Rapid Chiral HPLC Method Development

Transcription

1 Rapid Chiral HPLC Method Development Denise Wallworth, Sigma-Aldrich LCGC Webinar, November 19 th 2014

2 Introduction The role of chiral separations today Choices of solvents, CSPs Method development techniques Approaches to LC/MS

3 The Importance of Chiral Separations Traditionally pharma drug development Today. Biomarkers D,L-2-Hydroxyglutaric acid as a marker for brain tumours Drugs of abuse testing Source, metabolism Food Food adulteration, flavour and fragrance authenticity Environmental Enantioselective biodegradation of pollutants Not just chiral Positional isomers and closely related compounds

4 Metabolomics: Separation of Isoprenoid Pathway Metabolites Intens. x H 3 C CH 3 IP P - H 2 C CH 3 P - DMAP H H H 4 CYCLBND I HILIC Mode H 2 C CH 3 P H P H - H 3 C CH 3 P P - IPP H H H 2 R Köhling, R Meier, B Schönenberger and R Wohlgemuth, 9th Annual Conference of the Metabolomics Society, July 2013, Glasgow, Scotland. Poster P4-44 DMAPP Time [min]

5 Biodegradation of Phenoxyalkanoic Herbicides The R enantiomer was found to be more resistant to degradation in soil, but both enantiomers break down equally in plants. R(+) Day 7 S(-) Day 28 Dichlorprop (R is active) Dichlorprop extracted from soil CHIRBITIC T, RP Mecoprop (R is active) J.M.Schneiderheinze, D.W.Armstrong and A.Berthod; Chirality, 11 (1999),

6 Drugs of Abuse Testing: Levamisole (Tetramisole) Apart from its use as an anthelmitic therapeutic treatment for horses, Levamisole is also used as an adulterant in cocaine. 1. Aminorex 2. Dexamisole 3. Levamisole 4. Cocaine CYCLBND I 2000 DMP Reversed Phase Retention Time (min)

7 Chiral Separation Approaches for HPLC Pre-column derivatisation Chiral mobile phase additive Direct separation with a chiral stationary phase (CSP) Relies on reversible formation of transient diastereomeric complexes on the surface of the adsorbent The difference in energies, strengths of these transient complexes will be very subtle

8 Predicting Chiral Selectivity To achieve chiral recognition, multiple point interactions must take place the 3-point interaction rule still applies CSP Biphenyl derivative

9 Predicting Chiral Selectivity The approach of using experience is not as effective as it is with typical reversed-phase method development although published literature methods can guide a column selection Why Not?

10 Achiral Reversed Phase Chemistries L Si L L Si δ - F δ - F δ + F δ - F F δ - δ - Ascentis Phenyl Discovery HS F5 CH 3 Ascentis RP Amide (CH 2 ) 14 NH L Si L L

11 Typical Chiral Stationary Phases (CSPs) PLYSACCHARIDES (Chiral Technologies) H H H H H H H H H H CH 3 H H H 2 N CH 3 H H H H H H H H H N N N N N H H H CCH NH 3 HN 2 H H H H H H H CH 3 H CHIRBITIC R (Ristocetin A) CYCLBND

12 Typical Chiral Stationary Phases (CSPs) Polymeric Small Molecule Ligands Synthetic Polytartramides Natural Cellulose π-complex Cyclodextrins, Cyclofructans Methacrylate Amylose Ligand Exchange Crown Ether Polycyclic Amines Proteins Macrocyclic Glycopeptides Anion Exchange

13 Types of interactions that favour chiral selectivity Hydrogen bonding π π Ionic Dipole Steric Inclusion The strength of the diastereomeric complex formed between the solute and CSP surface changes in different solvents, and on different CSPs. Screen several solvents, several columns for best choice of method Avoids missing optimum solutions

14 Key points Interactions need to be balanced Leading, anchoring interaction (dominant, long range) Supporting interactions (often the chiral initiating, directional, short range) H-bonding, pi-pi, dipole stacking are directional and short range, and so, have a narrow window in which to be effective likely then to be the driving force in chiral selectivity. A dilemma... Multiple interaction types = peak broadening/less efficiency Single set of interactions = 1 column/few applications» A balancing act!

15 Proposed Structure of Macrocyclic Glycopeptide CSPs Vancomycin (CHIRBITIC V/V2) ( Teicoplanin (CHIRBITIC T/T2) ionic site H 3 C π-acceptor NH H H H NH H Cl NH A NH 2 NH B NH Cl CH 3 CH 3 H H H NH 2 hydrogen bonding & dipole stacking sites C NH NH H H CH ionic site HNCCH 3 H N HC H H H H CH 2 H Cl N H H A B H H N H H NHR N H H H C H H Cl H N CH 2 H H N H H D H H NH 2 Key interaction sites; A, B,C and D are cavities

16 Solvent choices Normal Phase Reversed Phase Polar Mobile Phases Polar

17 Polar Mobile Phases With and without additives: Polar organic mode (MeH, ACN, IPA and combinations) perates using similar retention mechanisms to normal phase Use for analytes that are capable of hydrogen bonding Works well in preparative chiral HPLC when solubility can be improved Polar ionic mode (MeH with acid/base or salt, sometimes with ACN) Use for ionisable analytes Used extensively for LC/MS applications Works well on CSPs with ionisable functional groups

18 Polar Ionic Mode (CHIRBITIC, Ionic Effects) A. Acid/Base Ratio Effects example: Mianserin column: CHIRBITIC V mobile phase: Methanol/HAc/TEA B. Salt Effects example: Atrolactic acid column: CHIRBITIC T mobile phase: Methanol/NH 4 salts mau /0.1/0.1 mau /0.1% (v/w) MeH/NH 4 TFA mau /0.15/0.05 min mau /0.1% (v/w) MeH/NH 4 Formate min mau /0.05/0.15 min mau /0.1% (v/w) MeH/NH 4 Ac min min min Additives dictate selectivity in the polar ionic mode.

19 Retention Behavior of Fluoxetine in Reversed- Phase Mode on CHIRBITIC CSP S-enantiomer R-enantiomer alpha Retention (min) Selectivity % MeH

20 Polar Ionic Mode to Reversed Phase Mode Separations column: CHIRBITIC V2, 250 cm x 4.6mm sample: Bupivacine Mobile Phase: 100/0.1w%: MeH/NH 4 TFA N H3 NH C CH 3 CH 3 mau α = 1.44 P1: 5.33 P2: min Add water to change to reversed phase mechanisms (retention increases) mau 15 Mobile Phase: 90/10: MeH/10mM NH 4 Ac ph 4.1 α = P1: P2: min min

21 Choice of Mobile Phase Mechanisms are different in different mobile phases ften worth screening more than one type of mobile phase on a single CSP Some CSPs exhibit strong chiral separations in one mobile phase type Choice of mobile phase may be determined by The CSP compatibilty The purpose of the method (analytical or prep, LC/MS or UV)

22 Chiral Column Screen: Matrix used in Supelco Labs Column Chromatographic Modes PI RP1 P NP1 NP2 CHIRBITIC TAG X X X CHIRBITIC V2 X X X CHIRBITIC T X X X CYCLBND I 2000 X X CYCLBND I 2000 HP-RSP X X CYCLBND I 2000 DMP X X X Cyclofructan (LARIHC) X X Kromasil CelluCoat, Amycoat X X X Kromasil TBB, DMB X X X Astec Cellulose DMP X X X Astec (R,R) P-CAP X X X

23 Chiral Column Screen: Mobile Phases For primary screening purposes, the mobile phase compositions are as follows: PI = 100:0.1:0.1, methanol:acetic acid: triethylamine RP1 = 70:30, 20 mm ammonium acetate (ph 4.0):acetonitrile P = 95/5/0.3/0.2 ACN/MeH/AcH/TEA (2 nd choice: 95/5/0.1/0.1 ACN/IPA/TFA/TEA) NP1 = 80:20, heptane: isopropanol (with 0.1% TEA and 0.1% TFA) NP2 = 70:30, heptane: ethanol

24 Chiral Column Screen: Protocols copies available on sigma-aldrich.com/chiral

25 ptimisation Techniques PLAR INIC MDE (CHIRBITIC) Test acid:base ratios from 1:4 to 4:1 Change from acid/base to dissolved volatile acid (optimise via concentration) Test adding ACN (to 50%) or water (to 10%) PLAR RGANIC Cellulosics: MeH/EtH blends, add 5-10% other RH or ACN CHIRBITIC: Test combinations of MeH with ACN, EtH, MTBE CYCLBND: Test acid:base ratios from 1:4 to 4:1 Cyclofructans: Change ACN:MeH ratio, adjust acid base ratio

26 ptimisation Techniques REVERSED PHASE CHIRBITIC: Change % and type organic modifier; adjust ph (increase for acidics, decrease for basics), buffer type (Amm Ac, Formate), ionic strength CYCLBND: as above, but decrease ph for acidic and neutral NRMAL PHASE Test alternative alcohols, add 0.1% TFA for acids, TEA or DEA for bases Test 50:25:25, heptane: isopropanol: MTBE (with 0.1% TEA and 0.1% TFA)

27 Knox plot of Fluoxetine CHIRBITIC V2 12 Reduced Plate Height ASCENTIS C Reduced Linear Velocity C18 h (data fit theoretically to Knox) V2 h (data fit theoretically to Knox) C18 h (experimental) V2 h (experimental) The optimal reduced linear velocity on the CHIRBITC V2 is 0.61, which translates into a flow rate of 0.15 ml/min on the analytical (4.6mm) column used in this study.

28 Using CHIRBITIC CSPs for LC/MS Fluoxetine on CHIRBITIC V2 Polar ionic mode Time (min) Warfarin on CHIRBITIC V Reversed phase mode

29 Methamphetamine in Polar Ionic Mode on CHIRBITIC V2 NH CH 3 NH CH 3 CHIRBITIC V2 10 cm x 3.0mm, 5 µm Polar Ionic Mode: methanol:acetic acid:ammonium hydroxide CH 3 (+)-Methamphetamine 1 2 CH 3 (-)-Methamphetamine Time (min)

30 Chiral bioanalysis Serine (a potential biomarker for psychiatric diseases) by LC-MS/MS CHIRBITIC TAG, 250 x 4.6mm, 5µm Mobile phase: EtH/H2, 20/80 Flow rate: 1.0 ml/min LD: 100ng/ml Reproduced with permission of Chris Gregory, HFL, Newmarket UK. Poster at BioForum, Guildford, July 2005 Bupivacaine in plasma by LC/MS-ESI CHIRBITIC V2, 150x2.1 mm, 5µm Mobile Phase: 90/10, MeH/10mM NH4Ac, ph 4.1 Flow Rate: 0.2 ml/min

31 High Throughput Bioanalytical Chiral LC/APCI/MS/MS: examples Compound Column Column size Mobile phase Flow rate t R enantiomers Ritalinic acid 1 CHIRBITIC T 50x4.6mm 100 MeH/0.03% ATFA 1.2 ml/min 5.0, 10.4 min Methylphenidate 2 (2500 samples) CHIRBITIC V 150x4.6mm 100 MeH/0.03% ATFA 1.0 ml/min 6.1, 7.2 min Fluoxetine 1 CHIRBITIC V 50x4.6mm 100 MeH/0.09% ATFA 1.0 ml/min 6.2, 6.8 min Nicardipine 1 CHIRBITIC V 50x4.6mm 100 MeH/0.05% ATFA 1.0 ml/min 1.1, 1.6 min Metoprolol 1 CHIRBITIC T 150x4.6mm 100 MeH/0.025% ATFA 1.2 ml/min 7.0, 7.8 min Salbutamol 3 (4000 samples) CHIRBITIC T 250x4.6mm 100/0.5/0.1 MeH/AcH/NH4H 2.0 ml/min 3.2, 3.7 min (metab at 2.0) 1. R Bakhtiar & F L S Lee, Rapid Commun in MS, 14, (2000) 2. L Ramos, R Bakhtiar, T Majumdar, M Hayes & F Tse, Rapid Commun MS, 13, (1999) 3. K Joyce, A E Jones, R J Scott, R A Biddlecombe &m S Pleasance, Rapid Commun MS, 12, (1998)

32 Separation of classes of molecules

33 β-blocker Separation on CHIRBITIC T Clenbuterol CHIRBITIC T Polar Ionic Mode, MeH/15mM Ammonium Formate Sotalol BETA RECEPTRS T PI.ESP No. Name tr k' Selectivity 1 clenbuterol clenbuterol metoprolol metoprolol sotalol sotalol atenolol atenolol Retention Time (min)

34 Ephedrine/Pseudoephedrine Chiral AGP Column 10 mm ammonium phosphate/ 1 mm octanoic acid H 3 C NH CHIRBITIC T Methanol:Acetic Acid:TEA H 3 C H Time (min) Pseudoephedrine Time (min) H 3 C NH H 3 C H Time (min) Ephedrine Time (min)

35 Amino Acids Methionine CHIRBITIC T Reversed Phase (ACN/Water) The ionic functional groups of CHIRBITIC phases provide high selectivity for ionisable and zwitterionic compounds, including amino acids

36 Mechanism for Amino Acids on CHIRBITIC CSPs Carboxyl group must be free (esters are separated by a different method, or by GC) Amine can be free or blocked AQC, Benzoyl, tbc, CNZ, Dansyl, FMC Amino acid can be aromatic, cyclic or aliphatic, natural or synthetic, α, β, or γ α-hydroxycarboxylic acids also separate well Mobile phase is often simple MeH / Water

37 Clinical (non-chiral) Applications of Amino Acids Biomarkers for inborn metabolic disorders in children CHIRBITIC T, TAG found to offer effective alternative to C18 Glutamine and lysine, one of the isobaric pairs that are a problem for MS/MS, can be readily separated Methods for dimethylarginine (possible biomarker for alcoholic hepatitis) with creatinine have also been developed Rapid separation of urinary cystine from RN, LYS and ARG without derivatisation for the diagnosis of cystinuria C Turner & N Dalton, WellChild Trust Laboratory, Evelina Children s Hospital, St Thomas Hospital, London; Personal communication C Turner, H Y Wu, RN Swaminathan, S Bird, P Barnfield, R N Dalton, J Inherited Metabolic Diseases, 2003, 26, Suppl 2, 197 (Presentation at the 9th Intl Congress on Inborn Errors of Metabolism).

38 Separating Molecules with Multiple Chiral centres

39 Structural Information of the Analytes Full structure is proprietary, however: 3 Chiral Centers, 8 Stereoisomers Functional Groups Include: Carboxylic acid 3 Aromatic rings Secondary Amine

40 Development of the separation of 8 stereoisomers on CHIRBITIC V2 in RP Instrument: Agilent 1100 Column: CHIRBITIC V2, 10 cm x 4.6 mm, 5 µm From this first try Time (min).to this result T im e (m in ) Columns: CHIRBITIC V2, 10 cm x 4.6 mm I.D., 5 µm, coupled with Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 µm.

41 Separation of Diastereomers on the Ascentis Phenyl in RP Mode Instrument: Agilent 1100 Column: Ascentis Phenyl, 15 cm x 4.6 mm I.D., 5 µm particles Temperature: 25 C Flow Rate: 1 ml/min Mobile Phase: 75:25, 20 mm ammonium acetate (ph 4.0): acetonitrile Detection: UV at 287 nm Inj. Vol.: 10 µl Sample: 8 component mix, 100 µg/ml in methanol Time (min) Ref: A Novel Approach in HPLC Chiral Method Development: Dealing with Multiple Chiral Centers, D.S.Bell, A Fridstrom and G Parmar, HPLC 2013, Amsterdam, Netherlands

42 Injection of 8 Stereoisomers on CHIRBITIC V2 in RP Instrument: Agilent 1100 Column: CHIRBITIC V2, 10 cm x 4.6 mm, 5 µm Temperature: 25 C Flow Rate: 1 ml/min Mobile Phase: 50:50, 20 mm ammonium acetate (ph 4.0): methanol Detection: UV at 287 nm Inj. Vol.: 10 µl Sample: 8 component mix, 100 µg/ml in methanol Time (min)

43 Summary & Conclusions Chiral HPLC can be versatile! Systematic method development protocols and optimisation guidelines are important and need to be used to provide the optimum method, fit for purpose Important to screen a variety of CSPs and mobile phases that offer different mechanisms in order to obtain an optimum solution

44 Acknowledgements With thanks to: Dave Bell, Manager, Pharmaceutical and Bioanalytical Research at Supelco, Bellefonte Paul Rodwell, Applied Applications Laboratory, Sigma-Aldrich, Cambridge, UK Gaurang Parmer, HPLC Product Manager, Supelco, Bellefonte

45 Live Q&A sigma-aldrich.com/chiral