“Chiral Impurity Methods –Case Study” - HPLC

1 chiral Impurity Methods case study Ch. LAKSHMI NARAYANADAICEL chiral TECHNOLOGIES (INDIA) PVT LTDIPCIPC--USPUSP8th8thASM 2009 ASM 2009 Introduction: chiral Impurity Methods Types of chiral Stationary Phases (CSPs) for HPLCP olysaccharide-CSPs Coated CSPs immobilised CSPs case study : Oxaliplatin chiral HPLC method Lamivudine chiral HPLC method Clopidogrel Bisulphate chiral HPLC methodOverviewOverviewChiral Impurity Methods chiral Impurity Methods IntroductionIntroduction chiral GC chiral HPLC chiral HPCE chiral SFCT ypes of CSPs and their loading capacitiesTypes of CSPs and their loading capacitiesDrug discovery today, volume 10, Number 8, April 2005 TypeCSPsLoading capacity(mg solute / g CSP)IPirkle type (Brush type)1-50 IIPolysaccharide derivatives5 150 IIIM acrocyclic Crown Impurity Methods chiral Impurity Methods Types of CSPT ypes of CSP1999200120032005 Polysaccharide80 5%3%12%87 4%2%7%86 2%1%11%92 4%2% 2%1995-2003 Tetrahedron Asymmetry2005 : J.

2 Am. Chem. type of CSPW hich type of CSP PirkleProteinOthersChiral Impurity Methods chiral Impurity Methods Types of CSPT ypes of CSPOOORORROn OJ-H OD-H CH3 OCH3CH3 NOHC ellulose derivativesOOROROROn AD-H AS-H CH3CH3 NOHA mylose derivativesONHCH3 Coated polysaccharideCoated polysaccharide--derived CSPsderived CSPsChiral Impurity Methods chiral Impurity Methods Types of CSPT ypes of CSPI mmobilised polysaccharideImmobilised polysaccharide--derived CSPsderived CSPsCHIRALPAK IACHIRALPAK IBCHIRALPAK ICChiral Impurity Methods chiral Impurity Methods Types of CSPsTypes of CSPsChiral Impurity MethodsChiral Impurity Methods case StudyCase study case study -1:Oxaliplatin, Chemotherapy drug case study -2:Lamivudine, Anti retroviral drug case study -3:Clopidogrel Bisulphate, Anti platelet drugChiral Impurity Methods chiral Impurity Methods case study 1 case study 1 OxaliplatinOxaliplatinPharmacopeial method conditions:Pharmacopeial method conditions:Column: CHIRALCEL OC x 250mmColumn: CHIRALCEL OC x 250mmMobile Phase: Ethanol / MeOH (30/70)Mobile Phase: Ethanol / MeOH (30/70)Flow Rate: mL/min; Injection Volume:20 Flow Rate: mL/min; Injection Volume:20 99 LLDetection : 254 nm by UV; Temp: 40 Detection : 254 nm by UV.

3 Temp: 40 CCConcentration: mg/mLConcentration: mg/mLRs is NLT is NLT : : Impurity Methods chiral Impurity Methods case study 1 case study 1 OxaliplatinOxaliplatinCostCost effecive method conditions:effecive method conditions:Column: CHIRALPAK IC x 250mmColumn: CHIRALPAK IC x 250mmMobile Phase: Ethanol / MeOH (30/70)Mobile Phase: Ethanol / MeOH (30/70)Flow Rate: mL/min; Injection Volume:20 Flow Rate: mL/min; Injection Volume:20 99 LLDetection : 254 nm by UV; Temp: 40 Detection : 254 nm by UV; Temp: 40 CCConcentration: mg/mLConcentration: mg/mLRs > > : : Impurity Methods chiral Impurity Methods case study 1 case study 1 OxaliplatinOxaliplatinCostCost effective method conditions:effective method conditions:Column: CHIRALPAK IC x 250mmColumn: CHIRALPAK IC x 250mmMobile Phase: Ethanol / MeOH (30/70)Mobile Phase: Ethanol / MeOH (30/70)Flow Rate: mL/min; Injection Volume:20 Flow Rate: mL/min; Injection Volume:20 99 LLDetection : 254 nm by UV; Temp: 40 Detection : 254 nm by UV.

4 Temp: 40 CCConcentration: mg/mLConcentration: mg/mLInjection no: 1 Injection no: 1 Injection no: 100 Injection no: 100 chiral Impurity Methods chiral Impurity Methods case study 2 case study 2 LamivudineLamivudinePharmacopeial method conditions:Pharmacopeial method conditions:Column: L45 (Cyclobond I 2000 SP, x 250mm, 5 Column: L45 (Cyclobond I 2000 SP, x 250mm, 599))Mobile Phase: Ammonium acetate/MeOH (95/05)Mobile Phase: Ammonium acetate/MeOH (95/05)Flow Rate: mL/min; Conc: mg / mL; Inj Vol: 10 Flow Rate: mL/min; Conc: mg / mL; Inj Vol: 1099 LLDetection : 270 nm by UV; Temperature: 25 Detection : 270 nm by UV; Temperature: 25 CCRs is NLT is NLT limit: limit: effective method conditions:effective method conditions:Column: CHIRALPAK IC ( x 250) mm, 5 micronColumn: CHIRALPAK IC ( x 250) mm, 5 micronMobile Phase: Ethanol/2 Mobile Phase: Ethanol/2 Propanol/DEA (90/10 , v/v/v)Propanol/DEA (90/10 , v/v/v)Flow Rate: mL/min; Conc: mg/mL; Inj Vol: 10 Flow Rate: mL/min; Conc: mg/mL; Inj Vol: 1099 LLDetection : 270 nm by UV; Temperature: 25 Detection : 270 nm by UV; Temperature: 25 C; Diluent : MPC.

5 Diluent : MPChiral Impurity Methods chiral Impurity Methods case study 2 case study 2 LamivudineLamivudineRs > > : : effective method conditions:effective method conditions:Column: CHIRALPAK IC ( x 250) mm, 5 micronColumn: CHIRALPAK IC ( x 250) mm, 5 micronMobile Phase: Ethanol/2 Mobile Phase: Ethanol/2 Propanol/DEA (90/10 , v/v/v)Propanol/DEA (90/10 , v/v/v)Flow Rate: mL/minFlow Rate: mL/minDetection : 270 nm by UV; Temperature: 25 Detection : 270 nm by UV; Temperature: 25 CCChiral Impurity Methods chiral Impurity Methods case study 2 case study 2 LamivudineLamivudineInjection no: 200 Injection no: 200 Injection no: 1 Injection no: 1 Method objective: 1. Clopidogrel bisulphate related substances quantification including chiral impurity2.

6 Clopidogrel bisuphate assay determinationClopidogrel bisulphateClopidogrel bisulphateChiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Pharmacopeial method conditions:Pharmacopeial method conditions:Column: L 57 (Ultron ESColumn: L 57 (Ultron ES OVM ( x 150) mm)OVM ( x 150) mm)Mobile Phase: 10mM Phosphate buffer/ACN (75/25)Mobile Phase: 10mM Phosphate buffer/ACN (75/25)Flow Rate: mL/min; Conc: mg/mLFlow Rate: mL/min; Conc: mg/mLDetection : 220 nm by UVDetection : 220 nm by UVClopidogrel and its related compoundsImpImp--AALimit: Limit: ImpImp--CCLimit: Limit: ImpImp--BBLimit: Limit: chiral Impurity Methods chiral Impurity Methods case study 3 case study 3 chiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Pharmacopeial method Clopidogrel bisulphateClopidogrel bisulphateSS criteria: Rs between Imp B1 and Clopidogrel NLT criteria: Rs between Imp B1 and Clopidogrel NLT Impurity Methods chiral Impurity Methods case study 3 case study 3 Clopidogrel bisulphateClopidogrel bisulphatePharmacopeial method chiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Cost effective method conditions.

7 Cost effective method conditions:Column: CHIRALCEL OJColumn: CHIRALCEL OJ H ( x 250) mm, 5 micronH ( x 250) mm, 5 micronMobile Phase: Methanol/DEA (100 , v/v)Mobile Phase: Methanol/DEA (100 , v/v)Flow Rate: mL/min; Conc: mg/mL; Diluent : EthanolFlow Rate: mL/min; Conc: mg/mL; Diluent : EthanolDetection : 220 nm by UV; Temperature: 25 Detection : 220 nm by UV; Temperature: 25 CCClopidogrel bisulphateClopidogrel bisulphateChiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Clopidogrel bisulphateClopidogrel effective method conditions:effective method conditions:Column: CHIRALCEL OJColumn: CHIRALCEL OJ H ( x 250) mm, 5 micronH ( x 250) mm, 5 micronMobile Phase: Methanol/DEA (100 , v/v)Mobile Phase: Methanol/DEA (100 , v/v)Flow Rate: mL/min; Diluent : EthanolFlow Rate: mL/min; Diluent : EthanolDetection : 220 nm by UV; Temperature: 25 Detection : 220 nm by UV.

8 Temperature: 25 CCChiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Injection no: 200 Injection no: 200 Injection no: 1 Injection no: 1 CostCost effective method conditions:effective method conditions:Column: CHIRALCEL OJColumn: CHIRALCEL OJ H ( x 250) mm, 5 micronH ( x 250) mm, 5 micronMobile Phase: Methanol/DEA (100 , v/v)Mobile Phase: Methanol/DEA (100 , v/v)Flow Rate: mL/min; Diluent : EthanolFlow Rate: mL/min; Diluent : EthanolDetection : 220 nm by UV; Temperature: 25 Detection : 220 nm by UV; Temperature: 25 CCClopidogrel bisulphateClopidogrel bisulphateChromatographic CharacteristicsChromatographic CharacteristicsChiral Impurity Methods chiral Impurity Methods case study 3 case study 3 Clopidogrel bisulphateClopidogrel bisulphateNote: Column memory effects may influence the elution of Imp A, Note: Column memory effects may influence the elution of Imp A, hence recommended to dedicate the column for this application.

9 Hence recommended to dedicate the column for this application. ConclusionsConclusions chiral HPLC using polysaccharide derived CSPs is a versatile tool to estimate chiral impurities in drug substances . Perhaps, the chiral Impurity Methods described in Pharmacopeia for Oxaliplatin, Lamivudine & Clopidogrel bisulphate are utilising the columns, which are not so durable and hence results into high analytical cost. It would be beneficial to adapt new generation chiral column chemistry available today to develop an efficient & cost effective chiral Impurity Mr. Raghuram, General Manager, Hetero Drugs Ltd for his continuous co-operation during Lamivudine method development My Colleagues Mr. Thirupathi & Mr. Srinivasu for their significant contribution during method development USP India for the lecture opportunityTHANK YOU ALL