指示稳定性的反相超高效液相色谱评价原料药和制剂配方中奈必洛尔杂质的方法建立与验证

Nebivolol （Fig.1），α，α′［iminobis （methylene）］bis ［6-fluro-3，4-dihydro-2H-1-benzopyran-2-methanol］hydrochloride is an antihypertensive drug ［1，2］. Nebivolol hydrochloride occurs in two isomeric forms ［3］. （＋）Nebivolol acts as strong adrenergic β1 blocker whereas （-）nebivolol as vasodilator［4，5］.

Impurity profiling of active pharmaceutical ingredients （API）and pharmaceutical formulations is one of the most challenging tasks of pharmaceutical analytical chemists under industrial environment［6］. The presence of unwanted or in certain cases unknown chemicals，even in small amounts，may influence not only the therapeutic efficacy but also the safety of the pharmaceutical products［7］. For these reasons，all major international pharmacopoeias have established maximum allowed limits for related compounds for both bulk and formulated APIs. As per the requirements of various regulatory authorities，the impurity profile study of drug substances and drug products has to be carried out using a suitable analytical method in the final product［8，9］.

Fig.1 Chemical structures of nebivelol and its impurities

A detailed literature survey revealed that there were some analytical methods reported for estimation of nebivolol either individually or in combination with other drugs like high performance thin layer chromatography （HPTLC），spectrophotometric，by derivative spectrophotometry and by HPLC［10-31］. The route of synthesis of nebivolol and possible degradants resulted five known impurities which are not reported in any of the pharmacopeia.

Till date there is not a single method has been reported for the determination of the impurities either in bulk drugs or in pharmaceutical formulations of nebivolol. It is felt necessary to develop a stability indicating method for nebivolol related impurities in API and tablet dosage formulation.

Hence，an attempt has been made to develop an accurate，rapid，specific and reproducible method for determination of nebivolol impurities（Fig.1）in API and in pharmaceutical dosage forms along with method validation as per International Conference on Hormonization （ICH）norms［32，33］. The stability tests were also performed on both drug substances and drug products as per ICH norms［34，35］.

1 Experimental

1.1 Reagents and materials

Nebivolol tablets were received from formulation research and development laboratory of Dr.Reddy’s Laboratories Ltd.，Integrated Product Development Organization （IPDO），and Hyderabad，India. Nebivolol API and impurities were received from Dr. Reddy’s Laboratories Ltd.，Chemical Technical Operation Unit VI，and Hyderabad，India. Dipotassium hydrogen orthophosphate，1-octane sulphonic acid sodium salt monohydrate and hydrochloric acid were procured from Merck，Germany. HPLC grade methanol and ortho phosphoric acid were purchased from Merck，Germany，and high-purity water was prepared by Millipore Milli Q plus purification system.

1.2 Equipment

Waters UPLC system with a photo diode array（PDA）detector （Model，binary gradient）was used for method development and method validation. The output signal was monitored and processed using Waters Empower software. Weighing was performed with a Mettler XS 205 Dual Range（Mettler-Toledo GmbH， Greifensee， Switzerland）. Photo stability studies were carried out in a photo stability chamber （SUN TEST XLS＋，Atlas，USA）. Thermal stability studies were performed in a dry air oven （Merck Pharmatech，Hyderabad，India）.

1.3 Chromatographic system

UPLC measurements were carried out using an Acquity BEH C18 column （100 mm×2.1 mm，1.7 μm，Waters）operated at 30 ℃with gradient elution at 0.18 mL／min. The mobile phase buffer was a mixture of 0.02 mol／L dipotassium hydrogen orthophosphate and 0.005 mol／L of 1-octane sulfonic acid sodium salt monohydrate containing 5 mL of triethyl amine （pH adjusted to 6.5 with ortho phosphoric acid solution）. UV detection was set at 281 nm，and injection volume was 5 μL. The mobile phase A consisted of pH 6.5 buffer and methanol （60 ∶40，v／v）；mobile phase B consisted of pH 6.5 buffer and methanol （20 ∶80，v／v）.The LC gradient program was set as：time （min）／mobile phase A （%）／mobile phase B （%）（v／v）：0.01／70／30，1.0／70／30，4.2／60／40，7.0／60／40，18.2／55／45，25.0／20／80，42.0／5／95，46.0／5／95，47.0／70／30 and 55.0／70／30. Totally 0.01 mol／L hydrochloric acid and methanol（20 ∶80，v／v）was used as diluent for sample preparation.

1.4 Preparation of standard solution and system suitability solution

A stock solution of nebivolol （550 μg／mL）was prepared by dissolving an appropriate amount in diluent. Working solution was prepared from the above stock solution for related substances determination （2.75 μg／mL of nebivolol）in diluent. A mixture of all impurities （2.75 μg／mL）along with nebivolol （550 μg／mL）was prepared in diluent. Also impurity stock solutions were prepared in diluent.

1.5 Preparation of test solution

Twenty tablets （nebivolol labeled：2.5，5，10 and 20 mg per tablet）were weighed and the average weight of tablet was calculated. The tablet powder equivalent to 55 mg of active pharmaceutical ingredient （nebivolol）was transferred into a 100 mL volumetric flask. To this flask 70 mL of diluent was added and the flask was sonicated for 20 min with intermediate shaking. The solution was then diluted to 100 mL with diluent and centrifuged at 4 000 r／min for 10 min. The supernatant （550 μg／mL of nebivolol）was collected and used as sample solution.

1.6 Method validation

The proposed method was validated as per ICH guidelines［33］.

1.6.1 System suitability

System suitability parameters were performed to verify the system performance. System precision was determined by six replicate injections of standard preparation. All the important characteristics，including the relative standard deviation，peak tailing，and theoretical plate number，were measured. The resolution between impurities was measured by injecting system suitability solution.All these system suitability parameters covered the system，method and column performance.

1.6.2 Specificity

Stress studies were performed at an initial mass concentration of 550 μg／mL of nebivolol in APIs and formulated sample to provide the stabilityindicating property and specificity of the method.Intentional degradation was attempted by the stress conditions of exposed to acid （1 mol／L HCl for 3 h at 60 ℃），base （0.1 mol／L NaOH for 3 h at 60 ℃），oxidation （15% （v／v）peroxide for 9 h on bench top），water （refluxed for 6 h at 60 ℃），heat （exposed at 105 ℃for 6 h），humidity （exposed to 90% relative humidity （RH）for 7 d）and photolytic stress （1.2 million lux hours followed by 200 Wh／m2）.

1.6.3 Precision

The precision for the determination of the impurities was checked by injecting six individual preparations of nebivolol （550 μg／mL）spiked with 0.825 μg／mL of each （R*S*）NBV-1，（RRSS）NBV-3，（RSRS）NBV-3，（R*S*S*S*）NBV-2 impurities and 2.75 μg／mL of monodesfluoro impurity，and calculating the RSD of each impurity.The intermediate precision of the method was also evaluated by different analysts using a different instrument in the same laboratory on a different day. Also precision and intermediate precision study were performed by spiking 0.825 μg／mL nebivolol in placebo as per test preparation.

1.6.4 Accuracy

Accuracy of the method was demonstrated at five different concentration levels in triplicate.The samples were prepared in triplicate by spiking nebivolol hydrochloride impurities in test preparation with 50%，100%，150%，250% and 500% of 0.5% （v／v）of （R*S*）NBV-1，（RRSS）NBV-3，（RSRS）NBV-3，（R*S*S*S*）NBV-2 and monodesfluoro impurity with respect to test mass concentration （550 μg／mL）. Also accuracy study was performed by spiking nebivolol in placebo at the above mentioned levels. The percentage mean recoveries at each level for all the impurities and nebivolol were calculated.

1.6.5 LODs and LOQs

The LODs and LOQs for all the impurities and nebivolol were estimated at S／N ＝3 and S／N ＝10 respectively，by injecting a series of dilute solutions with known concentrations. Precision and accuracy study was also carried out at LOQ level.

1.6.6 Linearity

Linearity solutions were prepared from stock solutions at six concentration levels from LOQ to 1.0% of （R*S*） NBV-1， （RRSS） NBV-3，（RSRS）NBV-3，（R*S*S*S*）NBV-2，monodesfluoro impurity and 1.2% of nebivolol with respect to the analyte concentrations. The peak area versus concentration data were subjected to leastsquares linear regression analysis. The calibration curve was drawn by plotting impurity areas against the mass concentration expressed in μg／mL.

1.6.7 Robustness

To determine the robustness of the developed method，experimental conditions were deliberately changed. The resolution between nebivolol and its impurities，tailing factor and theoretical plates of nebivolol peak were evaluated.

To study the effect of the flow rate on the developed method， it was changed from 0.18 mL／min to 0.16 mL／min and 0.20 mL／min. The effect of column temperature on the developed method was studied at 25 ℃and 35 ℃（instead of 30 ℃）. The effect of pH was studied by varying ±0.2 pH units （i. e. 6.3 and 6.7）and the mobile phase composition was changed ±10% from the initial composition. In all the above varied conditions，the component of the mobile phase was held constant.

1.6.8 Stability in solution and mobile phase

Nebivolol spiked samples were prepared in the diluent and leaving the test solutions at room temperature. The spiked samples were injected at 0，24，48 h time intervals. The impurity content was calculated，and the consistency of each impurity at each interval was checked. The prepared mobile phase was kept constant during the study period. The mobile phase study was demonstrated by injecting the freshly prepared sample solution at different time intervals （0-2 d）.

2 Results and discussion

2.1 Optimization of chromatographic conditions

The main criterion for developing an RP-UPLC method for the determination of impurities in nebivolol pharmaceutical dosage form in a single run，with emphasis on the method being accurate，reproducible，robust，stability-indicating，linear，free of interference from other formulation excipients and convenient enough for routine use in quality control laboratories.

Individual stock solutions of nebivolol and its impurities were scanned in photo diode array detector in the range of 200 to 400 nm and the spectra of each component were checked. From the spectra，all the impurities were having maximum absorbance at about 281 nm. Hence 281 nm was selected for the estimation of nebivolol impurities.

Nebivolol sample preparation （550 μg／mL）spiked with all the impurities （2.75 μg／mL）and placebo preparations were subjected to separation by RP-UPLC. Initially，the separation of all peaks were studied using 0.005 mol／L dipotassium hydrogen phosphate of pH 7.0 （pH adjusted with dilute ortho phosphoric acid solution）as mobile phase A and mixture of acetonitrile，methanol ＆Milli Q water （45 ∶45 ∶10，v／v／v）as mobile phase B on a reverse phase BEH C18 column （100 mm×2.1 mm，1.7 μm）. Waters （UPLC）system with gradient elution was used at 0.18 mL／min flow rate. Poor separation was observed between nebivolol and one unknown impurity at relative retention time of 1.04. Resolution between all the remaining impurities were found to be satisfactory. To achieve better separation，different gradient programmes by changing different mobile phases were tried，but the separation was not up to the mark.

Further separation condition was tried by adding 1-octane sulfonic acid sodium salt monohydrate as ion pair reagent and triethyl amine as additive in the mobile phase with different pH of the mobile phase buffers. Finally the chromatographic separation was achieved by using an Acquity BEH C18 column （100 mm×2.1 mm，1.7 μm）operated at 30 ℃ with gradient elution at 0.18 mL／min using a mobile phase buffer of a mixture 0.02 mol／L dipotassium hydrogen orthophosphate and 0.005 mol／L of 1-octane sulfonic acid sodium salt monohydrate containing 5 mL of triethyl amine （pH adjusted to 6.5 with ortho phosphoric acid solution）；UV absorbance at 281 nm；injection volume 5 μL. The mobile phase A consisted of pH 6.5 buffer and methanol （60 ∶40，v／v）；and the mobile phase B consisted of pH 6.5 buffer and methanol （20 ∶80，v／v）. The LC gradient program was set as：time （min）／mobile phase A（%）／mobile phase B（%）（v／v）：0.01／

70／30，1.0／70／30，4.2／60／40，7.0／60／40，18.2／55／45，25.0／20／80，42.0／5／95，46.0／5／95，47.0／70／30 and 55.0／70／30. All the impurities were well separated with a resolution greater than 2.No chromatographic interference due to the blank（diluent）and other excipients （placebo）at the retention times of nebivolol or any of the impurities were observed.

2.2 Results from forced degradation studies

All forced degradation samples were analyzed with the aforementioned HPLC conditions using a PDA detector to monitor the homogeneity and purity of the nebivolol peak and its related impurities. Individual impurities，placebo and nebivolol were verified and proved to be non-interfering with each other and thus it proved the specificity of the method.

There is no interference at the retention times of nebivolol and all known impurities from the other excipients. Degradation was not observed in acid stress， base stress， water stress， heat stress，humidity stress and photo stress studies.Significant degradation was observed in peroxide stress study. It is interesting to note that all the peaks due to degradation were well resolved from the peaks of nebivolol and its impurities. Further the peak purity of nebivolol or its impurities was found to be homogeneous based on the evaluation parameters such as purity angle and purity threshold using Waters Empower networking software.The verification of peak purity indicates that there is no interference from degradants，facilitating error-free quantification of nebivolol impurities. Also the mass balance of stressed samples was found to be more than 98%. Thus，the method is considered to be“stability-indicating”. The specificity results are shown in Table 1.

2.3 Results from method validation

After development the method was subjected to validation as per ICH guideline［33］. The method was validated to demonstrate that it is suitable forits intended purpose by the standard procedure to evaluate adequate validation characteristics （system suitability，specificity，accuracy，precision，linearity，robustness，ruggedness，solution stability，LOD，LOQ and stability-indicating capability）.

Table 1 Forced degradation data for nebivolol tablets

2.3.1 System suitability

The RSD of peak area from six replicate injections was below 5.0% （diluted standard solution，2.75 μg／mL of nebivolol）. Low values of RSD for replicate injections indicate that the system is precise. The results of other system suitability parameters such as resolution，peak tailing and theoretical plates are presented in Table 2. The acceptable system suitability parameters would be as follows：the RSD of replicate injections was not more than 5.0%，resolution between impurities was 2.0，the tailing factor for nebivolol was not more than 1.5 and the theoretical plates were not less than 10 000.

Table 2 Results of system suitability

2.3.2 Precision

The RSD for the individual of all the impurities and nebivolol in method precision study was within 4.2%. The results obtained in the intermediate precision study for the RSD of the individual of all the impurities were well within 6.0%，conforming high precision of the method. The results are shown in Table 3.

Table 3 Results of the precision （n＝6）

2.3.3 Accuracy

The recoveries of all five impurities and nebivolol from finished pharmaceutical dosage form ranged from 85.0% to 115.0% （Table 4）.

Table 4 Accuracy of the method

2.3.4 Results of LOD and LOQ

LOD and LOQ values for all the impurities and nebivolol are shown in Table 5. The RSDs of precision at LOQ concentration for all the impurities and nebivolol were found below 5.0%. The results of precision at LOQ level are shown in Table 6.

Table 5 LODs，LOQs and regression statistics

Table 6 Results of the precision at the limits of quantification （n＝6）

2.3.5 Linearity

Linear regression analysis demonstrated acceptability of the method for quantitative determination range of LOQ to 6.6 μg／mL. The correlation coefficients were found to be more than 0.995.The regression statistics are shown in Table 5.

2.3.6 Robustness

No significant effect was observed on system suitability parameters such as resolution，RSD，tailing factor，or the theoretical plates of nebivolol when small but deliberate changes were made to chromatographic conditions. The results are presented in Table 2，along with the system suitability parameters of normal conditions. Thus，the method was found to be robust with respect to variability in applied conditions.

2.3.7 Stability in solution and in the mobile phase

No significant changes were observed in the content of impurities during solution stability and mobile phase stability experiments when performed the impurities method. The solution stability and mobile phase stability experiment data confirm that the sample solution and mobile phases used during the impurity determination were stable for at least 48 h.

3 Conclusions

The gradient UPLC method developed for the determination of nebivolol impurities in both bulk drug and pharmaceutical dosage form was precise，accurate and specific. The method was validated as per ICH guidelines and found to be specific，precise，linear，accurate，rugged，and robust. The developed method can be used for the stability analysis of both nebivolol API and formulated samples.

Acknowledgement

The authors wish to thank the management of Dr. Reddy’s group for supporting this work. Authors wish to acknowledge the formulation development group for providing the samples for our research.

［1］ Budavari S. The Merck Index. 14th ed. Hunterdon County：White House Station，2006：1112

［2］ Martindale W. Martindale：the Extra Pharmacopoeia. 30th ed. New York：Pharmaceutical Press，1993：637

［3］ Siebert C D，Hansicke A，Nagel T. Chirality，2007，20：103

［4］ Pauwels P J，Gommeren W，Van Lommen G，et al. Mol Pharmacol，1988，34：843

［5］ Cockcroft J R，Chowienczyk P J，Brett S E，et al. J Pharmacol Exp Ther，1995，274：1067

［6］ Gorog S. Talanta，1997，44：1517

［7］ Sanjay B B，Bharati R K，Yogini S J，et al. Eurasian J Anal Chem，2007，2：32

［8］ ICH，Q3A （R2），2006

［9］ ICH，Q3B （R2），2006

［10］ Shirkhedkar A A，Bugdane P M，Surana S J. Pharmacol Rev，2008：141

［11］ Rajeshwari K R，Sankar G G，Rao A L，et al. Asian J Chem，2005，17：1259

［12］ Aboul-Enein H Y，Ali I. Pharmazie，2001，56：214

［13］ Aboul-Enein H X. Biomed Chromatogr，2003，17：111

［14］ Thevis M，Opfermann G，Schanzer W. Biomed Chromatogr，2001，15：393

［15］ Ramakrishna N V S. J Pharm Biomed Anal，2005，39：1006

［16］ Sheshashena Reddy T，Sita Devi P. JPC-J Planar Chromat，2007，20：149

［17］ Patel L J，Suhagia B N，Shah P B. Indian J Pharm Sci，2007，69：594

［18］ Deepak S，Anurekha J，Alankar S. Pharm Methods，2011，2（1）：9

［19］ Chandnani V C，Gupta K R，Chopde C T，et al. Int J ChemTech Res，2010，2（1）：69

［20］ Khandelwal N，Ranawat M S，Amardeep A，et al. Int J ChemTech Res，2011，3（1）：290

［21］ Priyanka B P，Chetan B C，Dev A J，et al. Int J ChemTech Res，2012，4（3）：1241

［22］ Keyur B A，Emanual M P，Falgun A M. J Chromat Separation Techniq，2012，3（5）：152

［23］ Della G T P，Molly M，Anila J，et al. Int J Pharm Sci Rev Res，2010，3（2）：139

［24］ Anilkumar S，Bhavesh P，Rakshit P. IJPBS，2010，1（4）：339

［25］ Chetan M B，Hanumanthachar K J，Jayanthi C. IJRPBS，2012，3（4）：1594

［26］ Pradeep M，Kamal S，Alka G. Int J Pharm Pharm Sci，2009，1（2）：5

［27］ Gajbhiye A，Dwivedi N. Curr Trends Tech Sci，2012，1（2）：118

［28］ Rao A L，Rajeswari K R，Sankar G G. E-J Chem，2010，7（2）：445

［29］ Tripti S，Rajesh P，Dannana G S，et al. Asian J Pharm Clin Res，2012，5：69

［30］ Sirisha N，Haripriya A，Swetha B N，et al. Der Pharmacia Lettre，2013，5（2）：78

［31］ Siddartha B，Sudheer B I，Uttam P P. IJPRBS，2014，3（2）：215

［32］ Rajagopalan R. Separ Sci Technol，2004，5：27

［33］ ICH，Q2（R1），2005

［34］ ICH，Q1A（R2），2003

［35］ ICH，Q1B，1996