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 chiral LC,Advanced Separation Technologies,March 2003,Proposed Structure of Vancomycin,p-accept
2、or,ionic site,hydrogen bonding & dipole stacking sites,sugar moieties,amine,A, B, C are inclusion pockets (weak),Multiple use Covalent bonding Broad applicability,Proposed Structures of Glycopeptide CSPs,Teicoplanin,Teicoplanin Aglycone,Key sites,Proposed Structures of Glycopeptide CSPs,Ristocetin A
3、,Features of CHIROBIOTIC 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 total methodsColumn coupling possible Rapid, broad screening p
4、rotocolsEach column can be used in all 3 modes,Broad Selectivity Based on the Same Stereogenic Center: CHIROBIOTIC T: Amino Alcohols,Albuterol,Atenolol,Metoprolol,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?
5、,The Polar Organic Mode was developed originally by Dr D W Armstrong 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,Ionic,What is the
6、 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 (predominantly hydrogen bonding) for the POM on Daicel is different from that on the CHIROBIOTIC phases and
7、 the optimization process is different,Ionic,What is the Polar Organic Mode?,The mechanism for the POM on the CHIROBIOTIC 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
8、 with salt containing compoundsCompatible 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 use ammonium trifluoroacetate, acetate or formate (0.001 t
9、o 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 complementary to cellulosic and amylosic phasesMany separations that are done on Chiracel in normal phase can also be accomplished in the polar ionic mo
10、de on CHIROBIOTIC columnsIf a compound is polar, solubility may be 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 separations typica
11、lly 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,Peak 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
12、/0.01: MeOH/HOAc/TEA,CHIROBIOTIC TAG,Selectivity on the CHIROBIOTIC T can be enhanced with CHIROBIOTIC TAG (aglycone from teicoplanin) for certain molecular typesEnhanced resolution for , , and cyclic amino acids, neutrals, acids, sulfur containing molecules and certain primary amines,CHIROBIOTIC TA
13、G,Mobile phase possibilities: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 good for large number of chiral sulfoxides, coumarins,Selectivity of Methanol fo
14、r Neutral Molecules on CHIROBIOTIC TAG,5-Methyl-5-phenylhydantoin,4-Methyl-5-phenyl-2-oxazolidinone,100% MeOH 0.8 mL/min,100% MeOH 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 methy
15、l sulfoxide,7 7.6,5.9 9,Method Development 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 Columns,3 solvents systems, plus 3 different CHIR
16、OBIOTIC 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)Reversed 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 Mod
17、e from Coupled Column Screen for LC/MS,Trimipramine,t1: 11.27t2: 11.54,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 1Columns: CHIROBIOTIC R+
18、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 (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% TE
19、AA, pH 6.0Results:No selectivity observed,16.01,Sample C,Run 3 (on Sample C): Normal Phase,Column: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 Pha
20、se: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 D,Column:CHIROBIOTIC TMobile Phase:100/0.2/0.1: MeOH/AcOH/TEAFlow Rate:1.0 mL/min,22.48,2
21、4.96,Using CHIROBIOTIC Phases for SFC,A recent (subcritical) study:TAG, T R for series of 110 compounds including neutral 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 Arm
22、strong, In press,Using CHIROBIOTIC 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 spectrophotometric methods to LC/MS/MS with minimization of
23、ion suppressionLimited use of inorganic buffersSeparations in higher organic, lower aqueous mobile phases preferredMethods using flammable solvents such as hexane may require additional safety measures (eg, N2 and/or post column dilution with organic/aq)Possible adaptability to cassette dosing and m
24、ultiplexing LC/MS/MS techniques,Considerations for Using CHIROBIOTIC Chiral LC Columns 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 compatible
25、 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 Bakhtiar & F L S Lee, Rapid Commun in MS, 14, 1128-1135 (2000)2. L Ramos, R Bakhtiar, T Majumda
26、r, 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 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, Rebe
27、cca J. Scott, Robert A. Biddlecombe, 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/min3 minute assay/96 well SPE100 pg/mL LOQ for parent compoun
28、d, 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/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 CH
29、IROBIOTIC R,V,T Kit for Drug Metabolism: LC/APCI/MRM Chromatograms,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 NH4Ac bufferSimultaneous chir
30、al separations offer the possibility of parallel loading, multiplexing and cassette dosingColumn coupling provides fast, simple generic screening,Biocatalysis,Courtesy of DSM Fine Chemicals,Determination of the conversion and enantiomeric excess of substrate / reaction products in a D-hydantoinase /
31、 D-carbamoylase reaction,Column: CHIROBIOTIC T (250x4.6 mm, 5m)Eluent: 80/20 15mM NH4Ac pH 4.1/MeOHFlow: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 produ
32、cts from an epoxyhydrolase 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,CASE STUDY 1: Nicardipine,Column:CHIROBIOTIC V(250x4.6mm, 5) Mobile phase: 100/0.2/
33、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 for 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: Nicard
34、ipine,Column:CHIROBIOTIC V (250 x 22.1 mm), 5m) 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,Purity Peak 1: 99.67% Peak 2: 99.46%,CASE STUDY 2: Polar Ionic Mode,Column: CHIRO
35、BIOTIC 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: 2mL,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:
36、1 mL/min 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: 100/0.5/0.5,MeOH/AcOH/TEA Flow rate: 15 mL/min UV: 254nm Inj. 140mg (in 2ml MeOH),Peak 1: 10.22Peak
37、2: 12.54,CASE STUDY 3: N-Acetyl Typtophan,Column: CHIROBIOTIC TAG (250x4.6mm, 5m)UV: 254 nmFlow rate: 1 mL/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 Try
38、ptophan,Column:CHIROBIOTIC TAG (250x22.1mm, 5m) 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: Purificationof Isoleucine on CHIROBIOTIC TAG,ISOLEUCINE,Column: CHIROBIOTIC TAGSize: 250 x 22.1mmM
39、obile Phase:30/70: MeOH/H2OFlow Rate:20 mL/min.Injection:170 mg in 10 mLLoad:24mg/gm CSP/hr,Recovery of Warfarin 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,
40、250 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 :Separations 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 for prep in reversed and normal phase,Web Site ,Complete LC & GC applications,
