A Validated Chiral LC Method for Enantiomeric Separation of Nebivolol Stereoisomers in Bulk Drugs and Dosage Forms on Amylose-Based Stationary Phase

Journal of Chromatographic Science 2014;52:1051 1058 doi:10.1093/chromsci/bmt158 Advance Access publication October 29, 2013 Article A Validated Chiral LC Method for Enantiomeric Separation of Nebivolol Stereoisomers in Bulk Drugs and Dosage Forms on Amylose-Based Stationary Phase Karri Visweswara Rao 1,2 *, Kesareddy Padmaja Reddy 1 and Pranab Haldar 1 1 Dr. Reddy s Laboratories Ltd., Active Pharmaceutical Ingredients, IPDO, Bachupally, Hyderabad 500072, AP, India and 2 Department of Chemistry, J. N. T. University, Kukatpally, Hyderabad 500072, AP, India *Author to whom correspondence should be addressed at: Dr. Reddy s Laboratories Ltd., Active Pharmaceutical Ingredients, IPDO, Bachupally, Hyderabad 500072, AP, India. Email: visweswarark@drreddys.com, havishkarri@gmail.com Received 25 December 2012; revised 29 September 2013 A novel and reproducible isocratic normal phase liquid chromatographic method was developed for the quantitative determination of 10 stereoisomers of Nebivolol in pharmaceutical bulk drugs and dosage forms. The method was developed using an amylose-based chiral stationary phase, Chiralpak AD-3 (250 3 4.6 mm, 3 mm) column with mobile phase containing n-hexane ethanol isopropanol diethanolamine in the ratio 42:45:13:0.1 (v/v/v/v). The eluted compounds were monitored at 280 nm. Ten stereoisomers of Nebivolol were well separated with resolution >2.0 for all pair of components. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, linearity (R 2 value >0.999), limit of detection, limit of quantification, accuracy (recovery range 95.8 103.2%), precision (relative standard deviation, RSD, <2.5%) and robustness. Nebivolol sample solutions were found to be stable when characterized over a period of 48 h. Forced degradation studies were also performed to demonstrate the stability-indicating power of the developed HPLC method. The method was found to be rugged and robust. Introduction Nebivolol hydrochloride is chemically described as (+)- [2R*[R*[R*(S*)]]]-a,a 0 -[iminobis-(methylene)]-bis-[6-fluoro-3,4- dihydro-2h-1-benzopyran-2-methanol] hydrochloride. It is a cardio-selective third-generation, b-blocker. It blocks the b-adrenoreceptor effect of adrenaline and noradrenaline, reducing heart rate and the force of myocardial infarction, decreases systemic blood pressure and increases diastolic pressure. In addition to the adrenergic blocking property, it possesses additional vasodilating activity mediated by L-arginine nitric oxide pathway (1 3). The drug Nebivolol is marketed as a racemate of two enantiomers, SRRR and RSSS, which are biologically active. The chemical structure of Nebivolol contains four asymmetric carbon atoms (chiral centers), which gives rise the possibility of forming 16 theoretical isomers. Owing to the presence of plane of symmetry, some of these isomers are identical and exist in the meso form. Accordingly, only 10 stereoisomers are present. Isomer 3 (SRSR) and Isomer 9 (RRSS) are the two mesomers and the remaining four enantiomeric pairs are Isomers 1 and 2 (RSSR, SRRS), Isomers 4 and 5 (SRRR, RSSS), Isomers 6 and 8 (RSRR, SRSS) and Isomers 7 and 10 (RRRR, SSSS) (Figure 1). Literature survey reveals the chiral separation of several b-adrenergic blockers using SPE-HPLC (4) and HPLC (5, 6). Few spectrophotometry methods were reported for quantification of Nebivolol in combination with other drugs (7, 8). RP-HPLC methods in pharmaceutical preparations (9 13) and estimation of Nebivolol in human plasma by LC MS/MS (14) was reported. There are some LC methods that describe enantiomeric separation of only two stereoisomers of Nebivolol (15 17). According to our research, one chiral LC method reported on enantiomeric separation of drugs (nadolol, indenolol and Nebivolol) with multiple chiral centers (18). In this article, the chiral method available for Nebivolol demonstrates the separation of 10 stereoisomers. This method was not validated, separation between stereoisomers was not appropriate (the resolution between Isomers 2 and 3,1.0) and detection and quantification limits were not reported. In this method, sensitivity and peak shapes of stereoisomers were also poor. The major drawback of the method available for Nebivolol is that it is not suitable for quantification of all stereoisomers at lower level and run time is also high. In this method, stress studies were also not reported. In this reported method, only ethanol was used as the mobile phase and Chiralpak AD (more particle size 10 mm) was used as the column. The mobile phase selection is one of the critical parameters as it encourages the solute and the stationary phase interactions. Mobile phase strength, composition and organic modifier have shown important roles in the chromatographic separation of 10 stereoisomers of Nebivolol. n-hexane with secondary alcohols impart a greater resolution to the chiral stationary phase than primary alcohols. Chiral stationary phase particle size has also shown an important role in the chromatographic separation of 10 stereoisomers of Nebivolol. A wellpacked column with 3 mm packings produces outstanding separation efficiency of a comparable 10 mm column. Basic hydroxyl amine modifier diethanol amine played a significant role in improving peak symmetry, chromatographic efficiency and resolution of the stereoisomers. For the enantiomeric separation of 10 stereoisomers of Nebivolol on lower micron amylose-based chiral stationary phase (CSP) (Chiralpak AD-3), n-hexane ethanol isopropanol diethanolamine (42:45:13:0.1) is a better mobile phase. As per ICH guidelines, the method was validated. Currently, the enantiomeric separation of some drugs with multiple stereogenic centers is one of the most difficult tasks for pharmaceutical analysis during method development. In Nebivolol, separation of 10 stereoisomers is very critical and there is no chiral HPLC method reported in the literature that can adequately separate these 10 stereoisomers. It is therefore felt necessary to develop a new enantioselective chiral HPLC # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 1. Chemical structures and names of all the 10 stereo isomers of Nebivolol hydro chloride; Isomer 1, Isomer 2, Isomer 3, Isomer 4, Isomer 5, Isomer 6, Isomer 7, Isomer 8, Isomer 9 and Isomer 10. method for the determination and quantitative estimation of Nebivolol 10 stereoisomers in a short run time without compromising the resolution and sensitivity. Hence, a reproducible and robust enantioselective normal phase HPLC method was developed for the quantitative determination of Nebivolol stereoisomers (Isomers 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10). The stereoisomers were prepared synthetically by Dr Reddy s process research group. This method was successfully validated according to the ICH guidelines (19). Experimental Materials and reagents Active pharmaceutical ingredient standards and samples were supplied by Dr. Reddy s Laboratories Limited, IPDO, Hyderabad, India. Commercially available Bystolic 10 mg tablets (Forest Pharmaceuticals, Inc., NY, USA) were used for the dosage form analysis. The HPLC grade n-hexane, ethanol, isopropanol and diethanol amine were purchased from Merck, Darmstadt, Germany. 1052 Visweswara Rao et al.

Chromatographic conditions and equipment LC was carried out on a Waters HPLC (Waters, Milford, MA, USA) with a 2695 model binary solvent delivery pump and with a 2996 model photodiode array (PDA) detector. The output signal was monitored and processed using the Empower software. The chromatographic separation was achieved on Chiralpak AD-3 HPLC 250 4.6 mm, 3 mm column (Diacel, USA) using isocratic mode. The mobile phase consists of n-hexane ethanol isopropanol diethanol amine in the ratio of 42:45:13:0.1 (v/v/v/v). The flow rate of mobile phase was 0.8 ml/min. The column temperature was maintained at 408C and the detection was monitored at a wavelength of 280 nm. The diluent selected was methanol. The injection volume was 20 ml. (RSD%) of peak area for each stereoisomer was calculated. The intermediate precision study was performed by a second analyst on a different day using a different system. Limit of detection and quantification Limit of detection (LOD) and limit of quantification (LOQ) for 10 stereoisomers of Nebivolol were determined at signal-to-noise ratios of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. The precision study was also determined at the LOQ level by injecting six individual preparations and calculating the RSD% of the area for 10 stereoisomers of Nebivolol. Preparation of stock solutions An individual stock solution (100 mg/ml) of all stereoisomers (denoted Isomers 1 10) was prepared in diluent. Preparation of sample solution A solution of Nebivolol drug substance (1000 mg/ml) was prepared in diluent. To prepare the sample stock solution, 20 tablets of Bystolic, each containing 10 mg of Nebivolol, were accurately weighed and crushed to a fine powder. Tablet powder equivalent to 50 mg of drug was transferred into a 50 ml volumetric flask, dissolved in 25 ml of diluent and sonicated for 30 min to ensure the dissolution. Finally, the solution was made up to 50 ml mark. These solutions were filtered through a 0.22-mm nylon membrane filter. Accuracy The accuracy of the method was established by standard addition and recovery was calculated for each added concentration. The study was carried out for stereoisomers in triplicate using four concentration levels from LOQ, 0.075, 0.15 and 0.225% of the analyte concentration (1000 mg/ml) and the recovery of stereoisomers was calculated. Linearity of response The linearity of the detector response to different concentrations was evaluated for 10 stereoisomers of Nebivolol by injecting each separately prepared solution covering the range LOQ to 0.3% (LOQ, 0.0375, 0.075, 0.1125, 0.15, 0.1875, 0.225 and 0.30% of the analyte concentration). The correlation coefficients, slopes and Y-intercepts of the calibration curve were determined. Stress studies Specificity is the ability of the method to measure the analyte response in the presence of its potential stereo isomers. The specificity of the developed chiral LC method for Nebivolol (SRRR and RSSS) was carried out in the presence of its stereoisomers namely Isomer 1 (RSSR), Isomer 2 (SRRS), Isomer 3 (SRSR), Isomer 6 (RSRR), Isomer 7 (RRRR), Isomer 8 (SRSS), Isomer 9 (RRSS) and Isomer 10 (SSSS). Stress studies were performed at an initial concentration of 1000 mg/ml of Nebivolol to provide the stability-indicating property and specificity of the proposed method. Intentional degradation of Nebivolol was attempted by using the stress conditions of UV light at 254 nm, heat at 1058C, acid with 3.0 N HCl at 708C, base with 3.0 N NaOH at 708C, hydrolysis at 708C and oxidation with 12% H 2 O 2 at room temperature (RT) to evaluate the ability of the proposed method to separate the Nebivolol stereoisomers from its degradation products. For heat and light studies, the study period was 10 days, whereas for hydrolytic, base, acid and oxidation, it was 48 h. Method validation Precision The precision of the developed method was verified by repeatability and by intermediate precision. The repeatability of the method was checked by injecting six individual preparations of Nebivolol sample spiked with all stereoisomers at its specification limit (0.15%). The percentage of relative standard deviation Robustness To determine the robustness of the method, the experimental conditions were deliberately changed. A solution of Nebivolol drug spiked with all stereoisomers at specification level (0.15%) was used in this study and analyzed six times at each experimental condition. The resolution between 10 stereoisomers of Nebivolol was evaluated. The mobile phase flow rate was 0.8 ml/min; to study the effect of flow rate on resolution, it was changed to 0.6 and 1.0 ml/min. To study the effect of change in percentage of ethanol on resolution by varying from 21 to þ1%, the other mobile phase components were held constant and the effect of column temperature was studied at 35 and 458C (instead of 408C). Solution stability and mobile phase stability The solution stability of 10 stereoisomers of Nebivolol was carried out by leaving the solution of Nebivolol drug spiked with all stereoisomers at specification level in tightly capped volumetric flasks at RT up to a 48 h period. The contents of stereoisomers were determined at 12 h intervals and studied up to 48 h. The stability of mobile phase was determined by analysis of freshly prepared solution of Nebivolol drug spiked with all stereoisomers at specification level. The content of stereoisomers were determined at 12 h interval and studied up to 48 h. The mobile phase was prepared at the beginning of the study period and not changed during the experiment. Validated Chiral LC Method 1053

Results Method development and optimization The main objective of this work was separation and accurate quantification of 10 stereoisomers of Nebivolol in bulk drugs and dosage forms. A solution of Nebivolol racemic mixture spiked with all stereoisomers (1.5 mg/ml) prepared in methanol was used in the method development. Nebivolol stereoisomers have maximum absorbance at 280 nm. So the wavelength of 280 nm was selected for LC analysis. To develop a rugged and suitable LC method for separation of all the 10 stereoisomers of Nebivolol, different mobile phases and stationary phases were employed. In method development trials with different chiral columns like Chiralpak AD-H, Chiralcel OJ-H, Chiralpak IA, Chiralpak IC and Chiralpak AD-3, Figure 2. (A) Nebivolol spiked with all its isomers at specification level with column Chiralcel OJ-H. (B) Nebivolol spiked with all its isomers at specification level with column Chiralpak IC. (C) Nebivolol spiked with all its isomers at specification level with column Chiralpak IA. (D) Nebivolol spiked with all its isomers at specification level with column Chiralpak AD-H. (E) Nebivolol spiked with all its isomers at specification level with column Chiralpak AD-3. 1054 Visweswara Rao et al.

various experiments were conducted, to select the best stationary phase and mobile phases that would give optimum resolution and selectivity for all the 10 stereoisomers. No separation was found on Chiralcel OJ-H (Figure 2A) and Chiralpak IC (Figure 2B). A slight separation with broad peaks (USP tailing.2.0) was observed on the Chiralpak AD-H and Chiralpak IA (Figure 2C) with n-hexane ethanol isopropanol (42:45:13, v/v/v) as mobile phase. Further, trials have been continued by using n-hexane ethanol isopropanol (42:45:13, v/v/v) mobile phase with amylose-based CSPs and by changing basic modifiers diethyl amine, n-butyl amine and diethanol amine. An addition of basic modifier diethanol amine in mobile phase has played an important role in the enhancing chromatographic efficiency and resolution between the stereoisomers. Separation was achieved on chiralpak AD-H column with the mobile phase consisting of n-hexane ethanol isopropanol diethanol amine in the ratio of 42:45:13:0.1 (v/v/v/v) (20). But less resolution was observed between critical pairs (Isomers 4, 5 and Isomers 6, 7) (Figure 2D). To increase the resolution between critical pairs, low micron particle size CSP (Chiralpak AD-3 250 4.6 mm, 3 mm) has been used. An improvement in the peak shape and resolution of all the 10 stereoisomers of Nebivolol was observed within a run time of 55 min. The ratio of mobile phase composition was finalized as n-hexane ethanol isopropanol diethanol amine (42:45:13:0.1, v/v/v/v) with an isocratic elution. The flow rate of mobile phase was fixed as 0.8 ml/min. Methanol was selected as diluent, as the blank chromatogram was clean without any interference with all the 10 stereoisomers and excipients was observed. Under optimized conditions, 10 stereoisomers of Nebivolol were well separated with resolution.2.0 (Figure 2E). The structure of the CSP in LC played an important role in the separation of stereoisomers. Derivatized cellulose and amylosebased CSPs have been used for enantiomeric separation of a large variety of racemates by LC. The mechanism of separation in direct chiral separation methods is the interaction of the CSP with the analyte to form short-lived diastereomeric complexes as a result of hydrogen bonding, dipole dipole interactions, charge transfer complex ( p p) formation, electro static interactions and inclusion complexation. As discussed above, the 10 stereoisomers of Nebivolol could not be separated on Chiralcel OJ-H (a tris-4-methyl benzoate ester of cellulose) and Chiralpak IC (a tris-3,5 dichlorophenyl carbamate of cellulose). Chiralpak AD-3 is a 3,5-tris-dimethyl phenyl carbamate derivative of a amylose coated on silica gel. Amylose forms a helical structure with defined grooves, making it different from the corresponding cellulose analogs, which appeared to be more linear and rigid in nature. These polysaccharides contain a large number of chirally active sites and thus have a relative high probability of interaction with solute, leading to separation of the stereoisomers. Peak tailing is a result of more interactions between Figure 2. Continued Validated Chiral LC Method 1055

solute and stationary phase constituents. A small amount of basic modifier diethanol amine blocks such active sites and improves peak symmetry, chromatographic efficiency and resolution of the stereoisomers. System suitability parameters were evaluated for Nebivolol and all its stereoisomers in finalized chromatographic conditions using a solution of Nebivolol containing all stereoisomers at specification level. The below conditions were finalized for the analysis of Nebivolol and all its stereoisomers. The enantiomeric separation was achieved on the Chiralpak AD-3, 250 4.6 mm, 3 mm column using a mobile phase containing n-hexane ethanol isopropanol diethanol amine (42:45:13:0.1, v/v/v/v) with a flow rate of 0.8 ml/min. The column temperature was maintained at 408C and the detection was monitored at a wavelength of 280 nm. Tailing factor for all the 10 stereoisomers of Nebivolol was found to be,1.5. The USP resolution of 10 stereoisomers of Nebivolol was.2.0 in finalized chromatographic conditions. The results are presented in Table I. Validation of the method Precision In repeatability study, the RSD% for the area of stereoisomers was within 2.5%. In the intermediate precision study, the RSD% for the area of stereoisomers was well within 2.1%, confirming good precision of the method. The RSD% values are presented in Table II. Table I. System Suitability Results (RT, RRT, USP Resolution and USP Tailing) of Nebivolol Spiked with all Its Isomers at Specification Level with Column Chiralpak AD-3 S. no Name RT (min) a RRT b (n ¼ 6) a USP resolution c (n ¼ 6) a USP tailing (n ¼ 6) a 1 Isomer 1 8.04 + 0.16 0.48 + 0.21 1.16 + 2.02 2 Isomer 2 9.88 + 0.27 0.60 + 0.11 5.27 + 2.01 1.08 + 3.14 3 Isomer 3 10.88 + 0.28 0.66 + 0.10 2.46 + 2.40 1.05 + 3.13 4 Isomer 4 16.59 + 0.36 1.00 + 0.00 9.81 + 1.04 1.35 + 0.26 5 Isomer 5 19.32 + 0.43 1.17 + 0.07 3.23 + 0.42 1.39 + 0.56 6 Isomer 6 27.79 + 0.76 1.68 + 0.40 7.94 + 1.46 1.11 + 5.14 7 Isomer 7 32.77 + 0.65 1.98 + 0.31 3.97 + 6.54 1.08 + 5.96 8 Isomer 8 36.93 + 0.84 2.23 + 0.51 2.89 + 3.91 1.06 + 6.31 9 Isomer 9 43.40 + 0.77 2.62 + 0.43 3.99 + 3.05 1.04 + 6.31 10 Isomer 10 46.79 + 0.78 2.82 + 0.47 2.24 + 1.07 1.03 + 5.14 a Mean + RSD% (n ¼ 6). b Relative retention times (RRT) were calculated against the retention time (RT) of Isomer 4. c USP resolutions were calculated between two adjacent peaks. RSD, relative standard deviation; USP, United States Pharmacopeia. Limit of detection and quantification The determined limit of detection, limit of quantification and precision at LOQ values for 10 stereoisomers of Nebivolol are presented in Table II. Linearity Linearity calibration plot for the 10 stereoisomers of Nebivolol was obtained over the calibration ranges tested, i.e., LOQ to 0.3% of the analyte concentration. The correlation coefficients, slopes and Y-intercepts of the calibration curve were determined. The correlation coefficient obtained was.0.999 (Table II). Accuracy The recovery of the all stereoisomers of Nebivolol from pharmaceutical dosage forms ranged from 95.8 to 103.2%. The results are presented in Table III. Robustness In all the deliberately varied chromatographic conditions (flow rate, organic solvent strength and column temperature), all stereoisomers were adequately resolved and elution orders remained unchanged. The resolution between critical pairs, i.e., for Isomer 2, Isomer 3 and Isomer 9, Isomer 10, was.2.0. The results are presented in Table IV. Stability in solution and in the mobile phase No significant changes were observed in the stereoisomers content of Nebivolol samples during solution stability and mobile phase study experiments, which indicates that sample solutions and mobile phase used during the study was stable up to 48 h. The results are posted in Supplementary data, Table S1. Results from stress studies All stress study samples were analyzed at an initial test concentration (1000 mg/ml) of Nebivolol in the finalized method. The peak purity of Nebivolol was performed for all stressed conditions using PDA detector and conformed the peak purity. Significant degradation was not observed in all stressed conditions. The proposed chromatographic conditions were found to be selective to the Nebivolol sample subjected to the applied stress conditions. The purity of peaks obtained from stressed samples was checked by using the PDA detector. In peak purity testing, the Empower software compares the spectrum from each data point within the peak against the peak apex spectrum. Purity angle is the numerical value of the measured peak s apex Table II. Method Validation Results LOD, LOQ, Regression, Repeatability and Intermediate Precision Parameter Isomer 1 Isomer 2 Isomer 3 Isomer 4 Isomer 5 Isomer 6 Isomer 7 Isomer 8 Isomer 9 Isomer 10 LOD (mg/ml) 0.05 0.05 0.05 0.06 0.06 0.06 0.07 0.07 0.07 0.08 LOQ (mg/ml) 0.20 0.20 0.21 0.23 0.24 0.25 0.26 0.28 0.30 0.31 Regression equation (y) Slope (b) 3.92E þ 07 3.26E þ 07 3.70E þ 07 4.65E þ 07 4.60E þ 07 4.67E þ 07 4.91E þ 07 4.97E þ 07 4.96E þ 07 5.09E þ 07 Intercept (a) 1173 917 862 640 656 455 2649 22588 2759 21936 Correlation coefficient 0.9997 0.9997 0.9997 0.9999 0.9999 0.9999 0.9999 0.9997 0.9996 0.9995 R 2 -value 0.9993 0.9994 0.9994 0.9997 0.9998 0.9999 0.9999 0.9993 0.9992 0.9992 Repeatability (RSD%) 0.33 2.14 0.45 0.19 0.18 1.41 1.17 1.45 1.12 0.79 Intermediate precision (RSD%) 0.43 0.28 0.61 0.26 0.16 0.50 0.83 0.18 0.71 0.42 Precision at LOQ (RSD%) 1.62 0.71 2.50 1.07 1.29 1.06 2.27 1.58 2.22 1.89 1056 Visweswara Rao et al.

Table III. Method Validation Accuracy (Recovery) Data Amount spiked a % Recovery b Isomer 1 Isomer 2 Isomer 3 Isomer 4 Isomer 5 Isomer 6 Isomer 7 Isomer 8 Isomer 9 Isomer 10 LOQ 102.5 + 1.6 100.7 + 0.7 102.4 + 2.5 101.9 + 1.1 99.0 + 2.3 100.8 + 1.6 100.8 + 2.2 103.2 + 1.9 0.075% 100.6 + 2.3 101.0 + 0.7 98.8 + 1.5 100.8 + 0.3 99.6 + 0.3 99.5 + 0.2 99.9 + 0.8 97.7 + 1.7 99.9 + 1.3 95.8 + 2.1 0.15% 101.4 + 0.4 100.4 + 2.1 99.7 + 0.5 99.7 + 0.1 98.6 + 0.2 100.0 + 1.7 98.3 + 1.2 99.2 + 1.8 98.9 + 1.1 100.0 + 0.8 0.225% 101.5 + 0.8 99.4 + 0.58 99.5 + 1.0 99.5 + 0.2 98.4 + 0.1 100.0 + 0.9 98.8 + 1.2 98.8 + 0.7 100.4 + 1.1 99.9 + 0.6 a Amount of stereoisomers spiked with respect to analyte concentration (1000 mg/ml). b Mean + RSD%. Table IV. Method Validation Robustness Data (USP Resolution) Parameter Isomer 1 Isomer 2 Isomer 3 Isomer 4 Isomer 5 Isomer 6 Isomer 7 Isomer 8 Isomer 9 Isomer 10 Actual flow and temperature 5.65 2.55 9.89 3.30 7.95 4.11 3.12 4.41 2.24 Flow 0.6 ml/min 5.50 2.55 10.14 3.26 8.22 4.25 2.72 4.07 2.20 Flow1.0 ml/min 5.07 2.36 9.51 3.18 7.76 3.74 2.81 4.08 2.06 Temperature 358C 5.34 2.48 9.94 3.23 8.23 4.82 2.89 4.29 2.07 Temperature 458C 5.06 2.38 9.51 3.16 7.87 3.96 2.75 3.86 2.08 Ethanol 44% 5.10 2.25 9.41 3.22 7.91 3.85 2.72 4.10 2.04 Ethanol 46% 5.31 2.41 9.62 3.25 7.95 3.99 2.94 4.21 2.07 spectrum. Purity threshold is the numerical value of the minimum allowable variation range. A purity angle less than the purity threshold indicates that there is no evidence of co-elution of unknown impurity, while the purity angle greater than purity threshold is evidence of the co-elution of the unknown impurity. This demonstrates the analyte peak homogeneity and thus confirms the stability-indicating power of the developed method. Discussion The method conditions and forced degradation studies indicate that the developed method is stability indicating and is capable of separation and accurate quantification of 10 stereoisomers of Nebivolol with good resolution. The repeatability and intermediate precision results confirmed that the method was highly precise at an LOQ level to 150% of the specification limit. The linearity results show that an excellent correlation existed between the peak area and concentration of all the 10 stereoisomers of Nebivolol. The accuracy results indicate that the method is highly accurate from LOQ level to 150% of the specification limit. The robustness results (flow rate, organic solvent strength and column temperature) indicate that the method is highly robust. The results from solution stability and mobile phase stability experiments confirmed that standard solutions and solutions in the mobile phase were stable up to 48 h during experimentation. Conclusion The normal phase HPLC method developed for quantitative analysis of 10 stereoisomers of Nebivolol in pharmaceutical dosage forms is precise, accuratee, linear, robust and specific. Satisfactory results were obtained from validation of the method. This method exhibited an excellent performance in terms of sensitivity, speed and resolution compared with existing methods. The method is stability indicating and can be used for routine analysis of production samples and to check the stability samples of Nebivolol. Supplementary data Supplementary data are available at Journal of Chromatographic Science online. Acknowledgments The authors thank the management of Dr Reddy s group for supporting this work. References 1. Dollery, C.; Therapeutic Drugs, 2nd ed. Churchill Livingstone, UK, (1999), pp. 52 57. 2. Goodman, A.G., Gilman, L.S., Gilman, A.G., Rall, T.W., Nies, A.S., Taylor, P.; The Pharmacological Basis of Therapeutics, 8th ed. Pergamon Press, Oxford, (1990), pp. 286, 847. 3. Martindale, S.C.; The Complete Drug Reference, 34th ed. Pharmaceutical Press, London, (2005), pp. 907.3, 938.2. 4. Ali, I., Al-Othman, Z.A., Hussain, A., Aboul-Enein, H.Y.; Chiral separation of b-adrenergic blockers in human plasma by SPE-HPLC; Chromatographia, (2011); 73: 251 256. 5. Ali, I., Gaitonde, V.D., Aboul-Enein, H.Y., Hussain, A.; Chiral separation of beta-adrenergic blockers on CelluCoat column by HPLC; Talanta, (2009); 78: 458 463. 6. Schmid, M.G., Gecse, O., Szabo, Z., Kilar, F., Gubitz, G., Ali, I., Aboul-Enein, H.Y.; Comparative study of the chiral resolution of b-blockers on cellulose tris (3,5-dimthyl phenyl carbamate) phase in normal and reversed phase modes; Journal of Liquid Chromatography & Related Technologies, (2001); 24: 2493 2504. 7. Patel, S.A., Patel, H.M.; Simultaneous determination of Nebivolol and hydrochlorthiazide in tablets by derivative spectrophotometry; American Journal of PharmTech Research, (2011); 1: 421 429. Validated Chiral LC Method 1057

8. Shah, K.V., Tirgar, P.R., Sheth, D.B., Desai, T.R.; Simultaneous estimation of Nebivolol hydrochloride and hydrochlorthiazide in bulk and tablet dosage form by multicompount and simultaneous estimation method; An International Journal of Pharmaceutical Science, (2011); 2: 27 35. 9. Manzoor, A., Manohara, Y.N., Ravi, M.C.; Development and validation of PHPLC method for simultaneous estimation of Nebivolol hydrochloride and hydrochlorothiazide in combined tablet dosage form; International Journal of ChemTech Research, (2012); 4: 328 336. 10. Kokil, S.U., Bhatia, M.S.; Simultaneous estimation of Nebivolol hydrochloride and valsartan using RP-HPLC; Indian Journal of Pharmaceutical Science, (2009); 71: 111 114. 11. Sudhakar, M., Venkateshwara Rao, J., Devika, G.S., Ramesh Petchi, R.; A Validated RP-HPLC method for simultaneous estimation of Nebivolol hydro chloride and S-amlodipine Besylate in tablet dosage forms; International Journal of Chemical and Pharmaceutical Science, (2010); 1: 28 32. 12. Mayank, J., Sukriti, T., Vinay Kumar, M., Sugat, S., Saima, S.; Simultaneous estimation of amolodipine Besylate and Nebivolol hydrochloride in combined dosage form by RP-HPLC; International Journal of Pharmacy & Life Sciences, (2010); 1: 428 432. 13. Packirisamy, T.M., Saravanan, C., Kumar, M., Jayakar, B.; Analytical method development and validation of Nebivolol HCl in tablet dosage form by RP-HPLC method; International Journal of Chemical and Analytical Sciences, (2010); 1: 134 135. 14. Ramakrishna, N.V.S., Vishwottam, K.N., Koteshwara, M., Manoj, S., Santosh, M., Varma, D.P.; Rapid quantification of Nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis, (2005); 39: 1006 1013. 15. Al-Othman, Z.A., Ali, I.; Rapid and economic Chiral-HPLC method of Nebivolol enantiomers resolution in dosage formulation; Biomedical Chromatography, (2012); 26: 775 780. 16. Aboul-Enein, H.Y., Ali, I.; HPLC enantiomeric resolution of Nebivolol on normal and reversed amylose based chiral phases; Die Pharmazie, (2001); 56: 214 216. 17. Aboul-Enein, H.Y., Ali, I.; Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantio separation of Nebivolol by HPLC; Journal of Biochemical and Biophysical Methods, (2001); 48: 175 188. 18. Aboul-Enein, H.Y.; High-performance liquid chromatographic enantioseparation of drugs containing multiple chiral centers on poly saccharide-type chiral stationary phases; Journal of Chromatography A, (2001); 906: 185 193. 19. ICH Q2 (R1), Validation of Analytical Procedures: Text and Methodology, 2005. 20. Cecilia, B.C., Peter, W.C.; Fast enantioseparations of basic analytes by high-performance liquid chromatography using cellulose tris(3,5- dimethylphenylcarbamate)-coated zirconia stationary phases; Journal of Chromatography A, (2000); 904: 17 33. 1058 Visweswara Rao et al.