using the proposed methods Method . Drug . Type of analysis . Nominal value, μg/mL (method I) ng/mL (method II) . Found ± SD . RSD, % . Er,% . MECK BIS Within-day 20 19.96 ± 0.28 1.40 –0.20 50 49.66 ± 0.93 1.87 –0.68 70 70.09 ± 1.09 1.57 0.13 Between-day 20 20.15 ± 0.38 1.89 0.75 50 49.62 ± 0.79 1.59 –0.76 70 70.28 ± 1.37 1.95 0.40 AML Within-day 20 19.69 ± 0.31 1.57 –1.55 50 49.74 ± 0.57 1.15 –0.52 70 69.50 ± 0.93 1.34 –0.71 Between-day 20 20.05 ± 0.31 1.55 0.25 50 49.86 ± 0.47 0.94 –0.28 70 69.65 ± 0.90 1.29 –0.50 UPLC BIS Within-day 10 9.86 ± 0.04 0.41 –1.40 40 40.38 ± 0.23 0.57 0.95 80 80.34 ± 0.21 0.26 0.43 Between-day 10 9.90 ± 0.17 1.72 –1.00 40 40.76 ± 0.32 0.79 1.90 80 81.46 ± 1.15 1.41 1.83 AML Within-day 50 50.37 ± 0.91 1.81 0.74 300 300.19 ± 2.31 0.77 0.06 800 810.15 ± 6.22 0.77 1.27 Between-day 50 50.32 ± 0.49 0.97 0.64 300 299.39 ± 1.60 0.53 –0.20 800 809.24 ± 4.88 0.60 1.16 Method . Drug . Type of analysis . Nominal value, μg/mL (method I) ng/mL (method II) . Found ± SD . RSD, % . Er,% . MECK BIS Within-day 20 19.96 ± 0.28 1.40 –0.20 50 49.66 ± 0.93 1.87 –0.68 70 70.09 ± 1.09 1.57 0.13 Between-day 20 20.15 ± 0.38 1.89 0.75 50 49.62 ± 0.79 1.59 –0.76 70 70.28 ± 1.37 1.95 0.40 AML Within-day 20 19.69 ± 0.31 1.57 –1.55 50 49.74 ± 0.57 1.15 –0.52 70 69.50 ± 0.93 1.34 –0.71 Between-day 20 20.05 ± 0.31 1.55 0.25 50 49.86 ± 0.47 0.94 –0.28 70 69.65 ± 0.90 1.29 –0.50 UPLC BIS Within-day 10 9.86 ± 0.04 0.41 –1.40 40 40.38 ± 0.23 0.57 0.95 80 80.34 ± 0.21 0.26 0.43 Between-day 10 9.90 ± 0.17 1.72 –1.00 40 40.76 ± 0.32 0.79 1.90 80 81.46 ± 1.15 1.41 1.83 AML Within-day 50 50.37 ± 0.91 1.81 0.74 300 300.19 ± 2.31 0.77 0.06 800 810.15 ± 6.22 0.77 1.27 Between-day 50 50.32 ± 0.49 0.97 0.64 300 299.39 ± 1.60 0.53 –0.20 800 809.24 ± 4.88 0.60 1.16 Open in new tab Table 3. Precision and accuracy for determination of BIS and AML in bulk form using the proposed methods Method . Drug . Type of analysis . Nominal value, μg/mL (method I) ng/mL (method II) . Found ± SD . RSD, % . Er,% . MECK BIS Within-day 20 19.96 ± 0.28 1.40 –0.20 50 49.66 ± 0.93 1.87 –0.68 70 70.09 ± 1.09 1.57 0.13 Between-day 20 20.15 ± 0.38 1.89 0.75 50 49.62 ± 0.79 1.59 –0.76 70 70.28 ± 1.37 1.95 0.40 AML Within-day 20 19.69 ± 0.31 1.57 –1.55 50 49.74 ± 0.57 1.15 –0.52 70 69.50 ± 0.93 1.34 –0.71 Between-day 20 20.05 ± 0.31 1.55 0.25 50 49.86 ± 0.47 0.94 –0.28 70 69.65 ± 0.90 1.29 –0.50 UPLC BIS Within-day 10 9.86 ± 0.04 0.41 –1.40 40 40.38 ± 0.23 0.57 0.95 80 80.34 ± 0.21 0.26 0.43 Between-day 10 9.90 ± 0.17 1.72 –1.00 40 40.76 ± 0.32 0.79 1.90 80 81.46 ± 1.15 1.41 1.83 AML Within-day 50 50.37 ± 0.91 1.81 0.74 300 300.19 ± 2.31 0.77 0.06 800 810.15 ± 6.22 0.77 1.27 Between-day 50 50.32 ± 0.49 0.97 0.64 300 299.39 ± 1.60 0.53 –0.20 800 809.24 ± 4.88 0.60 1.16 Method . Drug . Type of analysis . Nominal value, μg/mL (method I) ng/mL (method II) . Found ± SD . RSD, % . Er,% . MECK BIS Within-day 20 19.96 ± 0.28 1.40 –0.20 50 49.66 ± 0.93 1.87 –0.68 70 70.09 ± 1.09 1.57 0.13 Between-day 20 20.15 ± 0.38 1.89 0.75 50 49.62 ± 0.79 1.59 –0.76 70 70.28 ± 1.37 1.95 0.40 AML Within-day 20 19.69 ± 0.31 1.57 –1.55 50 49.74 ± 0.57 1.15 –0.52 70 69.50 ± 0.93 1.34 –0.71 Between-day 20 20.05 ± 0.31 1.55 0.25 50 49.86 ± 0.47 0.94 –0.28 70 69.65 ± 0.90 1.29 –0.50 UPLC BIS Within-day 10 9.86 ± 0.04 0.41 –1.40 40 40.38 ± 0.23 0.57 0.95 80 80.34 ± 0.21 0.26 0.43 Between-day 10 9.90 ± 0.17 1.72 –1.00 40 40.76 ± 0.32 0.79 1.90 80 81.46 ± 1.15 1.41 1.83 AML Within-day 50 50.37 ± 0.91 1.81 0.74 300 300.19 ± 2.31 0.77 0.06 800 810.15 ± 6.22 0.77 1.27 Between-day 50 50.32 ± 0.49 0.97 0.64 300 299.39 ± 1.60 0.53 –0.20 800 809.24 ± 4.88 0.60 1.16 Open in new tab (e)Robustness.—Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, deliberate variations in method parameters and provides an indication of its reliability during normal usage (21). Robustness of the proposed MEKC method was evaluated by calculating SD and RSD% of both peak area ratios and migration times after slight changes in the experimental parameters. Robustness of the method was performed on a mixture of 50 µg/mL of each drug and 20 µg/mL of IS. The studied parameters include borate buffer concentration (0.01 ± 0.002 M), buffer pH (9.2 ± 0.2), SDS concentration (0.025 ± 0.002 M), percentage (v/v) of methanol (20 ± 2%), and detection wavelength (± 2 nm). Likewise, robustness of the UPLC-FD method was studied by analysis of a mixture of 50 ng/mL BIS and 500 ng/mL AML, then the calculation of SD and RSD% of both peak areas and retention times after slight changes in the experimental parameters. The considered changes include TFA concentration (0.1 ± 0.005%), flow rate (0.6 ± 0.05 mL/min), and excitation and emission wavelengths (±2 nm). In these experiments, analysis was performed in triplicate injections with one deliberate change studied each time. Both methods proved to be robust enough, as the studied changes did not significantly affect peak areas, migration, or retention times of the analyzed drugs, as shown by RSD% values assembled in Table 4. Table 4. Robustness evaluation for analysis of the BIS and AML mixture using the proposed methods Method I: MECK . Parameter . Drug . Peak area ratio ± SD . RSD, % . Migration time ± SD . RSD, % . Buffer concentration ± 0.002 M BIS 1.30 ± 0.02 1.54 3.15 ± 0.01 0.32 AML 1.60 ± 0.01 0.63 3.44 ± 0.05 1.45 Buffer pH ± 0.2 pH unit BIS 1.27 ± 0.02 1.58 3.17 ± 0.03 0.95 AML 1.61 ± 0.02 1.24 3.38 ± 0.06 1.78 SDS concentration ± 0.002 M BIS 1.27 ± 0.02 1.58 3.12 ± 0.02 0.64 AML 1.62 ± 0.03 1.85 3.40 ± 0.03 0.88 %v/v methanol ± 2% BIS 1.28 ± 0.02 1.56 3.02 ± 0.07 2.32 AML 1.62 ± 0.03 1.85 3.32 ± 0.07 2.11 Wavelength ± 2 nm BIS 1.26 ± 0.02 1.59 — AML 1.58 ± 0.03 1.90 — Method II: UPLC-FD Parameter Drug Peak area ± SD RSD,% Retention time ± SD RSD,% TFA concentration ± 0.05 % BIS 5173 ± 9.54 0.18 0.86 ± 0.01 1.16 AML 1575 ± 23.35 1.48 2.14 ± 0.04 1.87 Flow rate ± 0.05 mL/min BIS 5179 ± 33.81 0.65 0.85 ± 0.04 4.71 AML 1587 ± 30.55 1.93 2.11 ± 0.10 4.74 Excitation λ ± 2 nm BIS 5123 ± 75.06 1.47 — — AML 1553 ± 15.28 0.98 — — Emission λ ± 2 nm BIS 5185 ± 24.03 0.46 — — AML 1564 ± 12.66 0.81 — — Method I: MECK . Parameter . Drug . Peak area ratio ± SD . RSD, % . Migration time ± SD . RSD, % . Buffer concentration ± 0.002 M BIS 1.30 ± 0.02 1.54 3.15 ± 0.01 0.32 AML 1.60 ± 0.01 0.63 3.44 ± 0.05 1.45 Buffer pH ± 0.2 pH unit BIS 1.27 ± 0.02 1.58 3.17 ± 0.03 0.95 AML 1.61 ± 0.02 1.24 3.38 ± 0.06 1.78 SDS concentration ± 0.002 M BIS 1.27 ± 0.02 1.58 3.12 ± 0.02 0.64 AML 1.62 ± 0.03 1.85 3.40 ± 0.03 0.88 %v/v methanol ± 2% BIS 1.28 ± 0.02 1.56 3.02 ± 0.07 2.32 AML 1.62 ± 0.03 1.85 3.32 ± 0.07 2.11 Wavelength ± 2 nm BIS 1.26 ± 0.02 1.59 — AML 1.58 ± 0.03 1.90 — Method II: UPLC-FD Parameter Drug Peak area ± SD RSD,% Retention time ± SD RSD,% TFA concentration ± 0.05 % BIS 5173 ± 9.54 0.18 0.86 ± 0.01 1.16 AML 1575 ± 23.35 1.48 2.14 ± 0.04 1.87 Flow rate ± 0.05 mL/min BIS 5179 ± 33.81 0.65 0.85 ± 0.04 4.71 AML 1587 ± 30.55 1.93 2.11 ± 0.10 4.74 Excitation λ ± 2 nm BIS 5123 ± 75.06 1.47 — — AML 1553 ± 15.28 0.98 — — Emission λ ± 2 nm BIS 5185 ± 24.03 0.46 — — AML 1564 ± 12.66 0.81 — — Open in new tab Table 4. Robustness evaluation for analysis of the BIS and AML mixture using the proposed methods Method I: MECK . Parameter . Drug . Peak area ratio ± SD . RSD, % . Migration time ± SD . RSD, % . Buffer concentration ± 0.002 M BIS 1.30 ± 0.02 1.54 3.15 ± 0.01 0.32 AML 1.60 ± 0.01 0.63 3.44 ± 0.05 1.45 Buffer pH ± 0.2 pH unit BIS 1.27 ± 0.02 1.58 3.17 ± 0.03 0.95 AML 1.61 ± 0.02 1.24 3.38 ± 0.06 1.78 SDS concentration ± 0.002 M BIS 1.27 ± 0.02 1.58 3.12 ± 0.02 0.64 AML 1.62 ± 0.03 1.85 3.40 ± 0.03 0.88 %v/v methanol ± 2% BIS 1.28 ± 0.02 1.56 3.02 ± 0.07 2.32 AML 1.62 ± 0.03 1.85 3.32 ± 0.07 2.11 Wavelength ± 2 nm BIS 1.26 ± 0.02 1.59 — AML 1.58 ± 0.03 1.90 — Method II: UPLC-FD Parameter Drug Peak area ± SD RSD,% Retention time ± SD RSD,% TFA concentration ± 0.05 % BIS 5173 ± 9.54 0.18 0.86 ± 0.01 1.16 AML 1575 ± 23.35 1.48 2.14 ± 0.04 1.87 Flow rate ± 0.05 mL/min BIS 5179 ± 33.81 0.65 0.85 ± 0.04 4.71 AML 1587 ± 30.55 1.93 2.11 ± 0.10 4.74 Excitation λ ± 2 nm BIS 5123 ± 75.06 1.47 — — AML 1553 ± 15.28 0.98 — — Emission λ ± 2 nm BIS 5185 ± 24.03 0.46 — — AML 1564 ± 12.66 0.81 — — Method I: MECK . Parameter . Drug . Peak area ratio ± SD . RSD, % . Migration time ± SD . RSD, % . Buffer concentration ± 0.002 M BIS 1.30 ± 0.02 1.54 3.15 ± 0.01 0.32 AML 1.60 ± 0.01 0.63 3.44 ± 0.05 1.45 Buffer pH ± 0.2 pH unit BIS 1.27 ± 0.02 1.58 3.17 ± 0.03 0.95 AML 1.61 ± 0.02 1.24 3.38 ± 0.06 1.78 SDS concentration ± 0.002 M BIS 1.27 ± 0.02 1.58 3.12 ± 0.02 0.64 AML 1.62 ± 0.03 1.85 3.40 ± 0.03 0.88 %v/v methanol ± 2% BIS 1.28 ± 0.02 1.56 3.02 ± 0.07 2.32 AML 1.62 ± 0.03 1.85 3.32 ± 0.07 2.11 Wavelength ± 2 nm BIS 1.26 ± 0.02 1.59 — AML 1.58 ± 0.03 1.90 — Method II: UPLC-FD Parameter Drug Peak area ± SD RSD,% Retention time ± SD RSD,% TFA concentration ± 0.05 % BIS 5173 ± 9.54 0.18 0.86 ± 0.01 1.16 AML 1575 ± 23.35 1.48 2.14 ± 0.04 1.87 Flow rate ± 0.05 mL/min BIS 5179 ± 33.81 0.65 0.85 ± 0.04 4.71 AML 1587 ± 30.55 1.93 2.11 ± 0.10 4.74 Excitation λ ± 2 nm BIS 5123 ± 75.06 1.47 — — AML 1553 ± 15.28 0.98 — — Emission λ ± 2 nm BIS 5185 ± 24.03 0.46 — — AML 1564 ± 12.66 0.81 — — Open in new tab (f)Stability of solutions.—Stability of working solutions was examined and no chromatographic changes were detected within 24 h at room temperature upon application of the described methods. There was no significant change in migration or retentions times and peak areas of the analyzed drugs with RSD% values less than 2%. Additionally, stock solutions prepared in HPLC-grade methanol were found to be stable for at least 2 weeks when kept at 4°C. The calculated concentrations of newly prepared solutions and those aged for 2 weeks were determined using the proposed methods and differences in concentrations were less than 2.0%. Application of the Validated Methods (a)Analysis of synthetic mixtures.—Several synthetic mixtures of both compounds were prepared and analyzed using the proposed validated methods. Drugs were added in different ratios, both above and below the normal ratio expected in the dosage form (1:1). The recovery (found concentrations), RSD%, and Er% values shown in Table 5 were satisfactory, thus validating the applicability, precision, and accuracy of the described methods and demonstrating their ability to resolve and quantify both drugs in different proportions. Table 5. Determination of BIS and AML in laboratory-prepared mixtures using the proposed methods Method I: MECK . Nominal value, μg/mL . Found ± SDa, μg/mL . RSD, % . Er, % . BIS . AML . BIS . AML . BIS . AML . BIS . AML . 50 50 49.44 ± 0.97 49.17 ± 0.91 1.96 1.85 –1.12 –1.66 50 25 49.02 ± 0.71 25.22 ± 0.41 1.45 1.63 –1.96 0.88 25 50 24.74 ± 0.45 49.61 ± 0.96 1.82 1.94 –1.04 –0.78 100 100 99.36 ± 0.98 100.77 ± 0.93 0.99 0.92 –0.64 0.77 50 10 49.14 ± 0.33 9.94 ± 0.15 0.67 1.51 –1.72 –0.60 10 50 9.82 ± 0.14 50.85 ± 0.42 1.43 0.83 –1.80 1.70 Method II: UPLC-FD Nominal value, ng/mL Found ± SDa, ng/mL RSD, % Er, % BIS AML BIS AML BIS AML BIS AML 50 50 50.65 ± 0.23 49.87 ± 0.34 0.45 0.68 1.30 –0.26 100 100 101.78 ± 0.24 100.29 ± 1.16 0.24 1.16 1.78 0.29 50 100 50.51 ± 0.24 101.07 ± 1.21 0.48 1.20 1.02 1.07 100 50 99.83 ± 0.42 50.28 ± 0.33 0.42 0.66 –0.17 0.56 10 100 10.08 ± 0.05 99.45 ± 1.83 0.50 1.84 0.80 –0.55 100 1000 99.13 ± 0.31 999.28 ± 1.74 0.31 0.17 –0.87 –0.07 Method I: MECK . Nominal value, μg/mL . Found ± SDa, μg/mL . RSD, % . Er, % . BIS . AML . BIS . AML . BIS . AML . BIS . AML . 50 50 49.44 ± 0.97 49.17 ± 0.91 1.96 1.85 –1.12 –1.66 50 25 49.02 ± 0.71 25.22 ± 0.41 1.45 1.63 –1.96 0.88 25 50 24.74 ± 0.45 49.61 ± 0.96 1.82 1.94 –1.04 –0.78 100 100 99.36 ± 0.98 100.77 ± 0.93 0.99 0.92 –0.64 0.77 50 10 49.14 ± 0.33 9.94 ± 0.15 0.67 1.51 –1.72 –0.60 10 50 9.82 ± 0.14 50.85 ± 0.42 1.43 0.83 –1.80 1.70 Method II: UPLC-FD Nominal value, ng/mL Found ± SDa, ng/mL RSD, % Er, % BIS AML BIS AML BIS AML BIS AML 50 50 50.65 ± 0.23 49.87 ± 0.34 0.45 0.68 1.30 –0.26 100 100 101.78 ± 0.24 100.29 ± 1.16 0.24 1.16 1.78 0.29 50 100 50.51 ± 0.24 101.07 ± 1.21 0.48 1.20 1.02 1.07 100 50 99.83 ± 0.42 50.28 ± 0.33 0.42 0.66 –0.17 0.56 10 100 10.08 ± 0.05 99.45 ± 1.83 0.50 1.84 0.80 –0.55 100 1000 99.13 ± 0.31 999.28 ± 1.74 0.31 0.17 –0.87 –0.07 a Mean ± standard deviation for five determinations. Open in new tab Table 5. Determination of BIS and AML in laboratory-prepared mixtures using the proposed methods Method I: MECK . Nominal value, μg/mL . Found ± SDa, μg/mL . RSD, % . Er, % . BIS . AML . BIS . AML . BIS . AML . BIS . AML . 50 50 49.44 ± 0.97 49.17 ± 0.91 1.96 1.85 –1.12 –1.66 50 25 49.02 ± 0.71 25.22 ± 0.41 1.45 1.63 –1.96 0.88 25 50 24.74 ± 0.45 49.61 ± 0.96 1.82 1.94 –1.04 –0.78 100 100 99.36 ± 0.98 100.77 ± 0.93 0.99 0.92 –0.64 0.77 50 10 49.14 ± 0.33 9.94 ± 0.15 0.67 1.51 –1.72 –0.60 10 50 9.82 ± 0.14 50.85 ± 0.42 1.43 0.83 –1.80 1.70 Method II: UPLC-FD Nominal value, ng/mL Found ± SDa, ng/mL RSD, % Er, % BIS AML BIS AML BIS AML BIS AML 50 50 50.65 ± 0.23 49.87 ± 0.34 0.45 0.68 1.30 –0.26 100 100 101.78 ± 0.24 100.29 ± 1.16 0.24 1.16 1.78 0.29 50 100 50.51 ± 0.24 101.07 ± 1.21 0.48 1.20 1.02 1.07 100 50 99.83 ± 0.42 50.28 ± 0.33 0.42 0.66 –0.17 0.56 10 100 10.08 ± 0.05 99.45 ± 1.83 0.50 1.84 0.80 –0.55 100 1000 99.13 ± 0.31 999.28 ± 1.74 0.31 0.17 –0.87 –0.07 Method I: MECK . Nominal value, μg/mL . Found ± SDa, μg/mL . RSD, % . Er, % . BIS . AML . BIS . AML . BIS . AML . BIS . AML . 50 50 49.44 ± 0.97 49.17 ± 0.91 1.96 1.85 –1.12 –1.66 50 25 49.02 ± 0.71 25.22 ± 0.41 1.45 1.63 –1.96 0.88 25 50 24.74 ± 0.45 49.61 ± 0.96 1.82 1.94 –1.04 –0.78 100 100 99.36 ± 0.98 100.77 ± 0.93 0.99 0.92 –0.64 0.77 50 10 49.14 ± 0.33 9.94 ± 0.15 0.67 1.51 –1.72 –0.60 10 50 9.82 ± 0.14 50.85 ± 0.42 1.43 0.83 –1.80 1.70 Method II: UPLC-FD Nominal value, ng/mL Found ± SDa, ng/mL RSD, % Er, % BIS AML BIS AML BIS AML BIS AML 50 50 50.65 ± 0.23 49.87 ± 0.34 0.45 0.68 1.30 –0.26 100 100 101.78 ± 0.24 100.29 ± 1.16 0.24 1.16 1.78 0.29 50 100 50.51 ± 0.24 101.07 ± 1.21 0.48 1.20 1.02 1.07 100 50 99.83 ± 0.42 50.28 ± 0.33 0.42 0.66 –0.17 0.56 10 100 10.08 ± 0.05 99.45 ± 1.83 0.50 1.84 0.80 –0.55 100 1000 99.13 ± 0.31 999.28 ± 1.74 0.31 0.17 –0.87 –0.07 a Mean ± standard deviation for five determinations. Open in new tab (b)Assay of tablets dosage form.—The proposed validated methods were applied for the assay of this binary combination in tablet dosage form. Active ingredients were extracted with methanol, and then aliquots were diluted with distilled water (method I) or mobile phase (method II) to give final concentration within the previously mentioned linear ranges. Active ingredients eluted at their defined migration or retention times, and no interfering peaks, were observed from any of the inactive ingredients or dosage form matrix. Recoveries were calculated using both external standard and standard addition methods. Analysis results exposed satisfactory accuracy and precision as indicated from % recovery, SD, and RSD% values (Table 6). Method I enabled peak purity check using the DAD where both drugs showed perfectly pure and homogenous peaks. These results proved that the proposed methods are applicable for the analysis of both drugs in their combined formulations with minimum sample preparation and a satisfactory level of selectivity, accuracy, and precision. Table 6. Application of the proposed methods to the analysis of the BIS–AML mixture in tablet dosage form Laboratory-prepared tablet . Method I: MECK . Method II: UPLC-FD . BIS . AML . BIS . AML . External standard Recovery, % ± SDa 99.80 ± 0.63 99.66 ± 1.49 100.05 ± 1.14 99.92 ± 0.74 RSD% 0.63 1.50 1.14 0.74 Standard addition Recovery, % ± SDa 100.27 ± 0.97 99.98 ± 0.68 101.60 ± 1.36 100.04 ± 1.69 RSD, % 0.97 0.68 1.34 1.69 Laboratory-prepared tablet . Method I: MECK . Method II: UPLC-FD . BIS . AML . BIS . AML . External standard Recovery, % ± SDa 99.80 ± 0.63 99.66 ± 1.49 100.05 ± 1.14 99.92 ± 0.74 RSD% 0.63 1.50 1.14 0.74 Standard addition Recovery, % ± SDa 100.27 ± 0.97 99.98 ± 0.68 101.60 ± 1.36 100.04 ± 1.69 RSD, % 0.97 0.68 1.34 1.69 a Mean ± standard deviation for five determinations. Open in new tab Table 6. Application of the proposed methods to the analysis of the BIS–AML mixture in tablet dosage form Laboratory-prepared tablet . Method I: MECK . Method II: UPLC-FD . BIS . AML . BIS . AML . External standard Recovery, % ± SDa 99.80 ± 0.63 99.66 ± 1.49 100.05 ± 1.14 99.92 ± 0.74 RSD% 0.63 1.50 1.14 0.74 Standard addition Recovery, % ± SDa 100.27 ± 0.97 99.98 ± 0.68 101.60 ± 1.36 100.04 ± 1.69 RSD, % 0.97 0.68 1.34 1.69 Laboratory-prepared tablet . Method I: MECK . Method II: UPLC-FD . BIS . AML . BIS . AML . External standard Recovery, % ± SDa 99.80 ± 0.63 99.66 ± 1.49 100.05 ± 1.14 99.92 ± 0.74 RSD% 0.63 1.50 1.14 0.74 Standard addition Recovery, % ± SDa 100.27 ± 0.97 99.98 ± 0.68 101.60 ± 1.36 100.04 ± 1.69 RSD, % 0.97 0.68 1.34 1.69 a Mean ± standard deviation for five determinations. Open in new tab Comparison with Reported Methods A comparison of the two suggested methods with those reported for analysis of this drug mixture are presented in Table 7. Few methods were previously suggested for assay of combined dosage forms of AML and BIS (3, 4, 6, 12, 13). The proposed methods guaranteed comparable or even much better sensitivity of measurement in terms of linearity ranges than these reported methods. In addition, the proposed separation methods are more specific and reliable than the traditional spectrophotometric methods (13). Table 7. Comparison of the proposed methods with reported methods for determination of the AML and BIS mixture Reference . Applied method . Linearity range . Application . (3) HPLC-UV detection 8–33 μg/mL Assay of tablets (4) HPLC-UV detection — Assay of tablets (5) UHPLC-PDA detection — Impurity profiling (6) HILIC-UV detection 50–150 μg/mL Assay of tablets (7) HPLC-MS/MS 0.2–50 ng/mL Pharmacokinetic study in rats (8) HPLC-MS/MS AML 0.05–15 ng/mL BIS 0.5–75 ng/mL Bioequivalence and pharmacokinetics studies in Chinese subjects (9) HPLC-MS/MS AML 0.1–10 ng/mL BIS 0.5–50 ng/mL Pharmacokinetics study in human male volunteers (10) UPLC-MS/MS AML 1–204 ng/mL BIS 1.6–163 ng/mL Therapeutic drug monitoring for patients undergoing antihypertensive drug treatment (11) UPLC-MS/MS AML 5–400 ng/mL BIS 3–300 ng/mL Determination of illegally added chemical hypotensors in Chinese patent medicines and health foods (12) HPTLC-UV densitometric detection 200–1200 ng/spot Assay of tablets (13) Dual wavelength and absorption-corrected spectrophotometric methods AML 10–30 μg/mL BIS 5–15 μg/mL Assay of tablets This work MEKC-DAD 10–100 μg/mL Assay of tablets HPLC-fluorescence detection AML 20–1000 ng/mL BIS 1–100 ng/mL Reference . Applied method . Linearity range . Application . (3) HPLC-UV detection 8–33 μg/mL Assay of tablets (4) HPLC-UV detection — Assay of tablets (5) UHPLC-PDA detection — Impurity profiling (6) HILIC-UV detection 50–150 μg/mL Assay of tablets (7) HPLC-MS/MS 0.2–50 ng/mL Pharmacokinetic study in rats (8) HPLC-MS/MS AML 0.05–15 ng/mL BIS 0.5–75 ng/mL Bioequivalence and pharmacokinetics studies in Chinese subjects (9) HPLC-MS/MS AML 0.1–10 ng/mL BIS 0.5–50 ng/mL Pharmacokinetics study in human male volunteers (10) UPLC-MS/MS AML 1–204 ng/mL BIS 1.6–163 ng/mL Therapeutic drug monitoring for patients undergoing antihypertensive drug treatment (11) UPLC-MS/MS AML 5–400 ng/mL BIS 3–300 ng/mL Determination of illegally added chemical hypotensors in Chinese patent medicines and health foods (12) HPTLC-UV densitometric detection 200–1200 ng/spot Assay of tablets (13) Dual wavelength and absorption-corrected spectrophotometric methods AML 10–30 μg/mL BIS 5–15 μg/mL Assay of tablets This work MEKC-DAD 10–100 μg/mL Assay of tablets HPLC-fluorescence detection AML 20–1000 ng/mL BIS 1–100 ng/mL Open in new tab Table 7. Comparison of the proposed methods with reported methods for determination of the AML and BIS mixture Reference . Applied method . Linearity range . Application . (3) HPLC-UV detection 8–33 μg/mL Assay of tablets (4) HPLC-UV detection — Assay of tablets (5) UHPLC-PDA detection — Impurity profiling (6) HILIC-UV detection 50–150 μg/mL Assay of tablets (7) HPLC-MS/MS 0.2–50 ng/mL Pharmacokinetic study in rats (8) HPLC-MS/MS AML 0.05–15 ng/mL BIS 0.5–75 ng/mL Bioequivalence and pharmacokinetics studies in Chinese subjects (9) HPLC-MS/MS AML 0.1–10 ng/mL BIS 0.5–50 ng/mL Pharmacokinetics study in human male volunteers (10) UPLC-MS/MS AML 1–204 ng/mL BIS 1.6–163 ng/mL Therapeutic drug monitoring for patients undergoing antihypertensive drug treatment (11) UPLC-MS/MS AML 5–400 ng/mL BIS 3–300 ng/mL Determination of illegally added chemical hypotensors in Chinese patent medicines and health foods (12) HPTLC-UV densitometric detection 200–1200 ng/spot Assay of tablets (13) Dual wavelength and absorption-corrected spectrophotometric methods AML 10–30 μg/mL BIS 5–15 μg/mL Assay of tablets This work MEKC-DAD 10–100 μg/mL Assay of tablets HPLC-fluorescence detection AML 20–1000 ng/mL BIS 1–100 ng/mL Reference . Applied method . Linearity range . Application . (3) HPLC-UV detection 8–33 μg/mL Assay of tablets (4) HPLC-UV detection — Assay of tablets (5) UHPLC-PDA detection — Impurity profiling (6) HILIC-UV detection 50–150 μg/mL Assay of tablets (7) HPLC-MS/MS 0.2–50 ng/mL Pharmacokinetic study in rats (8) HPLC-MS/MS AML 0.05–15 ng/mL BIS 0.5–75 ng/mL Bioequivalence and pharmacokinetics studies in Chinese subjects (9) HPLC-MS/MS AML 0.1–10 ng/mL BIS 0.5–50 ng/mL Pharmacokinetics study in human male volunteers (10) UPLC-MS/MS AML 1–204 ng/mL BIS 1.6–163 ng/mL Therapeutic drug monitoring for patients undergoing antihypertensive drug treatment (11) UPLC-MS/MS AML 5–400 ng/mL BIS 3–300 ng/mL Determination of illegally added chemical hypotensors in Chinese patent medicines and health foods (12) HPTLC-UV densitometric detection 200–1200 ng/spot Assay of tablets (13) Dual wavelength and absorption-corrected spectrophotometric methods AML 10–30 μg/mL BIS 5–15 μg/mL Assay of tablets This work MEKC-DAD 10–100 μg/mL Assay of tablets HPLC-fluorescence detection AML 20–1000 ng/mL BIS 1–100 ng/mL Open in new tab Conclusions This study described two new separation methods for the analysis of the binary combination of amlodipine besylate and bisoprolol fumarate. 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TI - Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate
JF - Journal of AOAC INTERNATIONAL
DO - 10.1093/jaoacint/qsaa136
DA - 2020-09-30
UR - https://www.deepdyve.com/lp/oxford-university-press/application-of-mekc-and-uplc-with-fluorescence-detection-for-GeueyIqSwK
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -