Development of Stability Indicating HPLC method for the ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701regression curves. The LOD and LOQ for Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-Eand Impurity-F were determined by injecting a seriesof dilute solutions with known concentrations 12 .LinearityThe Linearity of peak areas verses differentconcentrations was evaluated for Zaleplon and all therelated substances using 6 concentration levelsranging from LOQ to 150% of the specification level.The above tests were carried out for threeconsecutive days in the same concentration range forrelated substance method.AccuracyThe accuracy of the method for all the relatedsubstances was determined by analyzing Zaleplonsample solutions spiked with all the relatedsubstances at three different concentration levels of50, 100 and 150% of each in triplicate at the specifiedlimit. The percentage of recoveries for the impuritieswas calculated from the slope and Y-intercept of thecalibration curve obtained from linearity studies.RobustnessTo determine the roubustness of the developedmethod, experimental conditions were deliberatelyaltered and the resolution between Zaleplon,Impurity-A, Impurity-B, Impurity-C, Impurity-D,Impurity-E and Impurity-F was recorded. Theparameters selected were mobile phase compositon(±2% of gradient composition), pH of the mobiliephase (±0.2 units), flow rate (± 10%), wavelength (±5 nm) and column temperature (± 5°C). The effect ofthe percent organic strength on the resolution wasstudied by varing acetonitrile by -5 to +5% whileother mobilie phase components were held constant.Solution stability and mobile phase stabilityTo determine the stability of sample solution, thesample solutions of Zaleplon spiked with relatedsubstances at specified level were prepared andanalyzed immediately after preparation and afterdifferent time intervals up to 15h, while maintainingthe sample cooler temperature at about 25°C. Theresults from these studies indicated, the samplesolution was stable at room temperature for at least15 h.RESULTS AND DISCUSSIONOptimization of chromatographic conditionsThe main objective of the chromatographic methodwas to seperate Zaleplon from Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-E and Impurity-F. Impurities were coeluted using different stationaryphases such as C18 phenyl and cyano as well asdifferent mobile phases. The chromatographicseparation was achieved on a YMC Pack C8 , (150 4.6 mm), 5 particles. The gradient LC methodemploys solution A and B as mobile phase. Thesolution A contains phosphate buffer pH 3.0 andacetonitrile as solution B. The flow rate of the mobilephase was 1.5 ml/min and the peak shape of theZaleplon was found to be symmetrical. In theoptimized chromatographic conditions of Zaleplon,Impurity-A, Impurity-B, Impurity-C, Impurity-D,Impurity-E and Impurity-F were separated with aresolution greater than 2.5, typical relative retentiontimes were approximately 0.15, 0.54, 0.59, 0.70,1.11, 1.60 with respect to Zaleplon eluted at 10.971(Figure 2). The system suitability results are given inTable 1 and the developed LC method was found tobe specific for Zaleplon and all of its six impuritiesnamely Impurity-A, Impurity-B, Impurity-C,Impurity-D, Impurity-E and Impurity- F (Figure 2).N-[3-(3-Cyanopyrazolo [1,5-a]pyrimidin-7-yl)phenyl]-N-ethylacetaamide (Zaleplon)H 2 NNNHCN3-Amino-4-Cyanopyrazole (Impurity-A)CH 3CH 3NOON CH 3CH 3N-[3-[3-(Dimethylamino)-1-oxo-2-propenyl]phenyl]-N-ethylacetamide (Impurity -B)Vol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1426

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701NNNC O N H 2ON C H 3C H 3N-[3-(3-Carboxamidopyrazolo [1,5-a]pyrimidin-7-yl)phenyl]-N-ethylacetamide (Impurity -C)NNNC NON C H 3HN-[3-(3-Cyanopyrazolo [1,5-a]Pyrimidin-7-yl)phenyl]acetamide (Impurity -D)CH 3OCH 3NNNNCNN-[3-(3-Cyanopyrazolo [1,5-a]pyrimidin -5-yl)phenyl]-N-ethylacetamide(Impurity -E)CH 3OCH 3NOON CH 3CH 3N-[3-(3-Cyano-6-[(E)-3-(N-ethyl-Nacetyl)amino)phenyl-3-oxoprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]phenyl]-N-ethylacetamide(Impurity -F)NNNCNFig. 1: Chemical structures of Zaleplon and itsimpuritiesValidation of the MethodForced DegradationTo demonstrate the specificity and stability indicatingcharacteristics of the method, samples of Zaleplonwere subjected to various stress conditions such as5M HCl acid, 5M NaOH base, 30% v/v H2O2peroxide, Heat ( 105°C), photolytic (10 K Lux, 144h) and humidity degradation (92% RH, 144 h). Therewas no degradation observed in Zaleplon bulksamples under stress conditions such as thermal,photolytic and humidity degradation. Thedegradation of drug substance was observed duringacid, base hydrolysis and oxidative stress condition(Figure 2). Some unknown degradation peaks wereobserved under acidic conditions (5M HCl/85°C/120min). Zaleplon was degraded to Impurity-A and someunknown degradation peaks under base conditions(5M NaOH / 10 min). Zaleplon was degraded toImpurity-B and some unknown degradation peaksunder oxidative environment (treated with 30%H2O2/85°C/120 min). No interference from blank atRetention time of zaleplon as seen in Figure 2 (a).Selectivity of the method was performed byseparately injecting individual impurities, and noneof these impurities interfere with the Zaleplon peakwith minimum resolution of 2.5 between any twopeaks.Peak purity test results obtained by using a PDAdetector confirmed that the Zaleplon peak ishomogenous and pure in all the analyzed stresssamples. The summary of the forced degradationstudies was given in Table 3.PrecisionIn the study of the precision of the Zaleplon relatedsubstance method RSD of peak area of impuritiesImpurity-A to F was 5.3%. In the intermediateprecision study, %RSD for the area of all the siximpurities were well within 3.8%, conforming goodprecision of the method.Limit of detection (LOD) and limit ofquantification (LOQ)The values of LOD and LOQ for Zaleplon were0.050 µg/ml, 0.101 µg/ml and they were for relatedsubstances, in the ranges; 0.075-0.101 µg/mlrespectively. The calculated LOD and LOQconcentrations were verified for precision. RSD wasin the range of 11.3-16.8 for LOD and 3.2-5.9 % forLOQ respectively. The results were depicted in Table2.LinearityLinear calibration plot for the related substancemethod was obtained over the calibration rangestested, i.e., LOQ to 150% for impurities, Impurity-A,Vol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1427

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Impurity -B, Impurity-C, Impurity-D, Impurity-E andImpurity-F. The correlation co-efficient obtained wasgreater than 0.9951. Linearity was checked forrelated substance method over the sameconcentration range for three consecutive days. Theabove result show that an excellent correlationexisted between the peak area and the concentrationof all six impurities.AccuracyThe Accuracy of all these related substances wasfound to be in between the predefined acceptancecriterion of 94.7-107.4 and the data was given Table2.RobustnessWhen the chromatographic conditions flowrate,column temperature amount of organic solvent in themobile phase, pH were deliberately varied andresolution between the critical pair, i.e. Impurity-Eand Impurity-F was greater than 2.5, illustrating therobustness of the method.Solution StabilityThere were no significant changes in the amounts ofthe impurities during solution stability experimentperformed using the related substances method. Theresults from the studies indicated, the sample solutionwas stable at room temperature for at least 15 h.Fig. 2(a): BlankFig. 2(b): Zaleplon SampleFig. 2(c): Zaleplon sample spiked with all impurities at 0.1% specification levelVol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1428

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Fig. 2(d): Zaleplon stressed with 5M HCl at 85°C for 120 minsFig. 2(e): Zaleplon stressed with 5M NaOH at room temperature for 10 minFig. 2(f): Zaleplon stressed with 30%H2O2 at 85°C for 120 minsFig. 2: Chromatograms of Zaleplon a) blank, b) sample, c) sample spiked with allimpurities at 0.1% specification level, d) acid, e) base and f) peroxide degradationVol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1429

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 1: System suitability resultsS. No. Impurity name USP Plate count USP Tailing USP Resolution1 Impurity-A 3427 1.11 -2 Impurity-B 24539 1.07 35.103 Impurity-C 34391 0.99 2.544 Impurity-D 36783 0.96 6.755 Zaleplon 53348 0.93 12.856 Impurity-E 76853 0.95 6.447 Impurity-F 125944 0.93 13.42Table 2: Linearity, LOD, LOQ, Precision and Accuracy data for Zaleplon and its impuritiesTest Parameter Impurity-A Impurity-B Impurity-C Impurity-D Zaleplon Impurity-E Impurity-FLinearityr 0.9992 0.9972 0.9970 0.9951 0.9988 0.9969 0.9968Slope 22619 16552 36383 50980 45249 29659 23142Intercept 60 66 -148 117 675 -256 -1854RF 2.00 2.73 1.24 0.89 1.00 1.53 1.96LODCon (µg/ml) 0.101 0.126 0.075 0.050 0.050 0.101 0.125%RSD 16.8 11.3 12.7 14.3 13.6 14.4 15.7LOQCon (µg/ml) 0.203 0.253 0.150 0.101 0.101 0.202 0.251%RSD 5.9 3.9 3.2 5.8 2.9 4.7 5.4Accuracy % 94.7-101.3 98.0-99.3 106.7-107.4 95.4-103.6 98.1-100.2 95.4-103.6 95.3-95.7Precision(%RSD n=6)5.3 2.0 1.0 1.9 1.0 1.0 0.5Table 3: Forced degradation studies dataDegradation Mechanism Degradation ConditionZaleplon DegradationPeak Purity(% Area) (%) Purity Angle Purity Threshold- Undegraded 99.86 - 0.063 0.248Acid degradation5M HCl / 85°C /120 min.92.59 7.3 0.049 0.244Base degradation 5M NaOH / RT / 10 min. 86.16 13.7 0.034 0.232Peroxide degradation30% H 2O 2 / 85°C /120 min.89.82 10.1 0.057 0.377Thermal degradation 105°C / 144 Hours 99.86 Nil 0.060 0.248Photolytic degradation 10K Lux / 144 Hours 99.87 Nil 0.071 0.250Humidity degradation92% RH / 25°C /144 Hours99.85 Nil 0.055 0.246CONCLUSIONA new, accurate and selective gradient RP-HPLCmethod is proposed for the determination of Zaleplonrelated substances in Zaleplon drug substance andvalidated as per the ICH guidelines. The method isfound to be simple, selective, precise, accurate androbust. Therefore, this method can be used for routinetesting as well as stability analysis of Zaleplon drugsubstance. All statistical results (Percentage, Mean,RSD, Percentage difference and recovery %) werewithin the acceptance criteria.ACKNOWLEDGEMENTSThe authors wish to thank the management ofAurobindo Pharma Limited Research Centre for theirsupport, encouragement and permitting this work toVol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1430

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701communicate for publication. Authors also wish tothank the Chemical research and Analytical researchdepartments for their cooperation.REFERENCES1. Sweetmann C S., Martindale-The completedrug reference, 2011; 37 th Edition: p.1142.2. Horstkotter C., Schepmann D., Blaschke G.,Separation and identification of zaleplonmetabolites in human urine using capillaryelectrophoresis with laser-inducedfluorescence detection and liquidchromatography-mass spectrometry. Journalof Chromatography A, 2003; 1014, 71-81.3. Metwally FH., Abdelkawy M., AbdelwahabNS., Application of spectrophotometric,densitometric, and HPLC techniques asstability indicating methods fordetermination of Zaleplon in pharmaceuticalpreparations. Spectrochim Acta A MolBiomol Spectrosc. 2007; 68(5):1220-30.4. Daniel T., Anderson and Julie L., ElbogenDetermination of Specific Absorbance (A ¦)for Zaleplon (Sonata ® ) bySpectrophotometry. J Anal Toxicol 2009;33(8): 478-480.5. Jing QU., Xiu zhen X., Xi ming W et al ;Determination of the content in zaleplontablets by ultraviolet spectrophotometrydetermination . Shanxi Yike Daxue Xuebao2003; 34(6): 572.6. Feng F., Juanjuan Jiang., Hui Dai., andJieWu., Development and Validation of aHigh-Performance Liquid Chromatography–Electrospray Ionization–Mass SpectrometryAssay for the Determination of Zaleplon inHuman Plasma. Journal of ChromatographicScience 2003; 41(1):17-21.7. Ming D., Sufen Z., Jianfang L., and HuichenL., Determination of Zaleplon in HumanPlasma by RP - HPLC with FluorescenceDetection. Yaowu Fenxi Zazhi 2004; 24(6):611-613.8. Beibei Z., Zunjian Z., Yuan T., Fengguo X.,Yun C., Determination of zaleplon and itspharmacokinetics in human plasma byHPLC-APCI-MS. Zhongguo YiyuanYaoxue Zazhi 2005; 25(10): 916-920.9. Bharathi Ch., Joseph Prabahar K., PrasadCh. S., Saravana Kumar M., Magesh S.,Handa V.K., Ramesh D., Naidu A., Impurityprofile study of zaleplon. Journal ofPharmaceutical and Biomedical Analysis2007; 44: 101-109.10. ICH, Q2A: Text on Validation of AnalyticalProcedures, International Conference onHarmonization. October 1994.11. ICH, Q3B: Validation of AnalyticalProcedures: Methodology, InternationalConference on Harmonization. November1996.12. ICH Harmonised Tripartite Guideline, Q2(R1): Validation of Analytical Procedures:Text and Methodology. November 2005.Vol. 3 (4) Oct – Dec 2012 www.ijrpbsonline.com 1431