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`Exhibit 1023-1
`IPR2016-00379
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`

`
`CHIRAL
`SEPARATIONS
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1023-2
`IPR2016-00379
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`
`CHIRAL
`SEPARATIONS
`
`Edited by
`D. Stevenson
`
`The Robens Institute of Industrial and
`Environmental Health and Safety
`University of Surrey
`Gulldford, Surrey, United Kingdom
`and
`I. D. Wilson
`
`lei Pharmaceuticals
`Macclesfield, Cheshire, United Kingdom
`
`PLENUM PRESS • NEW YORK AND LONDON
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1023-3
`IPR2016-00379
`
`

`
`Library of Congress Cataloging in Publication Data
`
`Chromatographic Society International Symposium on Chiral Separations
`(1987: University of Surrey)
`Chiral separations.
`
`"Proceedings of the Chromatographic Society International Symposium on
`Chiral Separations, held September 3-4, 1987, at the University of Surrey,
`Guildford, Surrey, United Kingdom"-T.p. verso.
`Bibliography: p.
`Includes index.
`1. Liquid chromatography-Congresses. 2. Enantiomers-Separation(cid:173)
`Congresses. I. Stevenson, D. (Derrick) II. Wilson, Ian D. III. Chromatographic
`Society. IV. Title. QD79.C454C485 1987
`543'.0894
`89-15949
`ISBN 978-1-4615-6636-6
`ISBN 978-1-4615-6634-2 (eBook)
`DOl 10.1007/978-1-4615-6634-2
`
`Proceedings of the Chromatographic Society International Symposium
`on Chiral Separations, held September 3-4, 1987, at the
`University of Surrey, Guildford, Surrey, United Kingdom
`
`© 1988 Plenum Press, New York
`A Division of Plenum Publishing Corporation
`233 Spring Street, New York, N.Y. 10013
`Softcover reprint of the hardcover I st edition 1988
`
`All rights reserved
`
`No part of this book may be reproduced, stored in a retrieval system, or
`transmitted in any form or by any means, electronic, mechanical,
`photocopying, microfilming, recording, or otherwise, without written
`permission from the Publisher
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
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`
`PREFACE
`
`This volume represents the proceedings of a two-day international meeting on chiral
`chromatography held at the University of Surrey between 3-4 September 1987. The meeting
`was jointly organized by the Chromatographic SOCiety and the Robens Institute of the
`University of Surrey in response to the burgeoning interest in this rapid maturing field of
`chromatography. Nowhere is this interest more evident than in the agrochemical and
`pharmaceutical industries where the implications of different pharmacological and
`toxicological activity for the individual enantiomers present in a racemic drug ol" insecticide
`is an increasing area of concern. Developments in the area of chiral separations are at last
`beginning to provide SCientists with the necessary tools to study how animals and man
`handle racemates and relate their obseIVations to the obseIVed biological effects of these
`substances. The development of robust and Simple methods for the separation of
`enantiomers will therefore have a profound Impact on safety evaluation and drug design.
`
`The meeting proved to be very successful. with over 160 delegates from thirteen
`countries in Europe and America present to learn from the experiences of experts in the field
`of chiral chromatography and to hear about the latest developments. Hopefully. in future
`symposia on chiral separations at the University of Surrey. some of the delegates who were
`at this first meeting mainly to learn will return to report on their use of chiral
`chromatography in one of the most difflcult areas facing modem analysts -
`the reliable and
`accurate quantification of enantiomers at trace concentrations in biological samples.
`
`D. Stevenson and I.D. Wason
`
`v
`
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`
`CONTENTS
`
`The Chromatographic Society
`
`The Robens Institute
`
`The Biological Importance of Chirality and Methods Available to
`Determine Enantiomers
`D. Stevenson and G. Williams
`
`Drug Analysis Using High-Performance Liquid Chromatographic (HPLC)
`Chlral Stationaxy Phases: Where to Begin and Which to Use
`Irving W. Wainer, Rose M. Stiffin and Ya-Qin Chu
`
`Systematic Studies of Chlral Recognition Mechanisms
`Wflliam H. Pirkle, Thomas C. Pochapsky, John A. Burke m
`and Kr1s C. Deming
`
`Separation of Enantiomers of Oxyphenonlum Bromide by High-Performance
`Liquid Chromatography
`Karla G. Feltsma, Ben F.H. Drenth and Rokus A de Zeeuw
`
`The Use of Pirkle High-Performance Liquid Chromatography Phases in the
`Resolution of Enantiomers of Polycyclic Aromatic Hydrocarbon
`Metabolites
`Michael Hall and Philip L. Grover
`
`The Role of Solvents and Sterlc Factors in the Resolution of /i-Blocker
`Drugs on Chlral Urea Phases
`Nagaraja K.R. Rao, Robert C. Towill and Bindu Todd
`
`Complexation of Dansyl Amino Acid Enantiomers by /i-Cyc1odextrin
`Studied by High-Performance Liquid Chromatography and
`Fluorescence Measurements
`David A Briggs, Roger B. Homer and Russell Godfrey
`
`Analytical and Preparative Chiral Resolution of Some Aminoalcohols
`by Ion-Pair High-Performance Liquid Chromatography
`R.M. Gaskell and B. Crooks
`
`Simultaneous Enantioselective Determination of Underlvat1zed
`/i-Blocking Agents and their Metabolites in Biological Samples
`by Chlral Ion-Pairing High-Performance Liquid Chromatography
`T. Leeman and P. Dayer
`
`Ix
`
`xl
`
`1
`
`11
`
`23
`
`37
`
`43
`
`55
`
`61
`
`65
`
`71
`
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`Chtral Gas and High-Perfonnance Liquid Chromatographic Analysis of
`Enantiomers of Fungicides and Plant Growth Regulators: Application
`in Fungal. Plant and Soil Metabolism Studies
`T. Clark, AH.B. Deas and K. Vogeler
`
`Recent Developments in Enantiomer Separation by Complexation Gas
`Chromatography
`Volker Schurig and Rainer Link
`
`Use of Various Commercially Available Chiral Stationary Phases in
`Supercritical Fluid Chromatography
`P. Macaudiere. M. Caude. R Rosset and T. Tambute
`
`Strategies for Optimising Chiral Separations in Drug Analysis
`Anthony F. Fell and Terence AG. Noctor
`
`Further Use of Computer Aided Chemistry to Predict Chiral Separations
`in Liquid Chromatography: Selecting the Most Appropriate
`Derivative
`Ulf NOrinder and E. Goran Sundholm
`
`An Optical Rotation Detector for High-Perfonnance Liquid Chromatography
`D.M. Goodall and D.K. Lloyd
`
`Prospects for Chiral Thin-Layer Chromatography
`J.D. Wilson and RJ. Ruane
`
`Appendix 1 - Chiral Chromatography Literature 1987-1988
`
`Appendix 2 -
`
`Some Manufacturers and Suppliers of Chiral Columns
`
`Appendix 3 - Abstracts
`
`Author Index
`
`Compound Index
`
`Subject Index
`
`79
`
`91
`
`115
`
`121
`
`127
`
`131
`
`135
`
`145
`
`179
`
`181
`
`197
`
`199
`
`203
`
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`
`THE CHROMATOGRAPHIC SOCIE1Y
`
`The Chromatographic Society is the only international organization devoted to the
`promotion of. and the exchange of information on. all aspects of chromatography and
`related techniques.
`
`With the introduction of gas chromatography in 1952. the Hydrocarbon Chemistry
`Panel of the Hydrocarbon Research Group of the Institute of Petroleum. recognizing the
`potential of this new technique. set up a Committee under Dr. S. F. Birch to organize a
`Symposium on 'Vapor Phase Chromatography' which was held in London in June 1956.
`Almost 400 delegates attended this meeting and success exceeded all expectation. It was
`immediately apparent that there was a need for an organized forum to afford discussion of
`development and application of the method and. by the end of the year. the Gas
`Chromatography Discussion Group had been formed under the Chairmanship of Dr. A. T.
`James with D. H. Desty as Secretary. Membership of this Group was originally by invitation
`only but. in deference to popular demand. the Group was opened to all willing to pay the
`modest subscription of one guinea and in 1957 AJ.P. Martin. Nobel Laureate. was elected
`inaugural Chairman of the newly-expanded Discussion Group.
`
`In 1958 a second Symposium was organized. this time in conjunction with the Dutch
`Chemical SOCiety. and since that memorable meeting in Amsterdam the Group, now SOCiety.
`has maintained close contact with kindred bodies in other countries. particularly France
`(Groupement pour I'Avancement des Methodes Spectroscopiques et Physico-chimiques
`d'AnalyseJ and Germany (Arbeitskreis Chromatographie der Gesellschaft Deutscher
`Chemiker) as well as interested parties in Eire. Italy. The Netherlands. Scandinavia. Spain
`and Switzerland. As a result Chromatography Symposia. in association with Instrument
`Exhibitions. have been held biennially in Amsterdam. Edinburgh. Hamburg. Brighton.
`Rome. Copenhagen. Dublin. Montreux. Barcelona. Birmingham. Baden-Baden. Cannes.
`London. Nurnburg. Paris and Vienna.
`
`In 1958 'Gas Chromatography Abstracts' was introduced in journal format under the
`Editorship of C.E.H. Knapman; first published by Butterworths. then by the Institute of
`Petroleum. it now appears as 'Gas and Liquid Chromatography Abstracts' produced by
`Elsevier Applied Science Publishers and is of international status -
`abstracts. covering all
`aspects of chromatography. are collected by Members from over 200 sources and collated by
`the Editor Mr. E. R. Adlard aSSisted by Dr. P. S. Sewell.
`
`Links with the Institute of Petroleum were severed at the end of 1972 and the Group
`established a Secretariat at Trent Polytechnic in Nottingham. Professor Ralph Stock
`playing a prominent part in the establishment of the Group as an independent body. At the
`same time. in recognition of expanding horizons. the name of the organization was changed
`to the Chromatography Discussion Group.
`
`In 1978. the 'Father' of Partition Chromatography. Professor A J. P. Martin was both
`honoured and commemorated by the institution of the Martin Award which is designed as
`
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`
`testimony of distinguished contribution to the advancement of chromatography. Recipients
`of the award include:-
`
`E.R Adlard
`Professor U.A.Th. Brinkman
`Professor J.C. Giddings
`Professor J.F.K. Huber
`C.E.H. Knapman
`DrE. Kovats
`Dr C.S.G. Phillips
`Dr RP.W. Scott
`and Dr. GA.P. Tuey
`
`Prof. E. Bayer
`Dr L.S. Ettre
`Professor G. Guiochon
`Dr C.E.R Jones
`ProfessorJ.H. Knox
`Professor A. Liberti
`Dr G. Schomburg
`Professor R Stock
`
`The Group celebrated its Silver Jubilee in 1982 with the 14th International Symposium
`held, appropriately, in London. To commemorate that event the Jubilee Medal was struck as
`means of recognising significant contributions by younger workers in the field. Recipients
`of the Jubilee Medal include: Dr H. Colin, Dr K. Grob Jr., Dr J. Hermannson, Dr P.G.
`Simmonds and Dr R Tijssen.
`
`In 1984 the name was once again changed, this time to The Chromagraphic Society,
`which title was believed to be more in keeping with the role of a learned society having an
`international membership of some 1000 scientists drawn from more than 40 countries. At
`that time, the Executive Committee instituted Conference and Travel Bursaries in order to
`assist Members wishing to contribute to, or attend major meetings throughout the world.
`
`The Society is run by an Executive Committee elected by its Members, in addition to the
`international symposia, seven or eight one-day meetings covering a wide range of subjects
`are organized annually. One of these meetings, the Spring Symposium, is coupled with the
`Society's Annual General Meeting when, in addition to electing the Society's Executive
`Committee, Members have the opportunity to express their views on the Society's activities
`and offer suggestions for future policy.
`
`Regular training courses in all aspects of chromatography are run in conjunction with
`the Robens Institute of the University of Surrey and it is hoped that this particular service
`will eventually include advanced and highly specialised instruction.
`
`Reports of the Society's activities, in addition to other items of interest to its members
`(including detailed summaries of all papers presented at its meetings), are given in the
`Chromatographic Society Bulletin which is produced quarterly under the editorship of I.W.
`Davies.
`
`At the time of writing three grades of membership are offered:
`
`Membership with Abstracts
`Membership
`Student Membership (includes Abstracts)
`
`£20.00 per year
`£11.00 per year
`£6.00 per year
`
`Members receive the Bulletin free of charge, benefit from concessionary Registration
`Fees for all Meetings and Training Courses and are, of course, eligible to apply for Travel
`and/ or Conference Bursaries.
`
`For further information please write to:
`
`Mrs. J. Challis
`Executive Secretary
`THE CHROMATOGRAPHIC SOCIE1Y
`Trent Polytechnic
`Burton Street
`Nottingham NG 1 4BU
`United Kingdom.
`
`x
`
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`
`THE ROBENS INSTITUTE OF INDUSTRIAL AND
`
`ENVIRONMENTAL HEALTH AND SAFE1Y
`
`The Robens Institute of Industrial and Environmental Health and Safety was
`established by the University of Surrey in 1978. It is housed on the University campus at
`Gulldford. The Institute carries out both academiC and contract research in health and
`safety related areas. including analytical chemistry. tOxicology. occupational health and
`hygiene. ergonomics and environmental health. In addition. The Robens runs a major
`programme of post-experience courses and symposia. In 1984 the Chromatographic Society
`set up a series of training courses (in GLC and HPLC) now run annually at The Robens.
`
`xi
`
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`
`CHIRAL SEPARATIONS
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
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`
`THE BIOWGICAL IMPORTANCE OF CHIRALI1Y AND METHODS
`
`AVAILABLE TO DETERMINE ENANTIOMERS
`
`D. Stevenson and G.A. Williams
`
`The Robens Institute of Industrial and Environmental Health and Safety
`University of Surrey. Guildford. Surrey GU2 5XH. UK
`
`SUMMARY
`
`Many compounds are marketed as racemic mixtures even though it is now known that
`enantiomers can have different biological activities. This can be due to differences in. for
`example. protein binding. transport. metabolism and clearance. Methods available to
`determine enantiomers include chromatography on chiral stationary phases. the use of
`chiral additives in the mobile phase. derivatization to yield diastereoisomers. chiral
`detectors. NMR and enantiomer specific immunoassays. The advantages of the various
`methods are discussed.
`
`THE BIOWGICAL IMPORTANCE OF CHIRALIlY
`
`Compounds that exist in two forms that are nonsuperimposable mirror images show
`optical activity. That is. they rotate the plane of plane-polarised light in opposite
`directions. This property is shown by an asymmetriC carbon atom (te. one with four
`different substituents) but also by other atoms such as sulphur. phosphorus. some metal
`atoms and can also occur when rotation around an atomiC bond is hindered by bulky
`functional groups. Compounds differing only in their ability to rotate plane-polarised light
`in opposite directions are known as enantiomers.
`
`Many compounds. such as drugs. agrochemicals and food additives have been
`marketed as racemic mixtures (that is approximately equal proportions of enantiomers)
`even though it has long been known that different enantiomers may show very different
`biological activity. Differences in biological activity of drugs. agrochemicals. etc .• may
`arise due to differences in:
`
`- protein binding and transport [I]
`- mechanism of action [2]
`- rates of metabolism
`- changes in activity due to metabolism
`- clearance rates (3)
`- persistence in the environment
`
`It must be remembered that the human body is a highly stereospecific environment (le.
`we have D-sugars and L-amino acids) and that in nature. asymmetry at the molecular level
`is the norm. not the exception(4). Regulatory authorities have become aware that the
`efficacy and toxicology of enantiomers may differ from each other and from the racemic
`mixture. and are increasingly asking for data on stereochemistry.
`
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`It has also been discovered that metabolism may produce chiral metabolites from
`non-chlral molecules[5). Some examples are shown below
`
`Ketone reduction
`
`Reduction of a double bond
`
`Hydroxylation
`
`Oxidation
`
`R
`
`"-c = 0
`/'"
`R
`
`H
`
`•
`
`R
`H
`""'c/
`R'/ "OH
`
`R
`R
`I
`I
`C=C
`I
`I
`R"
`H
`
`R
`I
`R-C-H
`I
`H
`
`H
`------+
`
`OH
`------+
`
`H
`R
`I
`I
`H-C-C-R
`I
`I
`R"
`H
`
`R
`I
`R'-C-OH
`I
`H
`
`R
`R
`I
`I
`R-C-OX
`R-C-X
`0
`I ~ I
`X
`X
`
`Oxidation of tertiary amine
`
`R
`R
`I
`I
`R-N: ~ R'-N-O
`I
`I
`R'
`R"
`
`The importance of chirality when investigating the mechanism of toxicity can be
`demonstrated with the general example below. The toxic effect of a hydrocarbon was
`thought to be caused by a metabolite.
`
`H
`I
`R--C-R
`I
`H
`
`OH
`I
`R--C---+R
`I
`H
`
`The metabolite was isolated, characterized and synthesized for toxicity testing for
`comparison with the pattern seen for the parent compound. The activity of the chemically
`synthesized (racemic) metabolite was different to the in-vivo formed metabolite when one
`enantiomer predOminated.
`
`It is clear, therefore, that restricting the study of chlral aspects of metabolism to
`comparing the fate of enantiomers in biological fluid, may be misleading. It has also been
`
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`discovered that enzymes capable of converting one enantiomer to its antipode exist. as has
`been shown with ibuprofen.
`
`Analytical methods capable of discriminating between enantiomers are thus required
`for a variety of reasons. including:
`
`- checking the purity of products where the aim ts to market only one enantiomer
`- monitoring asymmetriC synthesis
`- determination of enantiomeric drugs In body fluids for pharmacokinetic studies.
`- in-vivo metabolism studies
`- in-vitro metabolism studies
`- checking if the interconversion pathway is active for a particular compound
`- testing the fate of agrochemicals in the environment.
`
`METHODS AVAILABLE FOR CHIRAL DISCRIMINATION
`
`The separation of enantiomers is a very exacting analytical task but not a new one. In
`1848 Louts Pasteur carried out mechanical (te. hand and eye) separation of enantiomers and
`then in 1858 used bacteria to discriminate between enantiomers by selectively destroying
`one. At about that time Pasteur also used a chlral reagent to fonn diastereotsomeric salts
`which have different physical properties. thus allowing separation by fractional
`crystallization. In the 1930s Henderson and Rule used the selective adsorbtion of camphor
`derivatives onto D-Iactose as a means of chiral discrimination. In the 1960s GU-Av and
`others used gas liqUid chromatography (GLC) with chiral stationary phases and also
`derivatization with chiral reagents followed by achiral GLC or thin-layer chromatography
`(TLC).
`In the 1960s high perfonnance liqUid chromatography (HPLC) was used after
`derivatlzation with chiral reagents. then in the 1980s HPLC with chlral stationary phases
`such as those developed by Pirkle became available. The modem analyst therefore has
`several different approaches that can be tried for the separation and/ or detennination of
`enantiomers
`
`- Dertvatization using chiral reagents then HPLC. GLC. TLC
`- Ch1ral stationary phases for HPLC. GLC. TLC
`- Chlral mobile phases for HPLC and TLC
`- Chlral detectors for HPLC
`- Nuclear magnetic resonance (NMR) using chlral shift reagents
`- Enantiomer specific immunoassays.
`
`DERIVATIZATION USING CHIRAL REAGENTS
`
`Until the advent of chlral stationary phases, the most common means of achieving
`the separation of enantiomers was by reaction with a reagent containing another
`asymmetric centre to fonn a pair of diastereotsomers. As diastereotsomers differ fu. thelr
`physical properties. these can be separated by conventional (non-chiral) stationary phases.
`In order to use this approach successfully. the reagent must be readily available in a
`chemically and optically pure fonn. must react at the same rate with both enantiomers and
`racemtsation. decomposition and side reactions must not occur during the derivatlzation.
`Clearly. the analyte molecules must contain a functional group suitable for derivatization
`and for practical reasons it is preferable that the reaction is quick. Examples of reagents
`used include:
`
`- for aminoacids
`
`- for amines
`
`- for alcohols
`
`N-trifluoroacetyl prolylchloride
`a - chloroisovalenylchloride
`menthylchloroformate
`N-trifluoroacetyl prolylchlOride
`a - phenylbutyric anhydride
`2 - phenylpropionylchlOride
`1 - phenylethylisocyanate menthyl chlorofonnate
`
`3
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`- for aliphatic
`and alicycliC acids
`- for ketones
`
`desoxyephedrine I-menthol
`
`2,2,2-trifluoro-l-pentylethylhydrazine
`
`An excellent list of chiral derivatization reagents is given by Souter 1985[6).
`
`When using derivatizatlon for HPLC it is also possible to introduce a chromophore to
`enhance detection; for example. using (+)-I-(9-fluoroenyl) ethylchloroformate[7). The
`advent of chiral stationary phases, particularly for HPLC means that this approach is
`usually favored ahead of diastereoisomer formation. However, chiral chromatography is
`often unsuccessful and so diastereoisomer formation will continue to receive attention[8)
`as a quick and robust solution. Enantiomer resolution using this approach is usually
`enhanced when bulky groups are attached to the chiral centre and when the chiral centres
`of both the reagent and the analyte are in close proximity in the resulting diastereoisomer.
`
`CHIRAL STATIONARY PHASES FOR HPLC. GLC AND TLC
`
`HPLC
`
`Presently. the use of HPLC with chiral stationary phases is the most common 'first
`approach' for enantiomer separation. A large number of different phases are commercially
`available; all are fairly expensive and, in some case, of questionable stability and
`reproducibility from column-to-column. In many ways this mirrors the early development
`of bonded phases for HPLC and hopefully the situation will improve. Most HPLC chiral
`stationary phases have quite specific structural requirements for successful enantiomer
`separation and therefore apply only to a limited range of compounds. Several authors have
`classified columns into groups and have attempted to deSCribe the mechanism of
`separation and thus the structural requirements on the analyte [9-12).
`
`1)
`
`Donor-Acceptor (Pirkle) Type Phases
`
`The first commercially available chiral stationary phase for HPLC was R-N-(3,5 -
`dinitrobenzoyl) phenylglycine bound ionically to aminopropyl silica[13). Several other
`Similar phases have followed. The structure of this phase is shown in Fig. 1. It has four
`possible pOints of interaction with analyte molecules. three are necessary for chiral
`separation to occur. These interactions can include hydrogen-bonding, dipole-stacking,
`charge-transfer and steric effects. The 7t-acceptor dinitrobenzoyl group can attract 7t-donor
`groups such as aromatic rings or alkyl groups. Many examples of separations on these
`columns have been reported often requiring achiral derivatization to reduce analyte
`polarity (a disadvantage of these phases) or to introduce functional groups suitable to
`interact with the phase.[14). With the Pirkle type columns, both ioniC and covalently bound
`phases are available. These often give Similar results though occasionally selectivity is
`different. The ionic phases can only be used with organic eluents (even then not more than
`-20% propan-2-01 in hexane). Sometimes the columns can be regenerated using
`commercially available solutions of the stationary phases.
`
`ii) Chiral Cavity Phases
`
`Chiral cavity type phases such as cyclodextrinS bound to silica through a spacer are
`now also commercially available and in widespread use[15,16). The cyclodextrin structure
`can be viewed as a cone (Fig.2) open at both ends with the interior of the cone relatively
`hydrophobic. If a chiral molecule fits exactly into the cone and has functional groups that
`interact with the secondary hydroxyl groups at the wider opening of the cone, then chiral
`separation may occur. Different cyclodextrin and derivatized cyclodextrins with different
`sized chiral cavities are available. Small molecules totally included in the cone will not
`separate. The cyclodextrin columns are used in the reversed-phase mode using typical
`water/methanol or acetonitrile type mobile phases, the organic modifier competes with
`solute molecules for inclusion in the hydrophobiC cavity.
`
`4
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`
`Fig. 1. The structure of 3, 5-dinitrobenzoyl phenyl glycine, used in Pirkle type chiral
`stationary phases for HPLC.
`
`Fig. 2.
`
`Inclusion of analyte molecule in a cyclodextrin 'chiral cavity' to give chiral
`separation.
`
`Mobile phase additives such as buffer salts can help improve column effiCiency and
`resolution. The cyclodextrin columns are reactively cheap. Cyclodextrin bonded phases
`are also very useful for separating cis-trans isomers.(17).
`
`iii) Helical Polymer Phases
`
`A third class of chiral stationary phase are helical polymers such as cellulose esters
`(Fig.3) and poly-(triphenylmethlmetacrylates) whose chirality arises from helicity[l8-20).
`It is thought that the mechanism of separation on these columns involves a combination of
`attractive interactions and inclusion of the analyte in a chiral cavity. They are mostly
`used in the normal phase mode and can be used as stationary phases themselves or as lower
`molecular weight fractions adsorbed onto silica gel. These columns are relatively
`expensive and are also subject to pressure limitations.
`
`Iv)
`
`Ligand Exchange Columns
`
`Ligand exchange columns such as those developed by Davankov have been used for
`chiral separations for many years(21).
`Stationary phases such as proline or
`hydroxyproline bound to silica via a spacer are commercially available. The phases are
`treated with copper salts. In order to achieve chiral separation the analyte molecules must
`form a reversible complex with the copper ion.(22) (Fig.4). The analyte molecules must have
`two polar functional groups on (or adjacent to) the asymmetric carbon atom. Thus a-amino
`acids and amino alcohols are ideal for this type of column, but they are not applicable for
`other compound types. They are used with aqueous mobile phases.
`
`v)
`
`Protein Phases
`
`The fifth type of chiral stationary phases are proteins bound to silica.. Two are
`commercially available; bovine serum albumin (BSA) [23,24) and alpha acid glycoprotein
`(AGP)[25,26). These are used with aqueous buffers (and small amounts of organic modifers)
`
`5
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`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1023-16
`IPR2016-00379
`
`

`
`Fig. 3. Basic unit of cellulose type phase.
`
`n
`
`Fig. 4. Ligand exchange type phases requiring complexing with Cu ions.
`
`as the proteins are not stable with organic solvents or extremes of pH. These columns can
`give very good resolution of enantiomers through chromatographic efficiency is often poor.
`Separations are thought to be based on combinations of hydrophobic and polar
`interactions, although ionic interactions may also occur, structural requirements for
`chiral separation are therefore difficult to predict. Generally speaking, the protein
`columns are of low capacity and cannot be used for preparative work.
`
`A summary of some of the advantages and disadvantages of HPLC chiral stationary
`phases is shown in the table[27].
`
`GLC
`
`The direct resolution of enantiomers on chiral stationary phases is also possible,[28,29]
`though there are very few such phases commercially available. The most commonly used
`phase is the capillary column Chirasil-Val® (both D and L are available).The structure of
`this phase which is based on L-valine-tert butylamide is shown in Fig.5. The phase has
`been used to separate many drugs, hydroxy acids, amino acids and amino alcohols.
`Enantiomer separation by GLC is much less common than HPLC The technique is limited
`to relatively volatile analytes or their derivatives. The Chirasil-Val phase itself cannot be
`used above temperatures of about 230°C as racemization of the stationary phase can occur.
`
`TLC
`
`Chiral stationary phases for thin-layer chromatography are also known. The
`Chiraplate® made by treating octadecyl-modified silica with copper acetate and 4 hydroxy-
`1-(2-hydroxydodecyl) proline as a chiral selector is commercially available. These plates
`have been used to separate underivatized amino acids[30]. Separation is based on ligand
`exchange and only molecules possessing two functional groups on (or possibly adjacent to)
`the asymmetric carbon atom capable of complexing with the copper ion will separate. In
`
`6
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1023-17
`IPR2016-00379
`
`

`
`Table 1.
`
`Some Advantages and Disadvantages of Chiral Stationary Phases for HPLC
`
`Ligand Exchange Columns
`Use aqueous mobile phases
`Require two polar functional groups on or near chiral centre
`
`Cyclodextrins
`Use aqueous mobile phases
`Fairly cheap
`
`Pirkle Type Phases
`Often require derivatization
`Wide range of applications with specific interactions
`Need to use non-polar mobile phases for ionic columns
`
`Helical Polymers
`Pressure limitations
`Solvent limitations
`
`Protein Columns
`Use hydrophobic and polar interactions, multiple binding sites
`Use aqueous mobile phase pH 5-9
`Tolerate only small amounts of organic modifier, column effiCiency low
`
`CH3 /CH3
`
`o \H H
`I
`I
`/I
`/CH /N, /CH3
`-CH2 /C
`'c
`~CH "'N
`""'C
`I
`I
`I '-CH3
`II
`CH3 H
`0
`CH3
`Fig. 5. The structure of Chirasil-Val.
`
`the authors' laboratory, separations were achieved in an unsaturated chamber with a
`development time of about 15 minutes for a distance of 10 cm. The mobile phases were
`mixtures of methanol/water/acetonitrile. The plates were easy to use and reproducible.
`Other stationary phases analogous to the HPLC phases such as Pirkle type phases and
`cyclodextrins have been reported(31) and may soon become commercially available.
`Chtral TLC could prove useful in metabolism studies and also as a guide to selection of
`highly expensive HPLC phases.
`
`CHIRAL MOBILE PHASE ADDITIVES
`
`The separation of enantiomers on chtral stationary phases involves the formation of
`reversible diastereomertc complexes. The same effect can sometimes be achieved by adding
`chiral reagents to the mobile phase and using non-chiral stationary phases. [32,33).
`Optically active ion-pair reagents have been used, particularly for basic drugs using pairing
`ions such as camphor sulphonic acid and Z-glycyl proline. Bovine serum albumin and
`cyclodextrin (34) (both more commonly used as a bound stationary phase) have also been
`used successfully in the mobile phase. In some cases it is quite likely that separation occurs
`via 'in-situ' coating of the column to form a temporary chiral stationary phase. Chiral
`mobile phase additives have the advantage that less expensive column packings can be used
`and that there is an even wider chOice than of stationary phases. They can also be used for
`preparative isolation, for example, when using chiral ion-pairing reagents. Mobile phase
`
`7
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1023-18
`IPR2016-00379
`
`

`
`additives are limited to compounds that will not affect the detection system. and the
`addition of large amounts of some additives can prove costly.
`
`CHIRAL DE'IEcroRS FOR HPLC
`
`The traditional means of distinguishing between enantiomers spectroscopically has
`involved using plane polartsed light and measuring optical rotation. This is a slmple
`method. ideal when dealing with bulk quantities of enantiomers of known speclflc
`rotation. A racemic mixture will give zero specific rotation. The technique at present is not
`sensitive enough for trace analysis. though sensitivity does vary from compound to
`compound Recently polarimetric detectors specifically designed for HPLC have become
`available[35.361 often used in series with a UV or refractive index detector to give both
`quantification and the ratio of enantiomers. A limit of detection as low as 10 ug is now
`possible. Such detectors allow chiral discrimination without chromatographic separation.
`excess of one enantiomer giving peaks in the opposite direction to the other.
`
`NMR
`
`Nuclear Magnetic Resonance (NMR) can often distinguish between diastereoisomers
`and can therefore be used directly after derivatization with a chiral reagent. More
`convenient is the use of chiral shift reagents which can also give a d