Analytical Method Development And Validation Of Bendamustine In Bulk .
Open Access Journal of Pharmaceutical ResearchISSN: 2574-7797Analytical Method Development and Validation ofBendamustine in Bulk Using RP-HPLCBhawani S*, Nageshwari HG, Mamatha G, Venu M, Sai Krishna M andResearch ArticleMurali Krishna KSVolume 2 Issue 2Department of analysis, MLR institute of pharmacy, IndiaReceived Date: May 31, 2018*Corresponding author: Sunkara Bhawani, Department of analysis, MLR institute ofPublished Date: August 01, 2018pharmacy, Dundigal, Quthubullapur Mandal, Hyderabad 500043, RR Dist, Telangana, Email: banu.sunkara@gmail.comAbstractA simple, sensitive, precise, accurate and economical method was developed and validated for the estimation ofBendamustine in bulk or formulation by RP-HPLC method using Inertsil column ODS of dimensions ODS -2, (150 x 4.6)mm, 5 µm. The mobile phase (trifluoroacetic acid and acetonitrile ) was pumped at a flow rate of 1.5ml/min in the ratio of68:32 and the eluents were monitored at 230 nm. Linearity was obtained in the concentration range of 10%-150% withR2 0.999. LOD and LOQ were found to be 2.9 µg/ml and 8.75 µg/ml. The method was statistically validated according toICH guidelines. RSD was found to be less than 2% indicating high degree of accuracy and precision of the proposed HPLCmethod. The percentage recovery was in good agreement with the labeled amount in the pharmaceutical formulations.Keywords: Treanda; HPLC; Trifluoroacetic acidIntroductionBendamustine bearing the name Treanda is achemotherapic medication used in the treatment ofchronic lymphocytic leukemia, multiple myeloma, andnon-hodgkins lymphoma. Bendamustine is a white, watersolublemicrocrystallinepowderwith amphoteric properties. It acts as an alkylating agentcausing intra-strand and inter-strand cross-linksbetween DNA bases. After intravenous infusion it isextensively metabolised in the liver by cytochrome p450.More than 95% of the drug is bound to protein –primarily albumin. Only free bendamustine is active.Elimination is biphasic with a half-life of 6–10 minutesand a terminal half-life of approximately 30 minutes. It iseliminated primarily through the kidneys. Combinationtherapy with bendamustine and rituximab hasdemonstrated superior efficacy to a standard rituximabcontaining chemotherapy regimen in patients withpreviously untreated indolent B-cell non-Hodgkinlymphoma, and it is currently being compared against thestandard first-line regimen in CLL: fludarabine,cyclophosphamide, and rituximab. Ongoing and plannedstudies are evaluating new strategies in whichbendamustine is being combined with existing agents andwith novel therapies to optimize use in different clinicalsettings [1-15].Analytical Method Development and Validation of Bendamustine in Bulk Using RP-HPLCJ Pharm Res
2Open Access Journal of Pharmaceutical ResearchMaterials and MethodsInstrument nameManufacturerShimadzu LC 20 10 CHT pump,HPLCPDA DetectorInertsil ODS -2, (150 x 4.6) mm,Column5 µmShimadzu, Thermo electronUV spectrophotometercorporationElectronic balanceSartoriousUltra sonicatorSpectral lab-model UCB50Pipettes, burettes,Borosil, class-BbeakersTable 1: Instruments used in the present tic acidManufacturerHetero laboratoriesMerck, HyderabadStartech labs, HyderabadTable 2: Chemicals and Reagents used in the presentwork.Method Development and Validation ofBendamustineSelection of Wavelength for DetectionSelection of solvent: The solubility of bendamustine wasdetermined in a variety of solvents as per Pharmacopoeialstandards. Solubility test was carried out in differentsolvents like distilled water, methanol, acetonitrile, diluteethanol. From the solubility studies, it was found thatbendamustine was soluble in methanol. Methanol wasselected as suitable solvent as there will be no solventinterference while scanning in UV.Determination of wave length Maxima: UV spectrum ofbendamustine in diluent (mobile phase composition) wasrecorded by scanning in the range of 200nm to 400nm.From the UV spectrum wavelength selected as 230 nm. Atthis wavelength bendamustine shows good absorbance.Preparation of mobile phase: The mobile phase iscomposed of a mixture of trifluoroacetic acid andacetonitrile in the ratio of 68:32(v/v). Prior to use, themobile phase was degassed and filtered via 0.45 μmmembrane filter.Preparation of Buffer: Add 1.0 mL of Trifluoroacetic acidin 1000 mL water, sonicate for 10 minutes and filter using0.45 µ filters.Chromatographic ConditionsInstrumentInjection VolumeMobile PhaseColumnWavelengthFlow RateRuntimeShimadzu LC 20 10 CHT pump,PDA Detector10 μlTrifluoroacetic acid : Acetonitrile(68:32)Inertsil ODS -2, (150 x 4.6) mm,5 µm230 nm1.5 ml/mim10 minTable 3: Variables in HPLC.Validation of Rp-Hplc MethodValidation is a key process for effective qualityassurance. “Validation” is established documentedevidence, which provides a high degree of assurance thata specific process or equipment will consistently producea product or result meeting its predeterminedspecification and quality attributes. The Validationparameters are: Specificity Linearity System Suitability Parameters Precision Accuracy or Recovery Assay Ruggedness Robustness LOD and LOQSpecificity: Specificity is the ability to measure accuratelyand specifically the analyte in the presence of componentsthat may be expected to be present in the sample. It is themeasure of degree of interference (or absence thereof) inthe complex sample mixtures such as the analyte mixedwith the formulation excipients, known impurities anddegradation product.Linearity: The linearity of an analytical procedure is itsability to obtain test results which are directlyproportional to the concentration of analyte in sample.System suitability: The System suitability is an integralpart of analytical procedure. The tests are based on theconcept that the equipment, analytical operations andsamples to be analyzed constitute an integral system thatcan be evaluated as such.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
3Open Access Journal of Pharmaceutical ResearchPrecision: Precision is the degree of closeness ofagreement among individual test results when themethod is applied to multiple samplings of a homogenoussample. It is a measure of either the degree ofreproducibility (agreement under different conditions) orrepeatability (agreement under same conditions) of themethod.Ruggedness: Ruggedness is the degree of reproducibilityof the results obtained under a variety of conditions.Robustness: The robustness of an analytical procedure isa measure of its capacity to remain unaffected by small,but deliberate variations in method parameters andprovides an indication of its reliability during normalusage. Robustness study is performed by analyzing thestandard at different conditions. The results obtainedwith altered conditions are compared against resultsobtained under normal chromatographic conditions.Variation in Flow Rate ( 0. 2 mL/min): The standardwas carried out by varying the flow rate of mobile phaseto 1.3 mL/min. and 1.7 mL/min. in place of actual flowrate 1.5 mL/min.Variation in Column Oven Temperature ( 2 C): Thestandard was carried out by varying the column oventemperature of 23 C and 27 C in place of actual columnoven temperature 25 C.Variation in Organic composition ( 2% of absolute):The standard was carried out by varying the mobile phaseorganic composition of 68:32 and 72:28 in place of actualMobile phase organic composition 70:30.Accuracy: The accuracy of an analytical procedureexpresses the closeness of agreement between the valuethat is accepted either as a conventional true value or anaccepted reference value and the value found. Todemonstrate the accuracy of assay test method, drugsubstance is spiked quantitatively in to placebo from 50%to 150% of working concentration of test concentration ateach level with triplicate preparation and analyzed usingthe test method. Typical chromatogram of Accuracy at100 % level for is shown in figure 4.Results and DiscussionSelection of Chromatographic MethodProper selection of the method depends on the natureof the sample (ionic or ionisable or neutral molecules), itsmolecular weight, pka value and stability. The drugselected in the present study is polar and so reversedphase or ion exchange chromatography can be used. Thereversed phase HPLC was selected for the initialseparation because of its simplicity and suitability. Fromthe literature survey and with the knowledge ofproperties of the selected drug, ODS column was tried.ODS 150 X 4.5 mm 5μcolumn was chosen as stationaryphase and mobile phase with different compositions suchas methanol and water was used. The separations werenot observed, so the combination of buffer and methanolwas finalized. The buffer used was trifluoroacetic acidbuffer.Effect of Ratio of Mobile PhaseUnder the chromatographic conditions mentionedabove, the different ratios of buffer and acetonitrile weretried i.e. for trifluoroacetic acid buffer and acetonitrile68:32 ratios were tried at which they gave good peaksand minimum retention time and good chromatogramwith proper resolution.System SuitabilityBendamustine PeakRetention 726142791754.7261418839Mean1426616% RSD0.6Tailing factor1.2Theoretical Plate5299Injection #Table 4: Results of system suitability for bendamustineAcceptance Criteria The Tailing factor for Bendamustine peak from firstinjection of standard solution should be not more than2.0. Theoretical Plates for Bendamustine peak from firstinjection of standard solution should be not less than2000. The relative standard deviation for Bendamustine peakfrom five replicate injections of standard solutionshould be not more than 2.0%.Conclusion: The results met the acceptance criteria;hence the method is system suitable for its intended use.Linearity: The linearity of an analytical procedure is itsability to obtain test results which are directlyBhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
4Open Access Journal of Pharmaceutical Researchproportional to the concentration of analyte in sample.The linearity of Bendamustine Hydrochloride isestablished by analyzing Linearity solutions of differentconcentrations from 10 % to 150 % of workingconcentration of method for Assay. The Linearity curve isplotted for area versus concentration. The results aresummarized in table 5. Typical chromatogram of Linearityat 100 % level is shown in figure1. The linearity graph ofis shown in figure 1. Typical chromatogram of Linearity at100 % level is shown in figure 2.Linearity LevelBendamustine HCl (µg/mL)10 %520 %1050%2580 %4090 %45100 %50125 %63150 %75Correlation coefficient (R) : 0.9999Slope : 519782111795Y-intercept: 7930.9Y-intercept bias at 100%level :0.6Table 5: Results for linearity of bendamustine.2500000y 27800x 7930.R² 7043Are 0080.00ConcFigure 1: Linearity Graph of Bendamustine HCl.Acceptance Criteria: Correlation coefficient should not be less than 0.999 forBendamustine Hydrochloride. Report the slope of regression line. Report the Y-intercept of regression line. Y-intercept bias at 100 % level should be between 5.0% for Bendamustine Hydrochloride.ConclusionThe results are within the acceptance criteria; hencethe analytical procedure is linear within the concentrationrange from 10 % to 150 % (5.057µg/mL to 75.849µg/mL)for Bendamustine Hydrochloride.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
5Open Access Journal of Pharmaceutical ResearchFigure 2: Typical chromatogram of linearity.PrecisionSample #123456Mean% RSDRetention Time (Average)4.1634.1704.1744.1714.1744.179% Assay101.0100.5100.5101.6101.8101.6101.20.6Table 6: Results of Intermediate Precision.Acceptance Criteria: The relative standard deviation of results obtainedfrom six sample preparations should not be more than2.0%Conclusion: The result meets the acceptance criteria andfound comparable, indicates that the method is precise.Figure 3: Chromatogram of Precision.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
6Open Access Journal of Pharmaceutical Research12Amount(mg added)47.23547.329Amount(mg 76298.92141.432140.18699.13141.301140.407% Recovery for 9 levels% RSD for 9 levelsAccuracy Level Sample #50%100%150%% Recovery100.6100.5Average% Recovery% RSD100.50.199.40.999.10.399.499.70.8Table 7: Results for accuracy of bendamustine.Accuracy (Recovery): To demonstrate the accuracy ofassay test method, drug substance is spiked quantitativelyin to placebo from 50% to 150% of workingconcentration of test concentration at each level withtriplicate preparation and analyzed using the test method.The result for Bendamustine HCl is tabulated in Table 7.Typical chromatogram of Accuracy at 100 % level for isexhibited in figure 4.Acceptance Criteria: % Recovery at each level and overall % recoveryshould be between 98.0 and 102.0 for BendamustineHCl. The % RSD at each level and overall recovery shouldnot be more than 2.0.Conclusion: The results are well within the acceptancecriteria; hence the method is accurate for its intended use.Figure 4: Typical Chromatogram for Accuracy.SpecificitySpecificity is the ability of the analytical procedure toassess unequivocally the analyte in the presence ofcomponents which may be expected to be present.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
7Open Access Journal of Pharmaceutical ResearchNameDiluentPlacebo 25mg per vialPlacebo 100mg per vialBendamustine Hydrochloride in Standard solutionBendamustine Hydrochloride in Sample solution25mg per vialBendamustine Hydrochloride in Sample solution100mg per vialBendamustine Hydrochloride in Spiked samplesolutionRetention time (min.)No Peak detectedNo Peak detectedNo Peak detected4.595ResultsNo interference observedNo interference observedNo interference observedN/A4.670N/A4.664N/A4.662NATable 8: Specificity Results for bendamustine.Acceptance Criteria: There should be no interference at the retention time ofBendamustine peak in the Chromatograms obtainedfrom the diluent and the placebo solutions.Conclusion:No Interference is observed at the retention time ofBendamustine peak in the chromatograms obtained fromthe diluent, placebo. The results met the acceptancecriteria; hence the method is specific for its intended use.Figure 5: Typical chromatogram for specificity.RuggednessRuggedness is the degree of reproducibility of theresults obtained under a variety of conditions. It waschecked that the results were reproducible underdifferences in conditions, analysts and instruments.Procedure: The standard solution and sample solutionwere injected by different analysts and the area forinjections in HPLC was measured.InjectionAnalyst 1Analyst 2RtPeak Area4.73414398824.7311426127Table 9: Results for ruggedness of bendamustine.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
8Open Access Journal of Pharmaceutical ResearchFigure 6: Typical chromatogram for Ruggedness.RobustnessThe robustness of an analytical procedure is ameasure of its capacity to remain unaffected by small, butdeliberate variations in method parameters and providesan indication of its reliability during normal usage.Robustness study is performed by analyzing the standardat different conditions. The results obtained with alteredInjection #12345Mean% RSDTailing factorTheoretical PlatesFlow Rate1.3 ions are compared against results obtained undernormal chromatographic conditions.Variation in Flow Rate ( 0. 2 mL/min.): The standardwas carried out by varying the flow rate of mobile phaseto 1.3 mL/min. and 1.7 mL/min. in place of actual flowrate 1.5 mL/min. The results are summarized in Table 10.Actual Flow Rate1.5 .72614188394.7261427917NA1426616NA0.61.25299Flow Rate1.7 .16912496764.1691242068NA1245158NA0.21.14842Table 10: Results of Robustness-Variation in Flow rate for Bendamustine.Variation in Column Oven Temperature ( 2 C): Thestandard was carried out by varying the column oventemperature of 23 C and 27 C in place of actual columnoven temperature 25 C. The results are summarized. Theresults are summarized in Table 11.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
9Open Access Journal of Pharmaceutical ResearchInjection #12345Mean% RSDTailing factorTheoretical plateColumn Oven Temperature23 CActual Column OvenTemperature 25 CColumn OvenTemperature 27 252991.25314Table 11: Results of Robustness-Variation in Column Oven Temperature for Bendamustine.Variation in Organic composition ( 2% of absolute)The standard was carried out by varying the mobile phaseorganic composition of 68:32 and 72:28 in place of actualInjection #12345Mean% RSDTailing factorTheoretical platesMobile phase composition(68:32)Mobile phase organic composition 70:30. The results aresummarized. The results are summarized in Table 12.Actual Mobile phasecomposition (70:30)Mobile phase 895410.21.256031.252991.24568Table 12: Results of Robustness-Variation in organic composition for Bendamustine Hydrochloride.Acceptance criteria: The Tailing factor for Bendamustine peak from firstinjection of standard solution should be not more than2.0. Theoretical Plates for Bendamustine peak from firstinjection of standard solution should be not less than2000. The relative standard deviation for Bendamustine peakfrom five replicate injections of standard solutionshould be not more than 2.0%.Conclusion: The system suitability meets for each alteredconditions. The results obtained with altered conditionsare comparable with the results obtained with normalconditions. The robustness result indicates that the testmethod is robust enough as demonstrated by altering theFlow rate ( 0.2 mL/min.), column temperature ( 0.2 C)and organic composition ( 2% of absolute).Sensitivity: Limit of Detection (LOD), Limit ofQuantification (LOD) was examined by injecting sixconsecutive injections of bendamustine solutions atlowest concentration (10 μg mL-1). LOD can be calculatedby using the formula, LOD 3.3 S.D/slope. LOQ can becalculated by using the formula, LOD 10 S.D/slope.The obtained LOD and LOQ values were found to be 2.9µg/ml and 8.75 µg/ml.Summary and ConclusionA simple, sensitive and reproducible HPLC method fordetermination of bendamustine has been developed inbulk and tablet dosage forms. The validation parametersBhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
10Open Access Journal of Pharmaceutical Researchwere found to be highly agreeable, indicating smallsample volume, short retention time, system suitability,specificity, linearity, limits of detection and quantification,precision, accuracy and robustness. Hence, the proposedmethod can be easily applied for the quantification ofbendamustine in routine quality control laboratories.al. (2007) Characterization of two phase I metabolitesof bendamustine in human liver microsomes and incancer patients treated with rmacology 59(6): 759-770.8.Rasschaert M, Schrijvers D, Van den Brande J, Dyck J,Bosmans J, et al. (2007) A phase I study ofbendamustine hydrochloride administered day 1 2every 3 weeks in patients with solid tumours. BritishJournal of Cancer 96: 1692-1698.9.Preiss R, Sohr R, Matthias M, Brockmann B, Huller H(1985) The pharmacokinetics of bendamustine(Cytostasane) in humans. Die Pharmazie 40(11): 782784.References1.2.3.4.5.Dubbelman AC, Rosing H, Darwish M, D'Andrea D,Bond M, et al. (2013) Pharmacokinetics and excretionof bendamustine in patients with relapsed orrefractory malignancy. Drugs R D 13(1): 17-28.Neil O, Maryadele J (2006) The Merck index: anencyclopedia of chemicals, drugs, and biologicals.Merck Research Laboratories, Whitehouse Station, NJ,Merck.Friedberg JW, Cohen P, Chen L, Sue Robinson K,Forero-Torres A, et al. (2008) Bendamustine inPatients With Rituximab-Refractory Indolent andTransformed Non-Hodgkin's Lymphoma: ResultsFrom a Phase II Multicenter, Single-Agent Study. JClinical Oncology 26(2): 204-210.Lissitchkov T, Arnaudov G, Peytchev D, Merkle KH(2006) Phase-I/II study to evaluate dose limitingtoxicity, maximum tolerated dose, and tolerability ofbendamustine HCl in pre-treated patients with Bchronic lymphocytic leukaemia (Binet stages B and C)requiring therapy. Journal of Cancer Research andClinical Oncology 132(2): 99-104.Teichert J, Sohr R, Baumann F, Hennig L, Merkle K, etal. (2005) Synthesis and characterization of some newphase ii metabolites of the alkylator bendamustineand their identification in human bile, urine, andplasma from patients with cholangiocarcinoma. DrugMetabolism and Disposition 33(7): 984-992.6.Matt Kalaycio (2008) Clinical Experience withBendamustine: A new treatment for patients withchronic lymphocytic leukemia. Clinical Leukemia 2(4):223-229.7.Teichert J, Baumann F, Qi Chao, Franklin C, Bailey B, et10. Ivanka Pencheva, Anita Bogomilova, Neli Koseva,Danka Obreshkova, Kolio Troev (2008) HPLC study onthe stability of bendamustine hydrochlorideimmobilized onto polyphosphoesters. Journal ofPharmaceutical and Biomedical Analysis 48(4): 11431150.11. Mathrusri Annapurna M, Pavani S, Anusha S, HarikaMahanti, Venkatesh B (2012) New analytical methodsfor the determination of Bendamustine hydrochloride:An anti-neoplastic drug. J Chem Pharm Res 4(3):1696-1701.12. (2005) ICH Validation of analytical procedures: Textand methodology Q2 (R1). International Conferenceon Harmonization.13. (2003) ICH Stability Testing of New Drug Substancesand Products Q1A (R2). International Conference onHarmonization.14. Grever MR, Andritsos LA, Lozanski G (2008) Chroniclymphoid leukemias, Abeloffs Clinical Oncology,Elsevier 4th (Edn.), Philadelphia, pp: 2293-2308.15. Sasi kiran goud E, Krishna reddy V (2013)Development and validation of RP-HPLC method fordetermination of related substances of Bendamustinehydrochloride in bulk drug. Der Pharmacia Sin 4(1):29-36.Bhawani S, et al. Analytical Method Development and Validation of Bendamustine in BulkUsing RP-HPLC. Pharm Res 2018, 2(2): 000158.Copyright Bhawani S, et al.
Analytical Method Development and Validation of Bendamustine in Bulk Using RP-HPLC J Pharm Res Analytical Method Development and Validation of Bendamustine in Bulk Using RP-HPLC . Table 3: Variables in HPLC.-Hplc Method Validation is a key process for effective quality assurance. "Validation" is established documented .
Validation guidelines 1. ICH Q2A Text on validation of analytical procedures: Definitions and terminology (March 1995) 2. ICH Q2B Validation of analytical procedures: Methodology (June 1997) 3. FDA (Draft) Guidance for Industry: Analytical procedures and methods validation 4. Pharmacopoeias USP and European Pharmacopoeia Guidelines
The protocol on the validation study should include the follow-ing points in the validation study: 1) the purpose and scope of the analytical method, 2) the type of analytical method and validation characteristics, 3) acceptance criteria for each validation character-istics. Consideration on the following points will be useful to pre-
Keywords: analytical validation, pharmaceutical analysis, analytical method INTRODUCTION Analytical methods play an essential role in the adequate fulfillment of product quality attributes. However, the proper quality can only be reached if the analytical method undergoes an appropriate validation pr
Non-pharmacopoeial methods 141 5. Method validation 142 6. Method verification 143 7. Method transfer 8.144 Revalidation 145 9. Characteristics of analytical procedures 146 147 1. PRINCIPLE 148 149 1.1 This appendix presents some information on the characteristics that should be considered 150 during validation of analytical methods. Approaches .
Cleaning validation Process validation Analytical method validation Computer system validation Similarly, the activity of qualifying systems and . Keywords: Process validation, validation protocol, pharmaceutical process control. Nitish Maini*, Saroj Jain, Satish ABSTRACTABSTRACT Sardana Hindu College of Pharmacy, J. Adv. Pharm. Edu. & Res.
All of the analytical validation parameters for this proposed method were determined according to ICH guidelines. Obtained validation parameters are presented in Table 1. Table 1. Analytical validation parameters (system suitability and linearity). Parameter Azelnidipine Telmisartan Linearity (μg/ml) 10-60 μg/ml 12.25-7.5 µg/ml
EPA Test Method 1: EPA Test Method 2 EPA Test Method 3A. EPA Test Method 4 . Method 3A Oxygen & Carbon Dioxide . EPA Test Method 3A. Method 6C SO. 2. EPA Test Method 6C . Method 7E NOx . EPA Test Method 7E. Method 10 CO . EPA Test Method 10 . Method 25A Hydrocarbons (THC) EPA Test Method 25A. Method 30B Mercury (sorbent trap) EPA Test Method .
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