Journal of Chromatographic Science 2014;52:1255– 1266 doi:10.1093/chromsci/bmt208 Advance Access publication February 9, 2014
Article
Sensitive and Robust Methods for Simultaneous Determination of Beclomethasone Dipropionate and Formoterol Fumarate Dihydrate in Rotacaps Vijaykumar K. Parmar*, Hetvi N. Patel and Bhavin K. Patel Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Ta. Petlad, Dist. Anand, Changa 388421, Gujarat, India *Author to whom correspondence should be addressed. E-mail:
[email protected];
[email protected] Received 31 May 2013; revised 8 December 2013
Fixed dose combination containing beclomethasone dipropionate (BDP) and formoterol fumarate dihydrate (FFD) is used in the treatment of asthma in form of dry powder inhaler. Two methods are described for the simultaneous determination of BDP and FFD in commercial rotacap formulation. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 220 nm. The separation was carried out on Merck HPTLC aluminum sheets precoated with silica gel 60F254 using hexane:ethyl acetate:methanol:formic acid (2.0:2.5:2.0:0.2, v/v/v/v) as mobile phase. The linearity was found to be in the range of 2.4 –8.4 mg/spot and 80–280 ng/spot for BDP and FFD, respectively. The second method was based on HPLC separation of the two ˚ drugs on the reversed phase Enable HPLC Analytical C18 G 120A (250 3 4.6 mm, 5 mm) column at ambient temperature using a mobile phase consisting of methanol:acetonitrile:phosphate buffer adjusted to pH 3.6 using orthophosphoric acid (65:25:10, v/v/v). Quantitation was achieved with UV detection at 220 nm based on peak area with linear calibration curves at concentration ranges of 10–200 and 0.3 –6.0 mg/mL for BDP and FFD, respectively. Both methods were validated in terms of precision, robustness, recovery and limits of detection and quantitation. The robustness of both methods was assessed using experimental design and results were analyzed by statistical and graphical approaches. Rotacaps formulation containing BDP (200/400 mg) and FFD (6 mg) were successfully quantified using the proposed methods. The proposed methods can be used as sensitive, precise, accurate and robust methods for quantification of BDP and FFD in Rotacaps.
Introduction Asthma is the commonest chronic disease in children in economically developed countries and is also common in adults (1). It is increasing in prevalence and severity. Asthma is associated with airway inflammation, airway hyperreactivity and acute bronchial constriction (2). The drugs used most commonly in the treatment of asthma, b2 adrenergic receptor agonists and glucocorticoids, have potentially serious side effects when delivered systemically (3). The aerosol delivery of these drugs produces a high local concentration in the lungs with a low systemic delivery, thereby significantly minimizing systemic side effects. Aerosol delivery in the forms of nasal sprays, metered-dose inhalers (MDI), dry powder inhalers (DPI) and nebulizers, are commonly used for treatment of asthma and chronic obstructive pulmonary disease (COPD) (4). The most of the aerosol products for asthma have been launched in combination like
b2-adrenoreceptor with corticosteroids for prolonged duration of action, more effectiveness and quick relief. A significant synergistic therapeutic effect can be obtained in the treatment of asthma by using a fixed dose combinations containing formoterol fumarate dihydrate (FFD) (6 mg) and beclomethasone dipropionate (BDP) (200/400 mg) in form of dry powder inhaler (5). BDP, 9a-chloro-11b-hydroxy-16b-methyl-3,20-dioxopregna1,4-diene-17,21-diyldipropionate (Figure 1a), is a glucocorticoid steroid (6). The anti-inflammatory effects of BDP in asthma include modulation of cytokine and chemokine production; inhibition of eicosanoid synthesis; marked inhibition of accumulation of basophils, eosinophils and other leukocytes in lung tissue; and decreased vascular permeability (2). The HPLC methods are reported for assay of BDP in bulk in Indian Pharmacopoeia (7), British Pharmacopoeia (8) and European Pharmacopoeia (9). BDP inhaler formulation is official in Indian Pharmacopoeia mentioning HPLC method for the assay of BDP. Dhudashia et al. reported stability indicating HPLC method for estimation of BDP from cream (10). The UPLC (11), HPLC (12) and spectrophotometric (13) methods were reported for simultaneous estimation of BDP and Salbutamol in bulk and Rotacaps. Also determination of BDP in biological fluids by liquid chromatography–mass spectrometry (14) and HPLC (15) methods has been reported. FFD, (RS)-20 -hydroxy-50 -[(RS)-1-hydroxy-2 [[(RS)p-methoxya-methylphenethyl]amino]ethyl] formanilide (Figure 1B), is a long-acting b2-agonist (6). It is used in the management of asthma and/or COPD (4). Inhaled formoterol works like other b2-agonists, causing bronchodilatation by relaxing the smooth muscle in the airway so as to treat the exacerbation of asthma (2). Potentiometric (7– 9), spectrophotometric (16, 17) and HPLC (18, 19) methods are reported in the literature for determination of FFD in bulk and pharmaceutical formulation. Several methods are reported for simultaneous determination of components of fixed dose combination product containing FFD and glucocorticoids such as fluticasone, budenoside and ciclesonide in the form of pulmonary product (20 –23). Determination of formoterol in urine by liquid chromatography –tandem mass spectrometry is also reported (24). From the literature survey, it was found that there is no known method available for the simultaneous determination of these two drugs in the commercially available formulation. Therefore, it was thought of interest to develop analytical methods for the simultaneous determination of BDP and FFD in Rotacaps. Two methods, HPTLC and HPLC, were developed and validated for determination of both drugs from marketed formulation.
# The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email:
[email protected]
Figure 1. Structure of (A) BDP and (B) FFD.
An experimental design approach was used for determination of robustness of the proposed methods as a part of an overall method validation strategy. The robustness testing is generally performed by evaluating the effects of individual factors by varying one variable at a time. The use of experimental design helps to predict the possible interactions between the factors with limited number of experiments. The design of experiment (DOE) approach supports ICH guidelines of applying quality by design (QbD) to predict the parameters negatively affecting the quality of product (25). The proposed methods are found to be sensitive and suitable for routine quality control of formulation containing BDP and FFD.
Experimental Instrumentation The HPTLC system (Camag Sonnenmattstr, Mutenz, Switzerland) consisting of a Linomat V semi-automatic spotting device, a glass twin-trough TLC chamber (20 10 cm), a TLC scanner-IV, a data station with winCATS (V 1.4.7) software and an HPTLC syringe (100 mL capacity; Hamilton Company, NV, USA) was used for thin layer chromatographic studies. The HPLC system (Shimadzu Corporation, Analytical Instruments Division, Kyoto, Japan) consisting of a Rheodyne syringe loading sample injector (20 mL), an LC-AT20 solvent delivery module, SPD M20A PDA detector, CTO-10AS column oven, LC solution work station was used for LC separation. The chromatographic separation was accomplished on an Enable HPLC Analytical C18 G 120 A˚ (250 4.6 mm, 5 mm) column protected with a guard column of the same stationary phase.
Chemicals BDP (Batch no. BMD 0160811) was procured as gift sample from Tripda Biotech Private Limited, Ahmedabad, India. FFD (Batch no. FF-0020610) was supplied as gratis sample from Cadila Healthcare Ltd., Ahmedabad, India. Drug substances were used without further purification and certified to contain 99.89% (w/w) purity for BDP and 99.20% (w/w) purity for FFD on dried basis. Acetonitrile, ethyl acetate, formic acid, hexane, methanol, orthophosphoric acid and potassium dihydrogen orthophosphate were procured from Loba Chemicals, Mumbai, India. The HPLC grade chemicals and reagents were used for HPLC method. Glass distillation assembly from Durga scientific, Vadodara was used to prepare triple distilled water. Marketed formulation containing BDP and FFD were procured from local market. 1256 Parmar et al.
Chromatographic conditions HPTLC method Separation was performed on precoated silica gel aluminium plate 60 F-254 (20 10 cm) with 250 mm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologists (Mumbai). The TLC plate was prewashed with methanol and dried in an oven at 1108C for 5 min. Samples were spotted on the TLC plate in the form of band leaving 10 mm from the bottom edge using Linomat V semi-automatic spotter and analyzed using following parameters: bandwidth, 6 mm; track distance, 10 mm; spraying rate, 150 nL/s; volume of mobile phase, 6.5 mL; temperature, 27 + 18C; chamber saturation time, 10 min; migration distance, 70 mm; slit dimension, 4.00 0.30 mm; scanning speed, 20 mm/s; detection wavelength, 220 nm. Mobile phase consisted of hexane:ethyl acetate:methanol:formic acid (2.0:2.5:2.0:0.2, v/v/v/v). HPLC method The mobile phase consisted of methanol:acetonitrile:phosphate buffer adjusted to pH 3.6 with orthophosphoric acid (65:25:10, v/v/v). Samples were analyzed using the following parameters: flow rate, 1.0 mL/min; injection volume, 20 mL; run time, 6 min; temperature, 27 + 28C; detection wavelength, 220 nm.
Preparation of stock solutions Accurately weighed 100 mg of BDP was transferred to 100 mL volumetric flask, dissolved and diluted up to the mark with methanol to get BDP stock solution containing 1 mg/mL of BDP. Accurately weighed 100 mg of FFD was transferred to 100 mL volumetric flask, dissolved and diluted up to the mark with methanol to get FFD stock solution containing 1 mg/mL of FFD.
Preparation of calibration curve For HPTLC method The combined working standard solution of BDP and FFD was prepared by diluting the stock solutions with methanol to prepare mixture of 300 mg/mL of BDP and 10 mg/mL of FFD. Aliquots of 8, 12, 16, 20, 24 and 28 mL of working standard solution (corresponding to 2.4, 3.6, 4.8, 6, 7.2, 8.4 mg/spot and 80, 120, 160, 200, 240, 280 ng/spot for BDP and FFD, respectively) were spotted on a TLC plate and analyzed. Calibration curve was
prepared by plotting peak area of BDP and FFD against their respective concentration. For HPLC method The working standard solutions of BDP and FFD were prepared by diluting stock solutions with mobile phase to prepare 400 mg/mL of BDP and 20 mg/mL of FFD solutions. Aliquots (0.25, 0.50, 1.0, 2.0, 4.0 and 5.0 mL) from working standard solution of BDP (400 mg/mL) and aliquots (0.15, 0.3, 0.6, 1.2, 2.4 and 3.6 mL) from working standard solution of FFD (20 mg/mL) were diluted up to 10 mL with mobile phase to prepare mixture of calibration standard solutions corresponding to 10, 20, 40, 80, 160 and 200 mg/mL of BDP and 0.3, 0.6, 1.2, 2.4, 4.8, 6.0 mg/mL of FFD. The calibration standard solutions were analyzed by the proposed procedure. Calibration curve was prepared by plotting peak area of BDP and FFD against their respective concentration. Sample preparation For assay To determine the content of BDP and FFD simultaneously in marketed formulations (formulation A: labeled claim: 200 mg BDP and 6 mg FFD in each capsule; Formulation B: labeled claim: 400 mg BDP and 6 mg FFD in each capsule), the 20 capsules were weighed and emptied. The capsule powder was transferred to 25 mL volumetric flask. Five milliliters of methanol was added. The capsule shells were rinsed twice with methanol (5 mL), dried and weighed. The methanolic extract was transferred to volumetric flask containing capsule powder. The solution was sonicated for 25 min, diluted up to the mark with methanol and filtered using 0.45 mm filters (Millipore, Milford, MA, USA) to get the sample solution containing 160 mg/mL of BDP and 4.8 mg/mL of FFD for formulation A and 320 mg/mL of BDP and 4.8 mg/mL of FFD for formulation B. For content uniformity Ten capsules were taken for content uniformity test. Each capsule was weighed and emptied. The content of each capsule was transferred to a series of 10 mL volumetric flasks. The capsule shells were rinsed with methanol and the rinsing solution was transferred to volumetric flask. The volume was made up to the mark with methanol. The mixtures were sonicated for 25 min. The resulting solution was filtered using 0.45 mm filters (Millipore, Milford, MA, USA) to get the sample solution containing 20 and 0.6 mg/mL of BDP and FFD (for formulation A) and 40 and 0.6 mg/mL of BDP and FFD (for formulation B), respectively. Validation of the proposed methods Both the methods were validated in compliance with ICH guidelines (26, 27). The following parameters were validated.
Specificity The specificity of the methods was ascertained by analyzing standard drug and sample solutions. HPTLC method The spot for BDP and FFD in sample solution prepared from marketed formulation was confirmed by comparing the Rf and
absorbance/reflectance spectrum with that of standard BDP and FFD. The peak purity of BDP and FFD was assessed by correlating the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot. HPLC method The chromatograms of standard and sample solutions containing BDP and FFD were compared to determine the specificity of the HPLC method. The retention time of BDP and FFD was found to check selectivity of the method. The PDA ( photo diode array) spectrum of drug peaks obtained from sample and standard solutions were compared. Peak purity analysis was performed using LC solution software. The specificity was further determined by the complete separation of BDP and FFD along with other parameters like retention time (Rt), capacity factor (k), tailing or asymmetrical factor (T), etc. Linearity Standard stock solution of the drug was diluted to prepare linearity standard solutions containing BDP and FFD in the concentration range of 2.4 –8.4 mg/spot, 80 –280 ng/spot and 10 – 200 mg/mL, 0.3 –6.0 mg/mL for HPTLC and HPLC method, respectively. Six sets of such solutions were prepared. Each set was analyzed to plot a calibration curve. Standard deviation (SD), slope, intercept and coefficient of determination (r 2) of the calibration curves were calculated to ascertain linearity of the method. Precision The precision is measure of either the degree of reproducibility or repeatability of analytical method. It is indication of random error. The precision of analytical method is expressed as a SD, relative standard error or coefficient of variance of series of measurement. HPTLC method Repeatability of measurement of peak area was carried out by repeated scan of the same spot (4.8 mg/spot of BDP and 160 ng/ spot of FFD) seven times without changing the plate position. The % RSD for peak area was calculated. Repeatability of sample application is based on seven-time application of combined standard solution. The % RSD for peak area was computed. Variations of results within same day (intraday precision) and among days (interday precision) are called as reproducibility. The intraday precision (% RSD) was determined by analyzing standard solution of BDP and FFD for three times on the same day. The interday precision (% RSD) was determined by analyzing standard solution of BDP and FFD for 5 days. The intra- and interday variation for determination of BDP and FFD was carried out at three different concentration levels 3.6, 4.8, 6.0 mg/spot of BDP and 120, 160, 200 ng/spot of FFD. HPLC method System suitability tests are used to verify that resolution and reproducibility of chromatography. It was performed by injecting 20 mL of standard solution, containing 200 mg/mL of BDP and 6 mg/mL of FFD, six times. Resolution (R), column efficiency (N), tailing factor (T) and precision of injection repeatability were analyzed. Sensitive and Robust Methods for BDP and FFD 1257
The intraday precision (% RSD) was determined by analyzing standard solution of BDP and FFD for three times on the same day. The interday precision (% RSD) was determined by analyzing standard solution of BDP and FFD for 5 days. The intra- and interday variation for determination of BDP and FFD was carried out at three different concentration levels 40, 80, 160 mg/mL of BDP and 1.2, 2.4, 4.8 mg/mL of FFD. Recovery studies The accuracy was determined by the standard addition method. To a fixed amount of pre-analyzed sample of BDP and FFD, increasing amount of standard BDP and FFD solution was added. HPTLC method Accuracy of an analysis is determined by calculating systemic error involved. The recovery studies were carried out by applying the method to drug sample to which known amount of BDP and FFD corresponding to 80, 100 and 120% of label claim had been added. At each level of the amount, three determinations were performed and the results obtained were compared with expected results. HPLC method Recovery study was performed by addition of known amounts of standard drugs to pre-analyzed commercial pharmaceutical product sample at three different concentration levels of drug (50, 100 and 150%). Along with sample solution of BDP (40 mg/mL) and FFD (1.2 mg/mL) working standard solution of BDP and FFD at each level were added to prepare mixtures of BDP (6080, 100 mg/mL) and FFD (1.8, 2.4, 3.0 mg/mL). The experiment was repeated three times. Robustness Robustness testing was performed by experimental design approach (28 –31). Plackett –Burman (PB) designs for testing of seven factors are the most commonly used designs for robustness testing of chromatographic methods. The proposed HPTLC method was tested for robustness using PB design with eight experiments. Seven HPTLC conditions were screened: (A) change in amount of hexane in mobile phase composition, (B)
change in amount of ethyl acetate in mobile phase composition, (C) change in amount of methanol in mobile phase composition, (D) change in saturation time, (E) change in detection wavelength, (F) change in bandwidth and (G) change in solvent run distance (Table Ia). Robustness of HPLC method was determined using two-level 24 – 1 fractional factorial design. The generator for this design, that has resolution IV, was D ¼ ABC. The procedure-related factors examined were: (P) change in mobile phase composition, (Q) change in pH of mobile phase, (R) change in flow rate and (S) change in detection wavelength (Table Ib). The DOEþþ (Reliasoft Corporation, AZ, USA; ver 1.0.7) and Design of Experiment (Stat Ease, MN, USA; ver 8.0.1) software were used to set up the experimental designs for HPTLC and HPLC methods, respectively. The % recoveries and Rf values for BDP and FFD were observed at each experiment designed for the HPTLC method, whereas % recoveries and resolution between FFD and BDP were observed for the HPLC method. The experiment was repeated three times. The experiments were executed in random order. The significance of the factor effects was determined statistically, using error estimates in the calculation of critical effects, and graphically, by means of half-normal plots and Pareto charts.
Limit of detection and limit of quantitation The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. The limit of detection (LOD) and limit of quantitation (LOQ) were separately determined at a signal-to-noise (S/N) ratio of 3 and 10. LOD and LOQ were experimentally verified by diluting known concentrations of BDP and FFD until the average responses were 3 or 10 times the SD of the responses for six replicate determinations.
Table I Factors and Their Levels for Robustness Testing of HPTLC and HPLC Methods Factors
Levels (2)
Nominal (0)
(þ)
(a) HPTLC method (A) Change in amount of hexane in mobile phase composition (mL) (B) Change in amount of ethyl acetate in mobile phase composition (mL) (C) Change in amount of methanol in mobile phase composition (mL) (D) Change in saturation time (min) (E) Change in detection wavelength (nm) (F) Change in bend width (mm) (G) Change in solvent run distance (cm)
1.8 2.25 1.8 9 219 4 6.5
2.0 2.50 2.0 10 220 6 7.0
2.2 2.75 2.2 11 221 8 7.5
(b) HPLC method (P) Change in amount of methanol in mobile phase composition (mL) (Q) Change in pH of mobile phase (R) Change in detection wavelength (nm) (S) Change in Flow rate (mL/min)
62 3.5 219 0.95
65 3.6 220 1.00
68 3.7 221 1.05
1258 Parmar et al.
Analysis of marketed formulations Assay
HPTLC method. Sample solution (25 mL) was spotted on TLC plate in triplicate. The plate was developed and analyzed. The peak area of the spots were measured for BDP and FFD and their concentrations in the samples were determined using multilevel calibration curves developed on the same plate under the same conditions using linear regression equation. HPLC method. From formulation A sample solution, a 20-mL volume of solution (4.8 and 160 mg/mL of FFD and BDP, respectively) was chromatographed in HPLC system, three times. The peak area of the BDP and FFD was measured at 220 nm, and their concentrations in the samples were determined using linear regression equation. From formulation B sample solution, 2 mL was pipette out to 10 mL volumetric flask and dilute up to the mark with mobile phase to get concentration 32 and 0.96 mg/mL of BDP and FFD, respectively. A 20-mL volume of the resulting solution was injected in HPLC system, three times. The peak area of the BDP and FFD was measured at 220 nm and their concentrations in the samples were determined using linear regression equation. Content uniformity A 20 mL volume of the sample solution for content uniformity was injected in HPLC system, three times, and analyzed. The peak area of the BDP and FFD was measured at 220 nm and their concentrations in the samples were determined using linear regression equation. The content uniformity for the marketed formulations was tested as per USP guidelines (32). Results Chromatographic separation HPTLC method Various solvent systems composed of toluene, methanol, ethyl acetate, hexane or mixture thereof were tried for optimization of mobile phase for HPTLC separation of BDP and FFD. But the best resolution and symmetrical peak shapes were achieved using mobile phase system consisting of hexane:ethyl acetate: methanol:formic acid (2.0:2.5:2.0:0.2 v/v/v/v). The Rf values were found to be 0.36 and 0.68 for FFD and BDP, respectively. The comparison of densitograms of standard drug solution and sample solution from formulation showed identical Rf values, i.e., 0.68 for BDP and 0.36 for FFD (Figure 2). Comparison of the spectra scanned at peak start (S), peak apex (M) and peak end (E) positions of individual spots of BDP and FFD obtained from sample solution showed a high degree of correlation (.0.99), confirmed the purity of the corresponding spots (Supplementary Figure 1). The spectrum of individual drug was also correlated with spectrum of standard BDP and FFD. The correlation obtained was 0.9998 and 0.9991 for BDP and FFD, respectively, confirmed the identity of the spots. HPLC method The mobile phase system consisting of methanol:acetonitrile: water (65:25:10, v/v/v) adjusted to pH 3.6 showed good separation, good peak shape and repeatable result. The elution
of both drugs from HPLC column was achieved at 1.69 min for FFD and 4.78 min for BDP (Figure 3). The results of the system suitability test (Table II) shows that the optimized chromatographic conditions are adequate for simultaneous determination. The resolution between BDP and FFD peaks was found to be 17.45. Chromatograms of standard drug solution and sample solution were compared and it showed identical retention times for both drugs (Figure 3). The peak purity indices for both drugs from standard and sample solution were found to be .0.999.
Method validation HPTLC method Representative calibration curve of BDP and FFD was obtained by plotting the peak area of BDP and FFD against concentration over the range of 2.4 –8.4 mg/spot and 80 –280 ng/spot, respectively. They were found to be linear over above-mentioned range with correlation coefficients of 0.9979 + 0.001 for BDP and 0.9978 + 0.0006 for FFD. The linear regression equations were y ¼ 1907.8x þ 2822.2 and y ¼ 13.21x 2 73.794 for BDP and FFD, respectively. The repeatability of measurement of peak area and sample application were expressed in terms of % RSD and were found to be 0.88, 0.82 and 1.47, 1.33 for BDP and FFD, respectively. The % RSD for intraday precision (Table III) was found to be 0.17 –0.85 and 0.17 –0.48 for BDP and FFD, respectively. The % RSD for interday precision (Table III) was found to be 0.32 –1.62 and 0.72–1.19 for BDP and FFD, respectively. The proposed HPTLC method when used for recovery studies for BDP and FFD from pharmaceutical formulation after spiking with additional standard drug afforded recovery between 98– 102% and mean recoveries for BDP and FFD from the marketed formulation are listed in Table IV. The LOQs and LODs were found to be 0.44 mg/spot, 0.14 ng/spot for BDP and 10.94 ng/spot, 3.61 ng/spot for FFD, respectively. The total eight experimental plans of PB design for robustness testing and the corresponding responses are summarized in Table V. The margin of errors (ME) or the critical effects were calculated for each response using algorithm of Dong (30). The values for ME are shown in Table V. Pareto charts evaluation of robustness data are depicted in Figure 4. The solution stability and spot stability studies were performed for the HPTLC method. Solutions of BDP (160 ng/mL) and FFD (4.8 ng/mL) were prepared from sample solution and stored at room temperature for 0.5, 1.0, 2.0, 4.0, 8.0 and 24 h, respectively. They were then applied on the TLC plate, after development of the densitogram was evaluated for additional spots if any. There was no indication of compound instability in the sample solution. The time the sample is left to stand on the solvent prior to chromatographic development can influence the stability of separated spots and are required to be investigated for validation. Two-dimensional chromatography using same solvent system was used to find out any decomposition occurring during spotting and development. In case, if decomposition occurs during development, peak(s) of decomposition product(s) shall be obtained for the analyte both in the first and second direction of the run. No decomposition was observed during spotting and development. Sensitive and Robust Methods for BDP and FFD 1259
Figure 2. Densitogram of (A) standard BDP (3.6 mg/spot, Rf: 0.68 + 0.02) and FFD (120 ng/spot, Rf: 0.36 + 0.02), (B) sample BDP (3.6 mg/spot, Rf: 0.68 + 0.02) and FFD (120 ng/spot, Rf: 0.36 + 0.02) measured at 220 nm, mobile phase hexane–ethyl acetate– methanol–formic acid (2.0:2.5:2.0:0.2, v/v/v/v).
HPLC method BDP and FFD showed good correlation coefficient in the concentration range of 10 –200 mg/mL (r ¼ 0.9980 + 0.001) and 0.3 –6.0 mg/mL (r ¼ 0.9976 + 0.0005) for the HPLC method, respectively. The linear regression equations were y ¼ 13,329x þ
1260 Parmar et al.
48,850 and y ¼ 54,558x þ 7,030 for BDP and FFD, respectively. The SD for slope and intercept calculated was 132.07 and 3742.65 for BDP and 397.94 and 806.68 for FFD, respectively. Intraday precision (%RSD, n ¼ 3) was found to be 0.21 –0.88 and 0.16– 0.95 for BDP and FFD, respectively. Interday precision
Rf 0.36 (for FFD) were observed in the densitogram of the drug samples extracted from capsules (Figure 2B). There was no interference from the excipients commonly present in the capsules. The HPLC chromatogram of the drug samples extracted from capsule showed peaks at retention time of 4.78 min (for BDP) and 1.68 min (for FFD) (Figure 3B). The HPLC method was further applied for content uniformity testing of marketed formulation. The steps of the test were adopted according to the USP procedure (32). The acceptance value (AV) was calculated for each of the Rotacaps and was found to be smaller than the maximum allowed acceptance value (LI). Comparison between HPLC and HPTLC methods Sample solutions were analyzed simultaneously by HPTLC and HPLC methods. Each sample was analyzed in triplicate. The results of both methods were compared by paired t-test. The data were treated as paired data. The statistical data are shown in Table VII.
Discussion By reviewing the literature in hand, it was found that no chromatographic methods were published for the simultaneous determination of binary mixture of BDP and FFD. Therefore, the aim of this work was to develop and validate chromatographic methods for simultaneous determination of the cited drugs.
Optimization of procedure Figure 3. HPLC chromatograms of (A) sample solution of BDP (160 mg/mL, Rt: 4.656 min) and FFD (4.8 mg/mL, Rt: 1.664 min), (B) working standard solution of BDP (160 mg/mL, Rt: 4.655 min) and FFD (4.8 mg/mL, Rt: 1.663 min) at 220 nm; mobile phase:methanol:acetonitrile: 25 mM phosphate buffer, pH 3.6, adjusted with orthophosphoric acid in ratio of 65:25:10 (v/v/v).
(%RSD, n ¼ 3) was found to be 1.10– 1.24 and 0.84 –1.25 for BDP and FFD, respectively. The intra- and interday precision result is depicted in Table III. The accuracy of the method was evaluated by recoveries of the added sample. The results of recovery studies for the HPLC method are presented in Table IVb. The LOQs were found to be 5.06 and 0.14 mg/mL for BDP and FFD, respectively, and the LODs were estimated to be 1.67 and 0.05 mg/mL for BDP and FFD, respectively. Standard solution of BDP (80 mg/mL) and FFD (2.4 mg/mL) were prepared from sample solution and stored at room temperature for 3 days. They were then injected into the HPLC system and no additional peak was found in the chromatogram indicating the stability of BDP and FFD in the sample solution. The statistical analysis of the robustness testing data was done by Design Expert software. The significance of the effects was evaluated by the statistical interpretation method (Algorithm of Dong) and graphical methods, namely a half-normal probability plot of the residuals and Pareto chart of the standardized effects. The half-normal plots showing effect of factor change on responses are depicted in Figure 5. The factor effects were calculated for each response and presented in Table VI. Analysis of the marketed formulation The proposed methods were applied for assay of BDP and FFD from marketed formulations. The spots at Rf 0.68 (for BDP) and
HPTLC method Initially, toluene and methanol in the ratio of 5:5 (v/v) were tried for separation of both drugs simultaneously. The spots were not developed properly and dragging was observed. Then toluene and methanol in the ratio of 3:7 (v/v) was tried. The developed spots were diffused and Rf was near to solvent front. Then the reverse ratio of the same mobile phase was tried. The distance travelled by developed spots was less and dragging was observed. To the above mobile phase, ethyl acetate was added with different ratios but the spot of BDP was diffused to solvent run, then ethyl acetate was replaced with ethanol but the developed spots lack compactness and were observed to be less persistent. Also the Rf values of FFD and BDP were not satisfactory because of less resolution between them. Then toluene was replaced by hexane and ethanol was again replaced by ethyl acetate. Different ratios of hexane, ethyl acetate and methanol were tried and finally 2.0:2.5:2.0 (v/v/ v) ratio of hexane, ethyl acetate and methanol was optimized. For improvement in peak shape of both the drugs, 0.2 mL of formic acid was added to hexane, ethyl acetate and methanol in the ratio of 2:2.5:2:0.2 (v/v/v/v). The densitometric scanning at 220 nm, the iso-absorptive point determined from overlain absorbance/reflectance spectra of FFD and BDP, resulted in increased sensitivity of the method. Well-defined spots were obtained when plate was activated at 1108C for 15 min and the chamber was saturated with the mobile phase for 10 min at room temperature. It was observed that drying of TLC plate after spotting and pre-saturation of TLC chamber with the mobile phase for 10 min ensured good reproducibility of Rf value. The proposed HPTLC method has the advantage of allowing determination of several samples at the same time. Sensitive and Robust Methods for BDP and FFD 1261
Figure 4. Representative Pareto charts to show the influence of variables studied in the response of BDP and FFD using PB experimental design for HPTLC method.
Table II System Suitability Tests for HPLC (n ¼ 6) Parameter
BDP
FFD
Retention time (min) Resolution Theoretical plates Tailing factor Injection repeatability (%RSD)
4.76 17.45 7701 1.22 0.86
1.69
1262 Parmar et al.
2342 1.65 1.40
HPLC method The main problem in the development of a method for the contemporary analysis of BDP and FFD was to find a suitable combination of mobile phases to separate the components. Preliminary isocratic studies on a reverse-phase C18 column with different mobile phase combinations were tried. According to the literature review, initially, acetonitrile and water were tried in the ratio of 80:20 (v/v). BDP showed good peak nature but the peak
phosphate buffer ( pH 3.6). Both the drugs showed typical peak nature and peaks were symmetrical at 220 nm (Figure 3). The proposed HPLC method utilizes an isocratic elution technique at room temperature with PDA detection for the simultaneous determination of both the drugs from rotacap formulation. The mobile phase with pH 3.6 gives greater stability to the analytical column. The real advantage of the HPLC method is low retention times: 1.69 and 4.78 min for FFD and BDP, respectively. It reduces total run time, leads to low solvent consumption and makes the method more economical.
of FFD was not separated. Then acetonitrile was replaced by methanol in the same ratio. Splitting was observed for both peaks. Then acetonitrile, methanol and water were tried in different ratios but there were problem in peak shape of both drugs and retention time of FFD was too long. Then the above mobile phase in different ratios were tried along with change in pH from 3.0 to 5.0 with the help of orthophosphoric acid. With 3.6 pH in 65:25:10 (v/v/v) ratio of methanol:acetonitrile:water, both the drugs were separated within 5 min and have good peak shapes. For stabilizing the pH, water was replaced by 25 mM
Table III Intra- and Interday Precisions of BDP and FFD (n ¼ 3) Drug
BDPa FFDb
HPTLC
Method validation The goal of this study was to develop sensitive and robust chromatographic methods for the simultaneous determination of BDP and FFD from their combined marketed formulation. The proposed methods were validated according to the ICH guidelines.
HPLC
Intraday precision
Interday precision
Intraday precision
Interday precision
SD of area
% RSD
SD of area
% RSD
SD of area
% RSD
SD of area
% RSD
64.40 7.60
0.51 0.34
109.36 19.59
0.87 1.00
6560.27 666.88
0.64 0.47
15083.29 1607.64
1.17 1.01
HPTLC method The linearity of calibration graphs and adherence of the system to Beer’s law was validated by high value of correlation coefficient and the SD for intercept value was ,2%. The repeatability studies ensured precision of densitometric scanner and spotting device. The % RSD for replicate sample solutions for intra- and interday study are ,2.0% for FFD and BDP which met the acceptance criteria established for the RP-HPLC method. This confirms that the method is precise. The % RSD values show that the proposed method provides acceptable intra- and interday variation of BDP and FFD. The signal-to-noise ratios of 3:1 and 10:1 were considered as LODs and LOQs, respectively. In this paper, PB design has been chosen to test the robustness of the HPTLC method. If the PB design is saturated, n trials enable to determine n 2 1 factor effects. In the current study, seven factors were tested with only eight experiments. The selection of factors was based on observations during method development and own experience. Each factor was studied at two levels. The different levels for each factor were selected symmetrically around the nominal value of the corresponding factor in the
a
Average of three concentrations 120, 160, 200 ng/spot and 1.2, 2.4, 4.8 mg/mL for HPTLC and HPLC, respectively. b Average of three concentrations 3.6, 4.8, 6 mg/spot and 40, 80, 160 mg/mL for HPTLC and HPLC, respectively.
Table IV Recovery Study of (a) HPTLC Method and (b) HPLC Method (n ¼ 3) Excess drug added to the analyte (%)
Theoretical content
% Recovery
% RSD
BDP (mg/spot)
FFD (ng/spot)
BDP
FFD
BDP
FFD
(a) HPTLC method 0 60 100 140
2.0 3.2 4.0 4.8
60 96 120 144
99.87 99.74 99.88 99.91
99.53 99.81 99.87 99.82
0.35 0.24 0.63 0.61
0.24 0.40 0.50 0.36
(b) HPLC method 0 50 100 150
40 60 80 100
1.2 1.8 2.4 3.0
99.98 98.53 99.99 99.39
99.78 99.79 99.66 99.98
0.27 1.15 0.28 0.42
0.32 0.25 0.47 0.31
Table V Eight Experiment PB Design to Examine the Seven Factors (A –G) Selected for Robustness Testing of HPTLC Method Experiments
Factors A
B
1 2 3 4 5 6 7 8
21 21 21 þ1 þ1 21 þ1 þ1
Responses
Effects of factors A
% Recovery (BDP) % Recovery (FFD) Rf values (BDP) Rf values (FFD)
Responses
21.01 0.34 20.018 0.0075
C
D
E
F
G
Rf values
% Recovery
þ1 21 21 21 þ1 þ1 21 þ1
þ1 þ1 21 þ1 21 21 21 þ1
þ1 þ1 21 21 þ1 21 þ1 21
21 þ1 21 21 21 þ1 þ1 þ1
þ1 21 21 þ1 21 þ1 þ1 21
21 þ1 21 þ1 þ1 þ1 21 21
B
C
D
E
F
G
BDP
FFD
BDP
FFD
100.46 99.77 101.65 98.79 100.94 100.74 98.59 100.25
99.25 99.54 99.73 100.10 99.68 99.91 100.25 99.77
0.67 0.67 0.64 0.64 0.64 0.68 0.66 0.65
0.35 0.36 0.34 0.33 0.34 0.33 0.38 0.36
Critical effect
0.90 20.25 0.007 20.0075
20.66 20.22 0.002 0.0025
20.42 20.20 0.008 0.0175
20.62 0.18 0.018 0.0175
21.01 0.20 0.013 20.0025
ME (a ¼ 0.05)
20.18 0.06 0.003 20.0175
1.729 0.525 0.028 0.029
Sensitive and Robust Methods for BDP and FFD 1263
Figure 5. Representative graphs to show the influence of variables studied in the response of BDP and FFD using 2421 experimental design for HPLC method.
Table VI Eight Experiment 2421 Fractional Factorial Design to Examine the Four Factors (P–S) Selected for Robustness Testing of HPLC Method Experiments
Factors P
Q
1 2 3 4 5 6 7 8
21 þ1 21 þ1 21 þ1 21 þ1
Responses
Effects of factors
% Recovery of BDP % Recovery of FFD Resolution
Responses R
S
% Recovery
21 21 þ1 þ1 21 21 þ1 þ1
21 21 21 21 þ1 þ1 þ1 þ1
21 þ1 þ1 21 þ1 21 21 þ1
P
Q
R
S
20.21 21.02 20.03
20.11 20.95 20.03
20.10 0.70 20.09
0.21 20.41 0.11
BDP
FFD
99.91 99.84 100.55 99.70 99.91 99.92 99.85 99.91
100.89 99.59 100.55 99.38 101.63 101.60 100.76 99.20
15.378 15.342 15.384 15.241 15.315 15.189 15.149 15.343 Critical effects
original method. The limits of the factors studied were selected according to error ranges which would be typically encountered in an analytical laboratory. All absolute factor effects on the quantitative responses, i.e., percentage recoveries of BDP and FFD, were found to be smaller than the corresponding critical effects, i.e., ME (a ¼ 0.05) ¼ 1.729 for BDP and 0.525 for FFD. All absolute effects on the response Rf values for both compounds were 1264 Parmar et al.
Resolution
PQ 20.18 20.35 0.05
PR 0.25 0.22 0.06
PS 20.14 20.68 0.02
ME (a ¼ 0.05) 0.42 1.66 0.15
smaller than their respective critical effects, i.e., ME (a ¼ 0.05) ¼ 0.028 for BDP and 0.029 for FFD. Concomitantly, graphical evaluation employing Pareto charts was done. It may be more comfortable viewing the Pareto Chart that has the significant effects selected. The Pareto graph (Figure 4) consists of bars with a length proportional to the absolute value of the estimated effect, divided by the pseudo standard error defined by Lenth (Lenth’s
Table VII Statistical Comparison of the Results Obtained by the Proposed HPLC and HPTLC Methods for the Analysis of Formulation A and B Parameters
Formulation A BDP HPTLC
Mean Variance d.f. t calculated t tabulated P-value*
between BDP and FFD, were found to be smaller than the corresponding critical effects. The % recoveries and resolution are not significantly affected by factor changes.
Formulation B FFD
BDP
FFD
HPLC
HPTLC
HPLC
HPTLC
HPLC
HPTLC
HPLC
100.07 99.97 0.12 0.07 2 0.31 2.92 0.39
99.54 0.06
99.96 0.09
99.85 0.02
100.06 0.16
99.68 0.12
100.01 0.01
2 22.21 2.92 0.08
2 20.68 2.92 0.28
2 22.24 2.92 0.08
*
P-value ¼ 0.05.
PSE). All main effects and interaction terms are found to be statistically insignificant as absolute values of main effects are below the critical t-value (a ¼ 0.05). Thus, in all situations, the % recoveries and Rf values for both the drugs are not significantly affected by factor changes. The method was found to be robust.
HPLC method The results of system suitability test (Table II) shows that the optimized chromatographic conditions are adequate for simultaneous determination. The resolution of BDP and FFD peaks is .1.5. Theoretical plates of BDP and FFD are .2,000, tailing factor of BDP and FFD is ,2.0, % RSD of injection repeatability is ,2.0%. The recovery studies for BDP and FFD from pharmaceutical formulation after spiking with additional standard drug afforded recovery between 98 and 102%. Robustness was assessed using experimental design approach. The results of robustness trials were evaluated by half-normal plots and statistical parameters. In half-normal plots, insignificant effects that tend to have a normal distribution centered near zero are normally distributed around the straight line, while significant effects deviate from it. Ideally, when the selection of statistically significant terms is complete, the Shapiro–Wilk P-value must be .0.10, indicating that the remaining (unselected) terms are normally distributed. In the current study, Shapiro–Wilk P-value of all the response parameters are .0.10 indicates that all unselected terms are normally distributed on straight line. It can be concluded from the half-normal plot that no effects are statistically significant as they all are normally distributed around the straight line. Although, we recommend using the half-normal plot of effects to choose the statistically significant effects, in some cases, a Pareto chart can also be very valuable. The Pareto chart is a useful tool for showing others the relative size of effects. There is standard t limits plotted on the graph to identify significant effects. After the initial selection of effects in half-normal plot, effects above the t-value limit (t (a ¼ 0.05) ¼ 2.445) are possibly significant and should be added if they are not already selected, effects below the t-value limit are not likely to be significant. So, in present state, all main effects and interaction terms are statistically in-significant. At the studied ranges, the effects of the factors were not statistically significant (a ¼ 0.05) for the response studied, recoveries of BDP and FFD and resolution between BDP and FFD. All absolute factor effects on the quantitative responses, i.e., percentage recoveries of BDP and FFD, and qualitative response, i.e., resolution
Analysis of the marketed formulation Both the drugs have good solubility in methanol; therefore, methanol was selected for the extraction from rotacap formulation. The formulation powder was sonicated with methanol for 25 min to ensure complete dissolution of both drugs. No interfering peaks were found in the chromatogram, indicating that formulation excipients did not interfere in the estimation of BDP and FFD by the proposed chromatographic methods. The low % RSD value indicated the suitability of this method for routine analysis of FFD and BDP in pharmaceutical dosage form. Content uniformity testing by HPLC Due to the high precision of the proposed method and its ability to rapidly estimate the concentration of the drugs in a single capsules extract with sufficient accuracy, the method is ideally suited for content uniformity testing which is a time-consuming process when using conventional assay techniques. The steps of the test were adopted according to the USP procedure (32). The AV was calculated for each of the rotacaps and was found to be smaller than the maximum LI. Comparison between HPLC and HPTLC method For four pairs of analysis, the Tcal was lower than the Ttab value obtained from Student’s distribution table for a risk factor of 5%, which showed that there was no statistically significant difference between HPLC and HPTLC analytical methods. HPTLC has developed to an extent that separation and quantitation can provide results that are comparable with another analytical method such as HPLC. Today, HPTLC has firm place among various analytical techniques as a reliable method for quantification at micro-, nano- and even at picogram level, even when present in complex formulations.
Conclusion The proposed HPTLC and HPLC methods provide precise, accurate and reproducible quantitative analysis for the simultaneous determination of BDP and FFD in rotacaps. Both the methods were validated as per the ICH guidelines. The robustness of the proposed methods was studied using DOEs and found to be robust at deliberate changes made in experimental conditions. Statistical tests indicate that the proposed HPTLC and HPLC methods reduce the duration of analysis and appear to be equally suitable for routine determination of BDP and FFD simultaneously in pharmaceutical formulation. The HPLC method is also applicable for the content uniformity test of rotacaps. Both the methods are suitable for routine analysis of BDP and FFD in their commercial dosage forms. The methods are also suitable and valid for application in quality control laboratories.
Supplementary material Supplementary data are available at Journal of Chromatographic Science online. Sensitive and Robust Methods for BDP and FFD 1265
Acknowledgments The authors thank Tripda Biotech Private Limited, Ahmedabad for gift sample of beclomethasone dipropionate and Cadila Healthcare Ltd., Ahmedabad for gift sample of formoterol fumarate dihydrate. Authors also thank to Charotar University of Science and Technology for providing the facilities for completion of the project.
References 1. Van den Berge, M., ten Hacken, N.H., Cohen, J., Douma, W.R., Postma, D.S.; Small airway disease in asthma and COPD: clinical implications; Chest, (2011); 139: 412–420. 2. Goodman, G.; Bronchial asthma. In Manuals of Pharmacology and Therapeutics, 11th ed. Mc-Graw Hill, New York, (2006), pp. 1031– 1061. 3. Tripathi, K.D. (ed); Essential of Medical Pharmacology, 4th ed. Jaypee Brothers, New Delhi, (2001), 201 pp. 4. Hickey, A.J.; Back to the future: inhaled drug products; Journal of Pharmaceutical Sciences, (2013); 102: 1165– 1170. 5. Tzani, P., Crisafulli, E., Nicoline, G., Aiello, M., Chetta, A., Clini, E.M., et al.; Effects of beclomethasone/formoterol fixed combination on lung hyperinflation and dyspnea in COPD patients; International Journal of Chronic Obstructive Pulmonary Disease, (2011); 6: 503–509. 6. O’Neil, M.J.; Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 12th ed. Merck & Co., Inc., Rathway, New Jersey, (2006), pp. 1419, 1941. 7. Indian Pharmacopoeia Commission. Indian Pharmacopoeia, vol. 2, 6th ed. Ministry of Health and Family Welfare, Government of India, Ghaziabad, India, (2010), pp. 873, 1386. 8. British Pharmacopoeia Commission. British Pharmacopoeia, Secretariat of the Medicines and Healthcare Products Regulatory Agency, London, UK, (2011), pp. 219, 963. 9. Council of Europe’s European Directorate for the Quality of Medicines and Healthcare (EDQM) and European Medicines Agency joint meeting on raw materials used for the production of cell-based and gene-therapy products, Council of Europe: European Directorate for the Quality of Medicines (EDQM). European Pharmacopoeia, vol. 2, Strasbourg, France, (2011), pp. 1449, 2067. 10. Dhudashia, K.R., Patel, A.V., Patel, C.N.; Development and validation of stability indicating RP- HPLC method for estimation of beclomethasone dipropionate in cream dosage form; Inventi Rapid: Pharm Analysis & Quality Assurance, (2012); 2013: 567–576. 11. Venkata, N.T., Gopinath, B., Bhaskar, V.V.; Development and validation of UHPLC method for simultaneous estimation of salbutamol sulphate and beclomethasone dipropionate; International Journal of Pharmacy and Biological Sciences, (2012); 2: 254– 268. 12. Martis, E.A., Gangrade, D.M.; Reverse phase isocratic HPLC method for simultaneous estimation of salbutamol sulphate and beclomethasone dipropionate in Rotacaps formulation dosage forms; International Journal of Pharmacy and Pharmaceutical Sciences, (2011); 3: 64– 67. 13. Lakshmi, P.B., Shetty, S.A., Priyatam, N.T., Gopinath, B., Manzoor, A.; Development of new spectrophotometric methods for the simultaneous estimation of levosalbutamol sulphate and beclomethasone dipropionate in bulk drug and pharmaceutical formulations (ROTACAP); International Journal of PharmTech Research, (2012); 4: 791–798. 14. Guan, F., Uboh, C., Soma, L., Hess, A., Luo, Y., Tsang, D.S.; Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine; Journal of Mass Spectrometry, (2003); 38: 823–832. 15. Foe, K., Brown, K.F., Seale, J.P.; Decomposition of beclomethasone propionate esters in human plasma; Biopharmaceutics and Drug Disposition, (1998); 19: 1 –8.
1266 Parmar et al.
16. Gousuddin, M., Raju, S.A., Sultan, U., Manjunath, S.; Development and validation of spectrophotometric methods for estimation of formoterol bulk drug and its pharmaceutical dosage forms; International Journal of Pharmacy and Pharmaceutical Sciences, (2011); 3: 300–309. 17. Gousuddin, M., Raju, S.A., Sultan, U., Manjunath, S.; Optimization and validation of quantitative spectrophotometric methods for determination of formoterol bulk drug and its pharmaceutical dosage forms; Journal of Analytical Chemistry, (2011); 1: 1 –5. 18. Ahmed, S., Jayakar, B., Aleem, M.A.; Development of reverse phase high performance liquid chromatography method and its validation for estimation of formoterol fumarate Rotacaps; International Journal of Pharmaceutical Science and Research, (2011); 2: 319–324. 19. Akapo, S.O., Asif, M.; Validation of a RP-HPLC method for the assay of formoterol and its related substances in formoterol fumarate dihydrate drug substance; Journal of Pharmaceutical and Biomedical Analysis, (2003); 33: 935– 945. 20. Trivedi, R.K., Chendake, D.S., Patel, M.C.; A rapid stability-indicating RP-HPLC method for the simultaneous determination of formoterol fumarate, tiotropium bromide, and ciclesonide in a pulmonary drug product; Scientia Pharmaceutica, (2012); 80: 1– 13. 21. Shah, B.D., Kumar, S., Yadav, Y.C., Seth, A.K., Ghelani, T.K., Deshmukh, G.J.; Analytical method development and method validation of tiotropium bromide and formoterol fumarate metered dose Inhaler (MDI) by using RP-HPLC method; Asian Journal of Biochemical and Pharmaceutical Research, (2011); 1: 145– 158. 22. Patil, A.T., Patil, S.D., Shaikh, K.A.; Sensitive LC method for simultaneous determination of ciclesonide and formoterol fumarate in dry powder inhaler; Journal of Liquid Chromatography and Related Technology, (2011); 34: 1568–1577. 23. Malik, K., Kumar, D., Tomar, V., Kaskhedikar, S., Soni, L.; Simultaneous quantitative determination of formoterol fumarate and fluticasone propionate by validated reversed-phase HPLC method in metered dose inhaler; Pelagia Research Library, (2011); 2: 77– 84. 24. Sardelaa, V.F., Deventerb, K., Pereiraa, H.M.G., de Aquino Netoa, F.R., Van Eenoob, P.; Development and validation of an ultra-high performance liquid chromatography–tandem mass spectrometric method for the direct detection of formoterol in human urine; Journal of Pharmaceutical and Biomedical Analysis, (2012); 70: 471–479. 25. ICH, Q8 (R2); Harmonised tripartite guideline, Pharmaceutical development, IFPMA. In Proceedings of the International Conference on Harmonization, Geneva, (2009), pp. 1 –28. 26. ICH, Q2A; Harmonised tripartite guideline, text on validation of analytical procedures, IFPMA. In Proceedings of the International Conference on Harmonization, Geneva, (1994), pp. 1–5. 27. ICH, Q2B; Harmonised tripartite guideline, validation of analytical procedure: methodology, IFPMA In Proceedings of the International Conference on Harmonization, Geneva, (1996), pp. 1–8. 28. Inglot, T., Gumieniczek, A., Maczka, P., Rutkowska, E.; New HPLC method with experimental design and fluorescence detection for analytical study of antihypertensive mixture, amlodipine and valsartan; American Journal of Analytical Chemistry, (2013); 4: 17– 23. 29. Enrique, M., Garcı´ a-Montoya, E., Min˜arro, M., Orriols, A., Tico´, J.R., Sun˜e´-Negre, J.M., et al.; Application of an experimental design for the optimization and validation of a new HPLC method for the determination of vancomycin in an extemporaneous ophthalmic solution; Journal of Chromatographic Science, (2008); 46: 828–834. 30. Barth, A.B., de Oliveira, G.B., Malesuik, M.D., Paim, C.S., Volpato, N.M.; Stability-indicating LC assay for butenafine hydrochloride in creams using an experimental design for robustness evaluation and photodegradation kinetics study; Journal of Chromatographic Science, (2011); 49: 512–518. 31. Gohel, N.R., Patel, B.K., Parmar, V.K.; Chemometrics-assisted UV spectrophotometric and RP-HPLC methods for the simultaneous determination of tolperisone hydrochloride and diclofenac sodium in their combined pharmaceutical formulation; Scientia Pharmaceutica, (2013); 81: 983–1001. 32. USP 30/NF 25; The United States Pharmacopoeia, 30th Rev, and The National Formulary, 25th ed., United States Pharmacopoeial Convention, Inc., Rockville, (2007).
