如何快速建立手性藥物分析的lc及l(fā)cmsms方法

1、Chiral Method Development Techniques for Analytical & Prep HPLC,AGENDA: Method development techniques for the CHIROBIOTIC series of chiral LC columns Techniques for chiral LC/MS/MS Advances in preparative

2、chiral LC,Advanced Separation Technologies,March 2003,Proposed Structure of Vancomycin,p-acceptor,,ionic site,,hydrogen bonding & dipole stacking sites,,,sugar moieties,,amine,,,A, B, C are inclusion pockets (weak),M

3、ultiple use Covalent bonding Broad applicability,Proposed Structures of Glycopeptide CSPs,Teicoplanin,Teicoplanin Aglycone,,,,,,,,Key sites,Proposed Structures of Glycopeptide CSPs,Ristocetin A,,,Features of CHIROBIOTI

4、C Phases,Reversed phaseLargest and most versatile number of separationsPolar Organic phaseFast kinetics, second major group of separationsBest suited for LC/MS systemsNormal phaseGenerally provides ca 10-15% of the

5、 total methodsColumn coupling possible Rapid, broad screening protocolsEach column can be used in all 3 modes,Broad Selectivity Based on the Same Stereogenic Center: CHIROBIOTIC T: Amino Alcohols,Albuterol,Atenolol,Me

6、toprolol,Mobile Phase:100/0.1/0.1:MeOH/HOAc/TEA @ 2.0 mL/minute,Oxprenolol,Propranolol,Sotalol,,,,11.82,12.55,7.08,7.83,What is the Polar Organic Mode?,The Polar Organic Mode was developed originally by Dr D W Armstrong

7、and Astec in ’85 for use with CYCLOBOND cyclodextrin technology100 % MeOH with added acid and base (typically 0.1%, each with a range of 1.0 to 0.001%), or equivalent saltsMethod development is very simple and fast,,

8、Ionic,What is the Polar Organic Mode?,The Polar Organic Mode terminology is now frequently applied to the Daicel phases, but in this case it means 100% organic only, usually MeOH, ACN or MeOH/EtOHThe mechanism (predomi

9、nantly hydrogen bonding) for the POM on Daicel is different from that on the CHIROBIOTIC phases and the optimization process is different,,Ionic,What is the Polar Organic Mode?,The mechanism for the POM on the CHIROBIOTI

10、C phases is predominantly ionic, making it essential for acid and base to be added. Thus, Polar Ionic Mode©,,Ionic,Advantages of Polar Ionic Mode© on CHIROBIOTIC V, T and R,No problem with salt containing compo

11、undsCompatible with column switching from C18 column to chiral columnScale-up is easy due to the volatility of mobile phase componentsComplementary to hexane/ethanol separations on polysaccharide CSPsFor LC/MS, can u

12、se ammonium trifluoroacetate, acetate or formate (0.001 to 0.5% w/v in methanol)Applicable to the use of gradients for method development,Normal phase vs Polar Ionic© Phase,CHIROBIOTIC columns appear to be complem

13、entary to cellulosic and amylosic phasesMany separations that are done on Chiracel in normal phase can also be accomplished in the polar ionic© mode on CHIROBIOTIC columnsIf a compound is polar, solubility may be

14、better in the PIM rather than normal phaseThe PIM can be advantageous and safer for prep,NOTE: If using CHIROBIOTIC columns in the Normal Phase, they need much higher EtOH levels than Daicel columns for most chiral sep

15、arations – typically 20-50% [EtOH],Metoprolol,CHIRACEL OD,Peak 1 – 11.9 min.Peak 2 – 18.2 min.,20/80/0.1: IPA/Hex/DEA,CHIROBIOTIC T,Peak 1 – 6.8 minPeak 2 – 7.5 min,100/0.2/0.1: MeOH/HOAc/TEA,Alprenolol,CHIRACEL OD,Pea

16、k 1- 12.4 minPeak 2 - 16.4 min,20/80/0.1: IPA/Hex/TFA,CHIROBIOTIC V,Peak 1 – 7.69 min.Peak 2 – 8.33 min.,100/0.01/0.01: MeOH/HOAc/TEA,CHIROBIOTIC TAG,Selectivity on the CHIROBIOTIC T can be enhanced with CHIROBIOTIC TA

17、G (aglycone from teicoplanin) for certain molecular typesEnhanced resolution for ?, ?, ? and cyclic amino acids, neutrals, acids, sulfur containing molecules and certain primary amines,CHIROBIOTIC TAG,Mobile phase poss

18、ibilities:Reversed phase – methanol as the organic modifierPolar ionic© phase – as for the CHIROBIOTIC TNeutral molecules use single solvent: 100% MeOH, EtOH, IPA, ACN. No acid or base requiredNormal Phase –

19、 good for large number of chiral sulfoxides, coumarins,Selectivity of Methanol for Neutral Molecules on CHIROBIOTIC TAG,,,,,,,,,,,5-Methyl-5-phenylhydantoin,4-Methyl-5-phenyl-2-oxazolidinone,100% MeOH @ 0.8 mL/min,100% M

20、eOH @ 0.8 mL/min,5.08 min9.62 min,5.35 min8.21 min,Chiral Sulfoxides on CHIROBIOTIC TAG,,Teicoplanin TAG,Mobile phase: Hexane/EtOH, 50/50Flow rate: 2 mL/min,p-Flurophenyl methyl sulfoxide,7 7.6,5.9 9,Method De

21、velopment Techniques Using Column Coupling,Generic screening methods for fast chiral method developmentColumn coupling utilized to enable simultaneous multi-column screening,Method Development Techniques Using Coupled

22、 Columns,3 solvents systems, plus 3 different CHIROBIOTIC columns (close coupled kit) provides 9 screens in 105 minutesSCREENING MOBILE PHASES:Polar ionic mode© (MeOH/AcOH/TEA, 100/0.02/0.01, 2.0 mL/min)Reverse

23、d phase mode (MeOH/TEAA (0.1%, pH 6.0), 25/75, 1.0 mL/min)Normal phase mode (Hex/EtOH, 40/60, 1.5 mL/min),Optimization in the Polar Ionic Mode© from Coupled Column Screen for LC/MS,Trimipramine,t1: 11.27t2: 1

24、1.54,t1:20.7t2:22.8,Screen,Optimised,R+V+T (10cm)MeOH/HOAc/TEA:100/0.02/0.012 mL/min.,V (25cm)100/0.02 v/w:MeOH/AFTA0.8 mL/min.,,CASE HISTORY: Generic Method Development Screen: 4 Unknowns,Polar Ionic Mode, Run

25、1Columns: CHIROBIOTIC R+V+T (100 x 4.6mm each)Mobile Phase: 100/0.1/0.05, MeOH/AcOH/TEAResults:,Sample A,Sample B,Sample C,Sample D,Run 2 (on Sample B and C): Reversed Phase Screen,Columns:CHIROBIOTIC R+V+T (

26、100x4.6mm each)Mobile Phase:20/80: MeOH/0.1% TEAA, pH 6.0Results:,Sample B,Sample C,Mobile Phase:40/60: MeOH/0.1% TEAA, pH 6.0Results:No selectivity observed,16.01,Sample C,Run 3 (on Sample C): Normal Phase,Colum

27、n:CHIROBIOTIC R+V+T (100 x 4.6mm each)Mobile Phase:1. 40/60: EtOH/Hexane2. 5/95: EtOH/HexaneResults:No selectivity (retention) observed,1.50,2.24,Optimized Results: Sample A,Column:CHIROBIOTIC VMobile Phas

28、e:100/0.2/0.1: MeOH/AcOH/TEAFlow Rate:0.7 mL/min,12.39,13.53,Optimized Results: Sample B,Column:CHIROBIOTIC TMobile Phase:40/60: MeOH/0.1% TEAA, pH 4.1Flow Rate:0.6 mL/min,16.54,17.71,Optimized Results: Sample

29、D,Column:CHIROBIOTIC TMobile Phase:100/0.2/0.1: MeOH/AcOH/TEAFlow Rate:1.0 mL/min,22.48,24.96,Using CHIROBIOTIC Phases for SFC,A recent (subcritical) study:TAG, T > R for series of 110 compounds including neutra

30、l ketones, sulfoxides, underivatized amino acids, carboxylic acids and b-blockersBaseline separation achieved within 15 minutes on at least one CHIROBIOTIC CSP,Ref: Y Liu & D W Armstrong, In press,Using CHIROBIOTIC

31、Phases for SFC,,Sulfoxide D6Mobile phases:7% MeOH in CO2A: CHIROBIOTIC TB: CHIROBIOTIC TAGC: CHIROBIOTIC R,A,B,C,,Chiral LC/MS/MS in Bioanalysis,Challenges for Chiral LC/MS/MS in Bioanalysis,Adaptability of spectr

32、ophotometric methods to LC/MS/MS with minimization of ion suppressionLimited use of inorganic buffersSeparations in higher organic, lower aqueous mobile phases preferredMethods using flammable solvents such as hexane

33、may require additional safety measures (eg, N2 and/or post column dilution with organic/aq)Possible adaptability to cassette dosing and multiplexing LC/MS/MS techniques,Considerations for Using CHIROBIOTIC Chiral LC Co

34、lumns in LC/MS/MS,High enantioselectivity in 100% MeOH (polar ionic mode©) with added acid/base or saltsLess toxic and lower boiling point than hexaneNon-corrosiveWorks especially well with ESIColumns are also

35、compatible with ammonium acetate or formate (aqueous mode)and with all principle solvents used in APCI (from alcohols to hydrocarbons, DMSO, DMF),High Throughput Bioanalytical Chiral by LC/APCI/MS/MS: Examples,1. R Bakh

36、tiar & F L S Lee, Rapid Commun in MS, 14, 1128-1135 (2000)2. L Ramos, R Bakhtiar, T Majumdar, M Hayes & F Tse, Rapid Commun MS, 13, 2054-2062 (1999)3. K Joyce, A E Jones, R J Scott, R A Biddlecombe &m S

37、 Pleasance, Rapid Commun MS, 12, 1899-1910 (1998),Application for Chiral LC/APCI/MS/MS in Pharmacokinetics: Salbutamol and its 4-O-sulfate Metabolite*,* Karina B. Joyce, Anne E. Jones, Rebecca J. Scott, Robert A. Bidd

38、lecombe, Stephen Pleasance, Drug Metabolism and Bioanalysis, Glaxo Wellcome R&D, Ware, UK, Rapid Communications MS, 12, 1899-1910 (1998),CHIROBIOTIC T (Teicoplanin), 250 x 4.6mmMeOH/AcOH/NH4OH: 100/0.5/0.1 @ 2 mL/mi

39、n3 minute assay/96 well SPE100 pg/mL LOQ for parent compound, 5 ng/mL for sulphate metabolism; 25 pg/mL LOQ for 80 mL injection4000 sample clinical study,,Chiral metabolite,Neat solution at 10 ng/mL of: Atenolol (m

40、/z 267.2 ? 145.0), Mianserin (m/z 265.2 ?208.2), Terbutaline (m/z 226.0 ? 152.2), Propranolol (m/z 260.2 ? 116.2). [Ref: R Bakhtiar, in press],Using the CHIROBIOTIC R,V,T Kit for Drug Metabolism: LC/APCI/MRM Chromatogra

41、ms,LC/MS – Conclusions,The use of the polar ionic mode© provides the fastest methods for chiral LC/MS/MS with widest applicabilityThe same column can be used for reversed phase separations in high organic with NH4

42、Ac bufferSimultaneous chiral separations offer the possibility of parallel loading, multiplexing and cassette dosingColumn coupling provides fast, simple generic screening,Biocatalysis,,Courtesy of DSM Fine Chemicals

43、,Determination of the conversion and enantiomeric excess of substrate / reaction products in a D-hydantoinase / D-carbamoylase reaction,,Column: CHIROBIOTIC T (250x4.6 mm, 5?m)Eluent: 80/20 15mM NH4Ac pH 4.1/MeOHF

44、low:1.0 mL/min,3,4: Product6,7: Starting material1,5: Intermediate2: Impurity,Biocatalysis,Courtesy of DSM Fine Chemicals,,,Determination of the enantiomeric excess of cis- and trans-diol reaction products from an ep

45、oxyhydrolase reaction,Column:CHIROBIOTIC R (250x4.6 mm, 5 ?m)Eluent:0.1% ammonia, pH 4.1 with formic acid / MeOH (50/50 %v/v)Flow:1.0 mL/min,,A,B,,,,A,,,,B,Using CHIROBIOTIC CSPs for Preparative LC Applications,CAS

46、E STUDY 1: Nicardipine,Column:CHIROBIOTIC V(250x4.6mm, 5?) Mobile phase: 100/0.2/0.1, MeOH/AcOH/TEA UV: 230 nm Flow rate: 1mL/min,Peak 1: 4.66Peak 2: 5.57,? =1.50,,CASE STUDY 1: Nicardipine,Optimization f

47、or prep,100/0.1w%, MeOH/NH4TFA,100/0.1w%, MeOH/NH4OAc,Peak 1: 4.07Peak 2: 4.69,Peak 1: 3.41Peak 2: 3.47,? = 1.50,? = 1.09,Column:CHIROBIOTIC VFlow rate: 1mL/min,,,CASE STUDY 1: Nicardipine,Column:CHIROBIOTIC

48、V (250 x 22.1 mm), 5?m) Load:20mg in 4mL Mobile phase: 100/0.1w%,MeOH/NH4TFA UV: 230 nm Flow rate: 12 mL/min Throughput: 1.2 mg/g CSP/hr,Peak 1: 8.26Peak 2: 9.51,,,,,,Fraction Collections,1,2,3,4,5,6,,P

49、urity Peak 1: 99.67% Peak 2: 99.46%,CASE STUDY 2: Polar Ionic Mode©,Column: CHIROBIOTIC V, 5mm 250 x 4.6mm Mobile phase: 100/0.2/0.1, MeOH/AcOH/TEA Flow rate: 0.9 mL/min UV: 254 nm Inj: 2

50、mL,Peak 1: 10.51 minPeak 2: 11.53 mina = 1.14,BASIC COMPOUND,CASE STUDY 2: Polar Ionic Mode ©,Column: CHIROBIOTIC V 5mm (Modified) 250 x 4.6mm Mobile phase: 100/0.5/0.5, MeOH/AcOH/TEA Flow rate: 1 mL/min

51、 UV: 254 nm Inj: 100mg,BASIC COMPOUND,Peak 1: 8.83 minPeak 2: 11.82 mina = 1.50,CASE STUDY 2: Polar Ionic Mode ©,BASIC COMPOUND,Column: CHIROBIOTIC V, 5mm (Modified) 250 x 22.1mm Mobile phase: 100/0.5/

52、0.5,MeOH/AcOH/TEA Flow rate: 15 mL/min UV: 254nm Inj. 140mg (in 2ml MeOH),Peak 1: 10.22Peak 2: 12.54,CASE STUDY 3: N-Acetyl Typtophan,Column: CHIROBIOTIC TAG (250x4.6mm, 5?m)UV: 254 nmFlow rate: 1 mL/

53、min,Mobile phase:100/0.1w%, MeOH/NH4OAc,Mobile phase:40/60 MeOH/0.1% TEAA, pH4.1,Peak 1: 3.80Peak 2: 15.59,Peak 1: 6.26Peak 2: 22.20,k1= 0.36, ? = 12.7,k1= 1.09, ? = 5.87,CASE STUDY 3: N-Acetyl Tryptophan,Column:

54、CHIROBIOTIC TAG (250x22.1mm, 5?m) Load: 200mg in 6mL Mobile phase: 100/0.1w%, MeOH/NH4OAc UV: 300 nm Flow rate: 35 mL/min Throughput: 20 mg/g CSP/hr,Peak 1: 2.86Peak 2: 4.53,,CASE STUDY 4: Puri

55、ficationof Isoleucine on CHIROBIOTIC TAG,ISOLEUCINE,Column: CHIROBIOTIC TAGSize: 250 x 22.1mmMobile Phase:30/70: MeOH/H2OFlow Rate:20 mL/min.Injection:170 mg in 10 mLLoad:24mg/gm CSP/hr,Recovery of War

56、farin from Aqueous Mobile Phase,,WARFARIN,UV Absorption,,Diluted Mobile PhaseCollection FromFraction 2,Elute withMeOH,Wash withH2O to remove buffer,Wash &equilibratewith H2O,Collection,,,,,Time,,,,,C18, 250

57、x 4.6mm2x dilution ofFraction 2 with H2O1.5 mL/min.UV-220nmInj. 35mg,Refer to pages 34/5 of the CHIROBIOTIC Handbook,Conclusions: Preparative LC on CHIROBIOTIC CSPs,Use of MeOH and the polar ionic mode© :Separ

58、ations are fast and efficientHigh sample throughputIncreases long term stability of CSPsLess toxic than traditional normal phase solventsLow volatility reduces product recovery timeThe same column can also be used f