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Chiral separation of amides using supercritical fluid chromatography
Accepted Manuscript Title: Chiral Separation of Amides Using Supercritical Fluid Chromatography Author: Yanqiao Xiang Joshua R. Dunetz Michael Lovdahl PII: DOI: Reference:
S0021-9673(13)00513-X http://dx.doi.org/doi:10.1016/j.chroma.2013.03.048 CHROMA 354202
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
27-11-2012 13-3-2013 18-3-2013
Please cite this article as: Y. Xiang, J.R. Dunetz, M. Lovdahl, Chiral Separation of Amides Using Supercritical Fluid Chromatography, Journal of Chromatography A (2013), http://dx.doi.org/10.1016/j.chroma.2013.03.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights 1. Baseline enantiomeric separation of nine amide analogs was achieved using SFC;
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2. Of the five CSPs investigated, the OD‐H and the IC columns showed the best results;
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3. Steric hindrance and hydrogen bonding have significant impact on the separations;
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4. Using optimized conditions, validation of a SFC chiral method was demonstrated.
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7 8 9 10 Chiral Separation of Amides Using Supercritical Fluid Chromatography
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Yanqiao Xiang,*,a Joshua R. Dunetzb, Michael Lovdahla
Analytical Research and Development, bChemistry Research and Development, Pharmaceutical Sciences, Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340
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Corresponding author. Current address: Analytical Development Small Molecule, Millennium: The Takeda Oncology Company, Cambridge, MA 02319, United States. Tel.:+1 617 444 1675; E-mail: [email protected]
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Abstract Nine amide derivatives bearing α-stereocenters as well as different substitutions on the amide nitrogen were synthesized via a n-propanephosphonic acid cyclic anhydride (T3P)-
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mediated coupling, and their enantiomeric pairs were separated using supercritical fluid
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chromatography (SFC). Five polysaccharide-based chiral stationary phases (CSPs), Chiralcel
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OD-H and OJ-H, and Chiralpak AD-H, AS-H and IC columns were explored for the chiral
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separation of these compounds. None of the compounds could be resolved on all five columns,
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and no single column could separate all nine pairs of enantiomers. Comparatively, the IC and
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OD-H columns showed the best results for this group of amides, yielding baseline separations for
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eight of nine pairs. The type of polar functional group and aromatic substitution in the CSPs and
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the substitutions on the amide nitrogen had a significant impact on the enantiomeric resolution of
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the compounds in the interaction between the analyte and the stationary phases. The potential
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separation mechanism and the effect of substitutions in the CSPs and amide solutes on the
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separation were discussed. The effects of the organic modifiers, modifier composition, mobile
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phase additives, and temperature were investigated for the separation of these amides on the IC
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or the OD-H column. Baseline resolution was achieved under optimized chromatographic
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conditions using an IC or an OD-H column. Linearity, reproducibility, and limit of quantitation
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were also demonstrated for the compound 9 (see Figure 1). Approximately three-fold
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improvement in signal-to-noise was observed using a SFC system with better instrument design.
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Keywords: SFC; enantiomeric separation; amide; pharmaceutical
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Introduction A survey within the past ten years has shown that 60-70% of the most frequently
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prescribed drugs and the drug candidates under development are single enantiomers [1]. For
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any enantiomeric drug or drug candidate, an enantiomeric impurity can produce different
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pharmacological, toxicological, metabolic, and pharmacokinetic effects within the chiral
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environment of a biological system [2-4]. As part of continuing efforts to improve the safety
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and efficacy of drugs, the attention of pharmaceutical companies and regulatory agencies has
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been focused on impurity control for active pharmaceutical ingredients (APIs). In order to
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develop robust processes and deliver high-quality products, reliable chiral separation
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methods have become necessary to assess the stereochemical integrity of enantiomers as
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well as examine the potential for interconversion of individual stereoisomers [5].
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An assessment of the enantiomeric purity of pharmaceuticals can be achieved using different chromatographic techniques including gas chromatography (GC) [6-8], supercritical
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fluid chromatography (SFC) [9-15], capillary electrophoresis (CE) [16], capillary
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electrochromatography (CEC) [17], and high performance liquid chromatography (HPLC) [18-
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20]. HPLC methods are most widely used for chiral separation in the pharmaceutical industry.
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However, taking advantage of diversified enantioselective HPLC chiral stationary phases
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(CSPs), SFC utilizes supercritical CO2 as a major mobile phase solvent, providing combined
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advantages of speed, efficiency, and environmental friendliness. The low viscosity and high
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diffusivity of supercritical CO2 allow higher flow rates resulting in faster separations, typically
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three to five times faster than HPLC. The higher diffusivity also leads to lower mass transfer
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resistance, therefore resulting in sharper peaks and better column efficiencies [21]. Together with
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improved instrumentation resulting in much better signal-to-noise, SFC is becoming an
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increasingly prevalent approach to chiral separation in pharmaceutical development. [22-23]. Amides are commonly found within compounds of pharmaceutical importance and were
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embedded within 25% of known pharmaceuticals at the start of the 21st century [24]. Not
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surprisingly, the condensation of an amine and carboxylic acid to form an amide bond has been
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the most frequently employed transformation for the synthesis of drug candidates [25]. Despite
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continuing advances in methods to prepare amide bonds, a recurring challenge for these
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couplings involves avoiding the epimerization of activated carboxylic acid substrates under
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reaction conditions. To this end, we developed a process mediated by T3P (n-propanephosphonic
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acid cyclic anhydride) to prepare a series of amides in high yield under conditions which
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suppress the epimerization of acid substrates that are particularly sensitive to racemization [26-
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27].
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In order to support the process development of this T3P-mediated amide coupling, chiral
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SFC methods were developed for nine pairs of amide enantiomers. Five commercially available
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polysaccharide chiral stationary phases were evaluated. The effects of the substituents at the
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amide nitrogen on the enantiomeric resolution of the compounds were explored. The effects of
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the organic modifiers, modifier composition, mobile phase additives, flow rate, and temperature
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on the separation of these amides were investigated. The method linearity, reproducibility, and
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limit of quantitation were evaluated for enantiomeric purity determination for both in-process-
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control development and the drug candidates produced using the T3P process.
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2. Materials and methods
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2. 1. Chemicals and Reagents
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HPLC grade methanol, ethanol (200 proof), isopropanol (IPA), acetonitrile (ACN), spectrophotometric grade trifluoroacetic acid (TFA), and reagent grade isopropylamine (IPAm)
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were purchased from Aldrich (Saint Louis, MO, USA). SFC-grade carbon dioxide used was
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purchased from Airgas (CT, USA)
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Chiralcel OD-H (OD-H) and OJ-H (OJ-H), and Chiralpak AD-H (AD-H), AS-H (AS-H)
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and IC columns were purchased from Chiral Technologies (West Chester, PA, USA).
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2.2. Sample Preparation
All amide samples used in this study were prepared as described in the references 26 and
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27, and the structures of their major enantiomers are shown in Figure 1.
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2. 3. Equipments
SFC experiments were performed on a Berger analytical system equipped with FCM-
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1200 dual pump fluid control module (Mettler-Toledo AutoChem, Columbia, MD) and a SFC
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Fusion™ A5 analytical instrument (Aurora SFC Systems, Inc. CA) connected to a 1100 HPLC
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system (Agilent Technologies, Palo Alto, CA). A high pressure flow cell was utilized with an
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HPLC PDA detector. Berger SFC ProNTo software (version 92.1) and Agilent ChemStation
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(version B.04.01) were used for the instrument control and the data collection. UV detection at
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both 210 nm and 254 nm was applied. Electrospray ionization mass spectrometer (MSD,
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Agilent), connected to the outlet of the UV detector flow cell, was used to verify the
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enantiomeric peak pairs.
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2.4. Chromatographic Conditions 6
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The screening methods as shown in Table 1 were used as a starting point for method development. Further method optimization was performed to improve separation by fine tuning
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the mobile phase/modifier composition, flow rate, and temperature for enantioselective SFC.
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3. Results and Discussion
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3. 1. Column Selection (screening results)
It is understood that the backbone structures of the CSPs examined in this study, which
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are formed from carbohydrate chains containing polar functional groups (e.g., esters, carbamates,
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residual hydroxyl groups), play a major role in chiral recognition. At the same time, the analyte
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structure and its functional groups are also very critical for the enantiorecognition, and multiple
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interactions between the amides and CSPs are possible [28]. For example, the C=O groups of the
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amides can hydrogen bond with the NH groups of the CSPs as well as form dipole-dipole
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interactions with the C=O groups of the CSPs. The NH moiety in some amides can hydrogen
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bond with the C=O groups of the stationary phases. Additionally, the phenyl and benzyl groups
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in the amides can form π-π interactions with the phenyl carbamate or ester moieties of the CSPs.
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Some CSPs can favor some interactions more than others depending on the stationary phase and
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solute structure. However, the detailed recognition mechanism for the polysaccharide based
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CSPs still has not been completely elucidated.
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Nine pairs of amide enantiomers were screened using five polysaccharide CSPs and three
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mobile phases using the methods described in Table 1, and some of the results are summarized in
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Table 2. It can be seen that eight of the nine pairs are baseline separated using an IC column, and
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the resolution order for the eighr separated pairs is 7>8>2>1>5>6>9>>3. The fact that amide 4
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was not separated while the highest resolution of amide 7 was achieved on the IC column could
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be due to differences in steric hindrance between 4 and 7. As the backbone and polar functional 7
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groups of the CSPs form a regular arrangement of grooves to serve as enantioselective binding
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pockets, the steric effect can determine the analyte accessibility to the binding sites, such as the
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hydrogen bonding sites buried inside of the cavity [28]. This explains why the least bulky amide
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7 can easily fit into the binding site to get the best enantiomeric resolution. However, amide 4
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shows higher steric hindrance effect to the binding site due to methyl groups at both ortho
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positions of the phenyl ring. Amide 3 contains a phenyl group like amide 4 but has a methoxy
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group at the para position instead of the ortho methyl groups, and it is baseline separated on the
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IC column as it represents less steric hindrance than amide 4. This result further confirms that the
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steric effect of the analyte can play a critical role in the separation of this group of amides on the
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IC column. In addition, the methoxy oxygen in amide 3, a hydrogen bonding acceptor, may form
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hydrogen bonding with the stationary phase to contribute a better separation of amide 3 than that
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of amide 4 [29-30]. Similar to the IC column, the OD-H column yielded baseline separations of
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eight of the nine enantiomeric pairs and the resolution order for the eight separated pairs on the
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OD-H column is 7>1>3>8>5>4>9>6. Amide 7 has the highest resolution, as high as 10.7, on the
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OD-H column, indicating steric effects can also play a critical role in separations on the OD-H
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column. However, unlike the separations on the IC column, the most sterically hindered amide 4
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is separated on OD-H column, but amide 2 is not. This could be due to the differences in the
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polar functional groups in the CSPs (3,5-dimethylphenyl carbamate for OD-H and 3,5-
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dichlorophenyl carbamate for IC column). Interestingly, amide 1 and 2 have similar structures
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except that amide 1 is a secondary amide whereas amide 2 is a tertiary amide with a methyl
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group attached to the nitrogen. This subtlety makes significant difference in the
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enantiorecognition as amide 1 is separated with the resolution as high as 7.4 while amide 2 is
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not resolved at all. In addition, amides 2 and 6 are the only two containing tertiary amine
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functionality in this series. Although 6 is almost baseline separated on the OD-H column, this
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amide shows the poorest resolution in this series. It can be deduced that the proton on the
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nitrogen in the amides also plays an essential role as an H-donor in the enantiorecognition of the
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amides on the OD-H column. On the other hand, the only structural difference between amide 2
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and 6 is a phenyl versus benzyl group, respectively, connected to the amide nitrogen. By
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comparison, the hydrogen-bonding ability of the carbonyl in amide 2 is decreased due to the
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delocalization of electron density from the amide into the adjacent phenyl group.
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The AD-H column separated seven of the nine amide pairs and the OJ-H column
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separated four of the nine enantiomeric pairs. The AS-H column showed the poorest selectivity
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for this series, separating only one of the nine amide pairs.
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Mobile phase additives are often used to improve enantiomeric separations and peak
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shapes via stationary phase modification and acidic or basic analyte ionization suppression.
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Methanol-based mobile phases without any additive, with basic additive, or with a mixture of
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TFA and IPAm additives were screened for this series of amides (results are not shown). Eight of
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the nine amide pairs are neutral. As expected, basic and a mixture of acidic and basic additives
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have little effect on the chiral separation of the neutral compounds as shown in the example in
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Figure 2: the separations of neutral 5 are almost the same using neutral, basic, or acid/basic
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methanol additives. However, the separation of the acid 9 is significantly affected by the acid
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and basic additives as shown in Figures 3 and 4. Using the OD-H column, the retention increases
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using a basic modifier, but the resolution decreases as shown in Figure 3 (b). It appears that a
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mixture of acidic and basic additives does not improve the resolution although it increases the
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retention (see Figure 3 (c)). Interestingly, on the IC column, the addition of IPAm to the mobile
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phase not only increases the retention of amide 9, but also improves the resolution as it increases
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from 2.2 in Figure 4 (a) to 3.5 in Figure 4 (b). IPAm is more effective at increasing the retention
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and resolution than the combination of IPAm and TFA. In addition, the eluting order of the
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enantiomers of 9 on the IC column was reversed relative to the OD-H column indicating a
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different chiral mechanism for the separation on these two columns. Comparatively, peak shape
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is significantly improved on the IC column.
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From the screening results, none of the amide pairs could be separated on all 5 columns
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and no column could resolve all nine pairs. Since the IC and the OD-H columns showed the best
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separations for this group of compounds, they were used for further method development.
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3.2. Effects of modifier and modifier composition
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As discussed in the “column selection” section, CSP provides chiral resolution through
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potential hydrogen bonding, dipole-dipole, dipole-induced dipole, and π-π interactions with the
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amides. Similar to the stationary phase, mobile phase plays an important role in chiral separation
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as it interacts with the amides as well as the stationary phases. The effects of organic modifier
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and modifier composition on the separation were investigated.
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Three organic modifiers, methanol, IPA, and ACN with the same percentage composition
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for each compound, were studied for the separation on IC column at 150 bar, 40 ºC and 4
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mL/min. The results showed that methanol was superior to IPA and ACN as eight of the nine
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pairs could be baseline resolved using methanol while six of nine pairs could be separated using
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ACN. Only amide 7 was separated using IPA as shown in Figure 5(b). It is suspected that the
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relatively lower polarity and higher viscosity of IPA may cause longer retention and poorer
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column efficiency. The change in the three-dimensional structure of the stationary phase due to
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IPA adsorption could lead to steric hindrance for chiral interaction for most of the compounds.
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Amide 7 is the least sterically hindered of the amides. Its enantiomeric separation is less affected
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by the three-dimensional structure changes caused by IPA. It turned out that amide 7 was
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resolved well using IPA (see Figure 5(b)). Comparing Figure 5(a) with Figure 5(c), it can be seen
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that the retention, selectivity, and resolution also significantly increase with ACN relative to
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methanol. The same trends were observed for all other compounds separated using both
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methanol and ACN. Comparing to methanol and IPA, ACN is less capable for hydrogen
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bonding. Therefore, the moiety of the analyte which potentially forms hydrogen bonding may be
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less favorable to interact with the ACN modified mobile phase than with the stationary phase. It
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explains the observation that retention, selectivity, and resolution were significantly higher for
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ACN compared to methanol and IPA. However, ACN caused relatively significant band
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broadening, especially for the amide 8 and 9. The lower column efficiency was observed for
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these amides. Overall, methanol as mobile phase modifier provides the best separations and
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reasonable enantiomeric resolution in the shortest time. Therefore, it was chosen for further
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method development.
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For all eight of the nine amides whose enantiomeric pairs were resolved on the IC
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column, and amide 4 which was resolved on the OD-H column, the retention and resolution
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decrease with an increase in the percentage of methanol in the mobile phase as shown in Figures
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6 and 7. In Figure 6，the retention plots for all but amide 9 are approximately linear in the
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methanol composition range studied. As methanol was changed from 15% to 10%, the retention
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time of the later eluting peak of amide 9 increased from 3.05 to 8.29 min. This dramatic retention
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increase indicated stronger interactions between amide 9 and CSP. A relatively small decrease in
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methanol content may allow for stronger hydrogen bonding between the solute and CSP, leading
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to longer retention as observed.
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In Figure 7, the resolutions for all but amide 7 increase linearly as methanol composition decreases from 30% to 10%. As methanol composition decreased from 30% to 10%, the
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resolution of amide 7 changed from 0.8 to 6.0 and the selectivity increased from 1.1 to 1.5.
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Therefore, it might be an indication that hydrogen bonding is not only a dominant interaction for
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retention but also a considerable contribution to chiral recognition of the amide 7 [28].
It is noted that with 10% methanol, the baseline separation was achieved for eight of the
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nine enantiomeric pairs on the IC column and also for the amide 4 on the OD-H column.
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However, 10% methanol causeed relatively long retentions for amides 8 and 9. Therefore, 20%
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methanol was chosen for these two pairs and 10% methanol was used for the rest.
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3.3. Effect of column temperature
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Temperature is a potential factor affecting chiral separation by changing the mobile phase
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viscosity and the diffusion coefficient of the solute as well as the thermodynamic parameters.
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The temperature effect on the separation was studied for the nine racemates at 25, 30, 35 and 40
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ºC. Except for amide 4, eight amides were studied on IC column using methanol as an organic
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modifier with the flow rate of 4 mL/min. Amide 4 was studied on OD-H column with a similar
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mobile phase and the same flow rate.
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According to van’t Hoff equation
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Where ΔH and ΔS are the standard molar enthalpy and molar entropy of transferring solute from
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mobile phase to stationary phases, respectively.
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Retention factor (lnk) as a function of temperature (1/T) is plotted as shown in Figure 8. As temperature increased, a linear relationship was demonstrated for all 9 amides as predicted by 12
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van’t Hoff equation. However, the retention increased for amides 1, 3, 4, 5, and 9 and decreased
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for amide 8. For amides 2, 6, and 7, their retention stayed the same. As temperatures increases, the density and thus solvating power of CO2 decreases,
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leading to an increase in retention. At the same time, the analyte solubility increases with
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increasing temperature, resulting in a decrease in retention. For amides 1, 3, 4 ,5 and 9, the first
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effect might be more pronounced and as a result retention increases with increasing temperature
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[10, 21, 31]. For the amide 8, the second effect might be more dominating, leading to a decrease
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in retention as temperature increases. For amides 2, 6 and 7, these two effects might be even out,
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and their retention stays consistent with increasing temperature.
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In our experiments, temperature had negligible impact on selectivity (data not shown) except for the case of amide 3 in which its selectivity decreased from 1.18 to 1.15 as the
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temperature increased from 25 to 40°C. The decreasing selectivity led to a decrease in the
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resolution of amide 3 at higher temperature although better column efficiency was achieved at
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higher temperature. However, except for amide 3, the resolution for the rest of the enantiomeric
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pairs increased with temperature, mainly due to the increasing column efficiency with
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temperature. For example, in the case of amide 9, as the temperature changes from 25 to 40ºC,
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the selectivity stayed almost constant, the retention for the late-eluting peak increased from 2.07
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to 2.14 min, and the average plate number for the pair increased from 3400 to 4200,
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corresponding to a resolution increase from 2.0 to 2.5. For amide 8, as the temperature changed
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from 25 to 40ºC, the selectivity stayed almost constant. Although the retention decreased from
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3.54 to 3.41 and 4.64 to 4.46 for the pair, the resolution enhanced from 4.2 to 4.7 as a result of
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increasing column efficiency from 3700 to 4900. Except for amide 3, higher temperature favors
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higher resolution for this group of amides. Therefore, 40ºC was chosen for further method
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development.
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3.4. Effect of additive The effect of mobile phase additive on the separation was discussed in “column
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selection” section. It was found that additives have more impact on amide 9 which bears a
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carboxylic acid, and IPAm provides better resolution and peak shape. The impact of increasing
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IPAm concentration in the mobile phase on the enantioselectivity of amide 9 was studied. As
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shown in Figure 9, greater concentrations of IPAm provide for longer retention times and better
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resolution. 0.2% IPAm was used for separation of amide 9 as it provided good resolution within
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a reasonable separation time.
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3.5. Applications
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Table 3 describes the final optimized method conditions, and Table 4 lists the retention, selectivity, and resolution using those conditions.
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For the application purpose, the method validation standards such as linearity,
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reproducibility, and limit of quantitation were determined for amide 9 using the optimized
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instrument conditions listed. Good linearity was achieved at both nominal (3.3 mg/mL) and low
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level concentration ranges. Within the concentration range from 2.4 to 4.3 mg/mL (n = 5), the
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linear equation y = 4.98 x 106 x – 2.78 x 105, (r2 = 0.998) was obtained, while within the
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concentration range from 9.9 to 99 µg/mL (n = 5), the linear equation y = 4.79 x 106 x -1.34 x 105
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(r2 = 0.998) was achieved for low level concentration range.
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The system reproducibility was investigated using six injections of 3.3 mg/mL and 9.9
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μg/mL of the compound 9 desired enantiomer, respectively. The RSD at 3.3 mg/mL was 0.46%,
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while the RSD at 9.9 μg/mL was 3.2%. 14
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All validation was carried out using a Berger SFC system. The relatively poor signal to
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noise in the SFC/UV detector only allowed quantitation to 9.9 µg/mL (0.3 area% of nominal
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concentration) of the undesired enantiomer as shown in Figure 10 (a) with acceptable signal to
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noise ratio of 10. With improved instrument design towards a less noisy baseline in an Aurora
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SFC system, the LOQ was re-evaluated as shown in Figure 10 (b) using 9.9 μg/mL of amide 9.
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The signal to noise ratio was improved from 10 to 25. The LOQ as low as 0.10% was achieved
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using the new instrument.
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4. Conclusions
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Our study showed that the enantiomers of nine amide analogs bearing an α-stereocenter
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and different substitutions on the amide nitrogen can be efficiently resolved using SFC. Of the
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CSPs investigated, OD-H and IC columns showed the best results with baseline resolution of
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eight of the nine enantiomeric pairs. However, amide 4 is separated by OD-H column but not by
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IC column, whereas amide 2 is separated by the IC column, but not on OD-H column. The
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elution order of some enantiomeric pairs was reversed on OD-H relative to IC column. These
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results indicate that different chiral recognition mechanisms are involved in the separations on
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the OD-H and IC columns based on the respective carbamate derivatives in the two CSPs. The
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effects of substitution on the amide nitrogen on the enantiorecognition were also investigated in
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this study. It was found that steric hindrance may play a critical role in the chiral separations on
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both OD-H and IC columns. This effect may be more significant on IC column for this series of
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amides since amides 3 and 4 with relatively bulky substitutions at the amide nitrogen were either
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poorly resolved or not resolved. A proton on the amide nitrogen was found necessary for the
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enantiorecognition on the OD-H column, indicating that the proton can hydrogen bond with the
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stationary phase, serving as an important enantiorecognition force for the amides on the OD-H
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column. In addition to CSPs, the organic modifier and organic modifier composition in the
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mobile phase have dramatic impacts on the chiral separation of amides. Compared to acetonitrile
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and IPA, methanol was found to provide the best separations as eight of nine amides were
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resolved. Also, reasonable resolutions in the shortest time were achieved using methanol.
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Retention, selectivity, and resolution increased as methanol content decreased, indicating that
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hydrogen bonding is not only a dominating interaction for retention but also a contributing
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attraction for chiral recognition, especially for amide 7. The effect of temperature on the
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retention varied among the amides. Although the temperature had negligible impact on
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selectivity, the resolution increased with temperature as a result of higher column efficiency at
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higher temperature for most of the amides investigated.
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Using optimized conditions, validation of a SFC chiral method for amide 9 was
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evaluated, confirming that the method can be used for quantitation. Finally, the substantial
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increase in signal to noise due to SFC instrument improvement enabled a method with lower
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detection and quantitation limits.
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Acknowledgments:
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The authors thank Hongyao Zhu for the fruitful discussion on the chiral interactions and Frederick J. Antosz for review comments.
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References:
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[1] H. Caner, E. Groner, L. Levy, I. Agranat, Drug Discov. Today 9 (2004) 105.
362
[2] L. Borgstrom, B. Kennedy, B. Nilsson, B. Angelin, Eur. J. Clin. Pharmacol.
363
38 (1990) 621.
364
[3] I. Wainer, (Ed) Drug Stereochemistry. Analytical Methods and Pharmacology, 2nd Ed.; 1993.
365
[4] D. B. Campbell, Eur. J. Drug Metab. Ph. 15 (1990) 109.
366
[5] W. H. De Camp, J. Pharm. Biomed. Anal. 11 (1993) 1167.
367
[6] Y. Xiang, G. W. Sluggett, J. Pharm. Biomed. Anal. 53 (2010) 878.
368
[7] V. Schurig, V. TrAC, 21 (2002) 647.
369
[8] O. Stephany, F. Dron, S. Tisse, A. Martinez, J.M. Nuzillard, V. Peulon-Agasse, J.
370
Chromatogr. A 1216 (2009) 4051.
371
[9] K. D. Klerck, D. Mangelings, D. Clicq, F. D. Boever, Y. V. Heyden, J. Chromatogr. A 1234
372
(2012) 72.
373
[10] Y. Zhao, W. A. Pritts, S. Zhang, J. Chromatogr. A 1189 (2008) 245.
374
[11] T. Q. Yan, C. Orihuela, J. P. Preston, Chirality 22 (2010) 922.
375
[12] L. Toribio, M. J. del Nozal, J. L. Bernal, J. Bernal, M. T. Martín, J. Chromatogr. A 1218
376
(2011) 4886.
377
[13] M.Ventura, B. Murphy, W. Goetzinger, J. Chromatogr. A 1220 (2012) 147.
378
[14] J. Qian-Cutrone, R. Hartz, V. T. Ahuja, V. M. Vrudhula, D. Wu, R.A. Dalterio, D. Wang-
379
Iverson, J. J. Bronson, J. Pharm. Biomed. Anal. 54 (2011) 602.
380
[15] A. Zhang, W. Gao, B. Ma, L. Jin, C. Lin, Anal Bioanal Chem 403 (2012) 2665.
381
[16] A. P. Kumar, J. H. Park, J. Chromatogr. A 1218 (2011) 1314.
Ac ce pt e
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M
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us
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360
17
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[17] Y. Gong, Y. Xiang, B. Yue, G. Xue, J. S. Bradshaw, H. K. Lee, M. L. Lee, J. Chromatogr.
383
A 1002 (2003) 63.
384
[18] M. M. Warnke, C. R. Mitchell, R. V. Rozhkov, D. E. Emrich, R. C. Larock, D. W.
385
Armstrong, J. Liq. Chromatogr. R. T. 28 (2005) 823.
386
[19] H. Nelander, S. Andersson, K. Öhlén, J. Chromatogr. A 1218 (2011) 9397.
387
[20] L. Zhou, V. Antonucci, M. Biba, X. Gong, Z. Ge, J. Pharm. Biomed. Anal. 51 (2010) 153.
388
[21] N. Wu, Adv. Chromatogr. 46 (2008) 213.
389
[22] R. Helmy, M. Biba, J. Zang, B. Mao, K. Fogelman, V. Vlachos, P. Hosek, C. J. Welch,
390
Chirality 19 (2007) 787.
391
[23] C. White, J. Burnett, J. Chromatogr. A 1074 (2005) 175.
392
[24] A. K. Ghose, V. N. Viswanadhan, J. J. Wendoloski, J. Comb. Chem. 1 (1999) 55
393
[25] S. D. Roughley, A. M. Jordan, J. Med. Chem. 54 (2011), 3451.
394
[26] J. R. Dunetz, Y. Xiang, A. Baldwin, J. Ringling, Org. Lett. 13 (2011) 5048.
395
[27] J. R. Dunetz, M. A. Berliner, Y. Xiang, T. L. Houck, F. H. Salingue, C. Wang, Y. Chen, S.
396
Wang, Y. Huang, D. Farrand, S. J. Boucher, D. B. Damon, T. W. Makowski, M. T. Barrila, R.
397
Chen, I. Martínez, Org. Process Res. Dev., 16 (2012) 1635.
398
[28] M. Lämmerhofer, J. Chromatogr. A 1217 (2010) 814.
399
[29] G. Ferguson, C. Glidewell, I. L. J. Patterson, Acta Cryst. C52 (1996) 420.
400
[30] M. D. Altman, A. Ali, G. S. K. K. Reddy, M. N. L. Nalam, S. G. Anjum, H. Cao, S.
401
Chellappan, V. Kairys, M. X. Fernandes, M. K. Gilson, C. A. Schiffer, T. M. Rana, B. Tidor,
402
JACS 130 (2008) 6099.
403
[31] P. Mourier, P. Sassiat, M. Caude, R. Rosse, J. Chromatogr. A 353 (1986) 61.
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404 405 Captions:
407
Figure 1. Chemical structure of amides (single enantiomer shown)
408
Figure 2. Separation of amide 5 on IC column (a) methanol; (b) methanol with 0.1% IPAm; (c)
409
methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in Table
410
1.
411
Figure 3. Separation of amide 9 on OD-H column (a) methanol; (b) methanol with 0.1% IPAm;
412
(c) methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in
413
Table 1.
414
Figure 4. Separation of amide 9 on IC column (a) methanol; (b) methanol with 0.1% IPAm; (c)
415
methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in Table
416
1.
417
Figure 5. The effect of organic modifiers on the separation of amide 7. Conditions: an IC
418
column, 40°C, 4 mL/min, back pressure of 150 bar and 10% organic modifier of (a) methanol;
419
(b) IPA; (c) ACN.
420
Figure 6. The effect of methanol composition on the retention. Conditions: IC and OD-H (for
421
amide 4 only) columns, 40°C, 4 mL/min, and back pressure of 150 bar.
422
Figure 7. The effect of methanol composition on the resolution. Same conditions as Figure 6.
423
Figure 8. The effect of temperature on retention. Conditions: IC and OD-H (for amide 4 only)
424
columns, 40°C, 4 mL/min, back pressure of 150 bar and 10% methanol (20% methanol for
425
amides 8 and 9).
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Figure 9. Separation of amide 9 at various isopropylamine additive concentrations (a) without
427
any additive; (b) with 0.1% IPAm; (c) with 0.2% IPAm.
428
Figure 10. Chromatograms of amide 9 at the concentration of 9.9 µg/mL (0.3% of nominal
429
concentration) using (a) Berger SFC system and (b) Aurora SFC, other conditions are listed in
430
Table 3.
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20
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure 8
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Figure 9
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Figure 10
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Table 1
Table 1. Chiral SFC Screening Method Conditions. Description
Modifier Gradient: Detector: Flow Rate: Injection Volume: Column Temperature: Sample Concentration: Back Pressure: Total Run Time: Modifiers:
Modifier from 5 to 50% gradient at 6.5%/min, hold for 1 min 210 nm and 254 nm 4.0 mL/min 10 µL 40°C Approximately 3 mg/mL sample in ACN 150 bar 8 min MeOH, 0.1% IPAm in MeOH 0.3% formic acid and 0.3% IPAm in 75/25 MeOH/ACN OD-H, OJ-H, AD-H , AS-H and IC
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Columns:
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Chromatographic parameters
Page 31 of 34
Table 2
Table 2. Selectivity and resolution results from method screening using methanol as a modifier AD-H Compound
Rs
1
AS-H
OD-H
OJ-H
IC
2
α
Rs
Α
Rs
α
Rs
α
Rs
α
3.03
1.11
1.65
1.07
0
1
7.36
1.29
0
1
2
1.28
1.06
0
1
0
1
0
1
3.16
1.11
3
1.65
1.06
0
1
6.95
1.25
1.7
1.06
1.5
1.05
4
2.03
1.11
0
1
2.15
1.09
1.3
1.07
0
1
5
1.40
1.06
1.61
1.08
3.60
1.12
6
0
1
0
1
1.42
1.07
7
0
1
0
1
10.7
1.60
0
8
2.80
1.14
0
1
4.45
1.18
9
1.60
1.08
0
1
1.95
1.07
Rs
1
2.11
1.08
0
1
1.71
1.05
us
cr 0
1
6.89
1.32
1.2
1.04
3.56
1.11
1.2
1.06
1.61
1.07
an
1
ip t
1
1.18(t 2 t1 ) , where t1 and t2 are the retention times for the less and more retained peaks, while W 0.5,1 W0.5,2 W0.5,1 W0.5, 2
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are the half peak width of the less and more retained peaks, respectively; t t 2 2 0 , where t0 is the void time. t1 t 0
Page 32 of 34
Table 3
Table 3. Optimized chiral SFC method conditions.
ip t
Outlet Pressure: Injection Volume: Flow Rate: Detection: Mobile Phase A: Mobile Phase B:
Chiralpak IC for the compounds except for the compound 4 OD-H for the compound 4, 4.6 x 250 mm 40 °C on the inlet side of the column, and 46.5 °C on the outlet side of the column 150 bar 10 µL 4 mL/min 254 nm bandwidth 16nm (210 nm for the compound 7) CO2 MeOH for the amides 1-8
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Column Temperature:
Description
0.2% IPAm in MeOH the amide 9
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ACN 8 minutes 90/10 Mobile Phase A / Mobile Phase B (v/v) for the compounds 1-7, while 80/20 Mobile Phase A / Mobile Phase B (v/v) for the compounds 8-9.
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Dissolving solvent: Run Time: Isocratic Conditions:
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Chromatographic parameters Column:
Page 33 of 34
Table 4
Table 4. Retention time, selectivity and resolution using the optimized conditions. Retention time
Retention time
Resolution1
Selectivity2
1
(t2, min) 2.4
(Rs) 3.8
(α) 1.3
2
(t1, min) 1.9 (t1) 2.6
3.3
3.8
1.3
3
3.0
3.5
2.3
1.2
4
3.0
3.6
1.9
1.2
5
2.2
2.6
2.4
6
3.7
4.3
1.5
7
1.3
2.0
6.0
8
3.4
4.4
4.7
9
2.9
4.0
cr 1.1
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an
Rs
1.2
5.0
1.5 1.3 1.4
1.18(t 2 t1 ) , where t1 and t2 are the retention times for the less and more retained peaks, while W 0.5,1 W0.5,2 W0.5,1 W0.5, 2
M
1
ip t
Amide
Ac ce p
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d
are the half peak width of the less and more retained peaks, respectively; t t 2 2 0 , where t0 is the void time. t1 t 0
Page 34 of 34
S0021-9673(13)00513-X http://dx.doi.org/doi:10.1016/j.chroma.2013.03.048 CHROMA 354202
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
27-11-2012 13-3-2013 18-3-2013
Please cite this article as: Y. Xiang, J.R. Dunetz, M. Lovdahl, Chiral Separation of Amides Using Supercritical Fluid Chromatography, Journal of Chromatography A (2013), http://dx.doi.org/10.1016/j.chroma.2013.03.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights 1. Baseline enantiomeric separation of nine amide analogs was achieved using SFC;
3
2. Of the five CSPs investigated, the OD‐H and the IC columns showed the best results;
4
3. Steric hindrance and hydrogen bonding have significant impact on the separations;
5
4. Using optimized conditions, validation of a SFC chiral method was demonstrated.
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7 8 9 10 Chiral Separation of Amides Using Supercritical Fluid Chromatography
12 13 14 15 16
Yanqiao Xiang,*,a Joshua R. Dunetzb, Michael Lovdahla
Analytical Research and Development, bChemistry Research and Development, Pharmaceutical Sciences, Pfizer Worldwide Research and Development, Eastern Point Road, Groton, CT 06340
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a
*
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Corresponding author. Current address: Analytical Development Small Molecule, Millennium: The Takeda Oncology Company, Cambridge, MA 02319, United States. Tel.:+1 617 444 1675; E-mail: [email protected]
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24 25 26 27 28 29 30 31 32 33 34
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40 41
Abstract Nine amide derivatives bearing α-stereocenters as well as different substitutions on the amide nitrogen were synthesized via a n-propanephosphonic acid cyclic anhydride (T3P)-
43
mediated coupling, and their enantiomeric pairs were separated using supercritical fluid
44
chromatography (SFC). Five polysaccharide-based chiral stationary phases (CSPs), Chiralcel
45
OD-H and OJ-H, and Chiralpak AD-H, AS-H and IC columns were explored for the chiral
46
separation of these compounds. None of the compounds could be resolved on all five columns,
47
and no single column could separate all nine pairs of enantiomers. Comparatively, the IC and
48
OD-H columns showed the best results for this group of amides, yielding baseline separations for
49
eight of nine pairs. The type of polar functional group and aromatic substitution in the CSPs and
50
the substitutions on the amide nitrogen had a significant impact on the enantiomeric resolution of
51
the compounds in the interaction between the analyte and the stationary phases. The potential
52
separation mechanism and the effect of substitutions in the CSPs and amide solutes on the
53
separation were discussed. The effects of the organic modifiers, modifier composition, mobile
54
phase additives, and temperature were investigated for the separation of these amides on the IC
55
or the OD-H column. Baseline resolution was achieved under optimized chromatographic
56
conditions using an IC or an OD-H column. Linearity, reproducibility, and limit of quantitation
57
were also demonstrated for the compound 9 (see Figure 1). Approximately three-fold
58
improvement in signal-to-noise was observed using a SFC system with better instrument design.
59
Keywords: SFC; enantiomeric separation; amide; pharmaceutical
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60 61 62 63 64 3
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65
Introduction A survey within the past ten years has shown that 60-70% of the most frequently
67
prescribed drugs and the drug candidates under development are single enantiomers [1]. For
68
any enantiomeric drug or drug candidate, an enantiomeric impurity can produce different
69
pharmacological, toxicological, metabolic, and pharmacokinetic effects within the chiral
70
environment of a biological system [2-4]. As part of continuing efforts to improve the safety
71
and efficacy of drugs, the attention of pharmaceutical companies and regulatory agencies has
72
been focused on impurity control for active pharmaceutical ingredients (APIs). In order to
73
develop robust processes and deliver high-quality products, reliable chiral separation
74
methods have become necessary to assess the stereochemical integrity of enantiomers as
75
well as examine the potential for interconversion of individual stereoisomers [5].
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An assessment of the enantiomeric purity of pharmaceuticals can be achieved using different chromatographic techniques including gas chromatography (GC) [6-8], supercritical
78
fluid chromatography (SFC) [9-15], capillary electrophoresis (CE) [16], capillary
79
electrochromatography (CEC) [17], and high performance liquid chromatography (HPLC) [18-
80
20]. HPLC methods are most widely used for chiral separation in the pharmaceutical industry.
81
However, taking advantage of diversified enantioselective HPLC chiral stationary phases
82
(CSPs), SFC utilizes supercritical CO2 as a major mobile phase solvent, providing combined
83
advantages of speed, efficiency, and environmental friendliness. The low viscosity and high
84
diffusivity of supercritical CO2 allow higher flow rates resulting in faster separations, typically
85
three to five times faster than HPLC. The higher diffusivity also leads to lower mass transfer
86
resistance, therefore resulting in sharper peaks and better column efficiencies [21]. Together with
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87
improved instrumentation resulting in much better signal-to-noise, SFC is becoming an
88
increasingly prevalent approach to chiral separation in pharmaceutical development. [22-23]. Amides are commonly found within compounds of pharmaceutical importance and were
90
embedded within 25% of known pharmaceuticals at the start of the 21st century [24]. Not
91
surprisingly, the condensation of an amine and carboxylic acid to form an amide bond has been
92
the most frequently employed transformation for the synthesis of drug candidates [25]. Despite
93
continuing advances in methods to prepare amide bonds, a recurring challenge for these
94
couplings involves avoiding the epimerization of activated carboxylic acid substrates under
95
reaction conditions. To this end, we developed a process mediated by T3P (n-propanephosphonic
96
acid cyclic anhydride) to prepare a series of amides in high yield under conditions which
97
suppress the epimerization of acid substrates that are particularly sensitive to racemization [26-
98
27].
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In order to support the process development of this T3P-mediated amide coupling, chiral
100
SFC methods were developed for nine pairs of amide enantiomers. Five commercially available
101
polysaccharide chiral stationary phases were evaluated. The effects of the substituents at the
102
amide nitrogen on the enantiomeric resolution of the compounds were explored. The effects of
103
the organic modifiers, modifier composition, mobile phase additives, flow rate, and temperature
104
on the separation of these amides were investigated. The method linearity, reproducibility, and
105
limit of quantitation were evaluated for enantiomeric purity determination for both in-process-
106
control development and the drug candidates produced using the T3P process.
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107
2. Materials and methods
108
2. 1. Chemicals and Reagents
109
HPLC grade methanol, ethanol (200 proof), isopropanol (IPA), acetonitrile (ACN), spectrophotometric grade trifluoroacetic acid (TFA), and reagent grade isopropylamine (IPAm)
111
were purchased from Aldrich (Saint Louis, MO, USA). SFC-grade carbon dioxide used was
112
purchased from Airgas (CT, USA)
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Chiralcel OD-H (OD-H) and OJ-H (OJ-H), and Chiralpak AD-H (AD-H), AS-H (AS-H)
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and IC columns were purchased from Chiral Technologies (West Chester, PA, USA).
115
2.2. Sample Preparation
All amide samples used in this study were prepared as described in the references 26 and
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27, and the structures of their major enantiomers are shown in Figure 1.
118
2. 3. Equipments
SFC experiments were performed on a Berger analytical system equipped with FCM-
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1200 dual pump fluid control module (Mettler-Toledo AutoChem, Columbia, MD) and a SFC
121
Fusion™ A5 analytical instrument (Aurora SFC Systems, Inc. CA) connected to a 1100 HPLC
122
system (Agilent Technologies, Palo Alto, CA). A high pressure flow cell was utilized with an
123
HPLC PDA detector. Berger SFC ProNTo software (version 92.1) and Agilent ChemStation
124
(version B.04.01) were used for the instrument control and the data collection. UV detection at
125
both 210 nm and 254 nm was applied. Electrospray ionization mass spectrometer (MSD,
126
Agilent), connected to the outlet of the UV detector flow cell, was used to verify the
127
enantiomeric peak pairs.
128
2.4. Chromatographic Conditions 6
Page 6 of 34
129
The screening methods as shown in Table 1 were used as a starting point for method development. Further method optimization was performed to improve separation by fine tuning
131
the mobile phase/modifier composition, flow rate, and temperature for enantioselective SFC.
132
3. Results and Discussion
133
3. 1. Column Selection (screening results)
It is understood that the backbone structures of the CSPs examined in this study, which
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134
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130
are formed from carbohydrate chains containing polar functional groups (e.g., esters, carbamates,
136
residual hydroxyl groups), play a major role in chiral recognition. At the same time, the analyte
137
structure and its functional groups are also very critical for the enantiorecognition, and multiple
138
interactions between the amides and CSPs are possible [28]. For example, the C=O groups of the
139
amides can hydrogen bond with the NH groups of the CSPs as well as form dipole-dipole
140
interactions with the C=O groups of the CSPs. The NH moiety in some amides can hydrogen
141
bond with the C=O groups of the stationary phases. Additionally, the phenyl and benzyl groups
142
in the amides can form π-π interactions with the phenyl carbamate or ester moieties of the CSPs.
143
Some CSPs can favor some interactions more than others depending on the stationary phase and
144
solute structure. However, the detailed recognition mechanism for the polysaccharide based
145
CSPs still has not been completely elucidated.
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Nine pairs of amide enantiomers were screened using five polysaccharide CSPs and three
147
mobile phases using the methods described in Table 1, and some of the results are summarized in
148
Table 2. It can be seen that eight of the nine pairs are baseline separated using an IC column, and
149
the resolution order for the eighr separated pairs is 7>8>2>1>5>6>9>>3. The fact that amide 4
150
was not separated while the highest resolution of amide 7 was achieved on the IC column could
151
be due to differences in steric hindrance between 4 and 7. As the backbone and polar functional 7
Page 7 of 34
groups of the CSPs form a regular arrangement of grooves to serve as enantioselective binding
153
pockets, the steric effect can determine the analyte accessibility to the binding sites, such as the
154
hydrogen bonding sites buried inside of the cavity [28]. This explains why the least bulky amide
155
7 can easily fit into the binding site to get the best enantiomeric resolution. However, amide 4
156
shows higher steric hindrance effect to the binding site due to methyl groups at both ortho
157
positions of the phenyl ring. Amide 3 contains a phenyl group like amide 4 but has a methoxy
158
group at the para position instead of the ortho methyl groups, and it is baseline separated on the
159
IC column as it represents less steric hindrance than amide 4. This result further confirms that the
160
steric effect of the analyte can play a critical role in the separation of this group of amides on the
161
IC column. In addition, the methoxy oxygen in amide 3, a hydrogen bonding acceptor, may form
162
hydrogen bonding with the stationary phase to contribute a better separation of amide 3 than that
163
of amide 4 [29-30]. Similar to the IC column, the OD-H column yielded baseline separations of
164
eight of the nine enantiomeric pairs and the resolution order for the eight separated pairs on the
165
OD-H column is 7>1>3>8>5>4>9>6. Amide 7 has the highest resolution, as high as 10.7, on the
166
OD-H column, indicating steric effects can also play a critical role in separations on the OD-H
167
column. However, unlike the separations on the IC column, the most sterically hindered amide 4
168
is separated on OD-H column, but amide 2 is not. This could be due to the differences in the
169
polar functional groups in the CSPs (3,5-dimethylphenyl carbamate for OD-H and 3,5-
170
dichlorophenyl carbamate for IC column). Interestingly, amide 1 and 2 have similar structures
171
except that amide 1 is a secondary amide whereas amide 2 is a tertiary amide with a methyl
172
group attached to the nitrogen. This subtlety makes significant difference in the
173
enantiorecognition as amide 1 is separated with the resolution as high as 7.4 while amide 2 is
174
not resolved at all. In addition, amides 2 and 6 are the only two containing tertiary amine
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8
Page 8 of 34
functionality in this series. Although 6 is almost baseline separated on the OD-H column, this
176
amide shows the poorest resolution in this series. It can be deduced that the proton on the
177
nitrogen in the amides also plays an essential role as an H-donor in the enantiorecognition of the
178
amides on the OD-H column. On the other hand, the only structural difference between amide 2
179
and 6 is a phenyl versus benzyl group, respectively, connected to the amide nitrogen. By
180
comparison, the hydrogen-bonding ability of the carbonyl in amide 2 is decreased due to the
181
delocalization of electron density from the amide into the adjacent phenyl group.
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The AD-H column separated seven of the nine amide pairs and the OJ-H column
183
separated four of the nine enantiomeric pairs. The AS-H column showed the poorest selectivity
184
for this series, separating only one of the nine amide pairs.
an
182
Mobile phase additives are often used to improve enantiomeric separations and peak
186
shapes via stationary phase modification and acidic or basic analyte ionization suppression.
187
Methanol-based mobile phases without any additive, with basic additive, or with a mixture of
188
TFA and IPAm additives were screened for this series of amides (results are not shown). Eight of
189
the nine amide pairs are neutral. As expected, basic and a mixture of acidic and basic additives
190
have little effect on the chiral separation of the neutral compounds as shown in the example in
191
Figure 2: the separations of neutral 5 are almost the same using neutral, basic, or acid/basic
192
methanol additives. However, the separation of the acid 9 is significantly affected by the acid
193
and basic additives as shown in Figures 3 and 4. Using the OD-H column, the retention increases
194
using a basic modifier, but the resolution decreases as shown in Figure 3 (b). It appears that a
195
mixture of acidic and basic additives does not improve the resolution although it increases the
196
retention (see Figure 3 (c)). Interestingly, on the IC column, the addition of IPAm to the mobile
197
phase not only increases the retention of amide 9, but also improves the resolution as it increases
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9
Page 9 of 34
from 2.2 in Figure 4 (a) to 3.5 in Figure 4 (b). IPAm is more effective at increasing the retention
199
and resolution than the combination of IPAm and TFA. In addition, the eluting order of the
200
enantiomers of 9 on the IC column was reversed relative to the OD-H column indicating a
201
different chiral mechanism for the separation on these two columns. Comparatively, peak shape
202
is significantly improved on the IC column.
ip t
198
From the screening results, none of the amide pairs could be separated on all 5 columns
204
and no column could resolve all nine pairs. Since the IC and the OD-H columns showed the best
205
separations for this group of compounds, they were used for further method development.
us
3.2. Effects of modifier and modifier composition
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206
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203
As discussed in the “column selection” section, CSP provides chiral resolution through
208
potential hydrogen bonding, dipole-dipole, dipole-induced dipole, and π-π interactions with the
209
amides. Similar to the stationary phase, mobile phase plays an important role in chiral separation
210
as it interacts with the amides as well as the stationary phases. The effects of organic modifier
211
and modifier composition on the separation were investigated.
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207
Three organic modifiers, methanol, IPA, and ACN with the same percentage composition
213
for each compound, were studied for the separation on IC column at 150 bar, 40 ºC and 4
214
mL/min. The results showed that methanol was superior to IPA and ACN as eight of the nine
215
pairs could be baseline resolved using methanol while six of nine pairs could be separated using
216
ACN. Only amide 7 was separated using IPA as shown in Figure 5(b). It is suspected that the
217
relatively lower polarity and higher viscosity of IPA may cause longer retention and poorer
218
column efficiency. The change in the three-dimensional structure of the stationary phase due to
219
IPA adsorption could lead to steric hindrance for chiral interaction for most of the compounds.
220
Amide 7 is the least sterically hindered of the amides. Its enantiomeric separation is less affected
10
Page 10 of 34
by the three-dimensional structure changes caused by IPA. It turned out that amide 7 was
222
resolved well using IPA (see Figure 5(b)). Comparing Figure 5(a) with Figure 5(c), it can be seen
223
that the retention, selectivity, and resolution also significantly increase with ACN relative to
224
methanol. The same trends were observed for all other compounds separated using both
225
methanol and ACN. Comparing to methanol and IPA, ACN is less capable for hydrogen
226
bonding. Therefore, the moiety of the analyte which potentially forms hydrogen bonding may be
227
less favorable to interact with the ACN modified mobile phase than with the stationary phase. It
228
explains the observation that retention, selectivity, and resolution were significantly higher for
229
ACN compared to methanol and IPA. However, ACN caused relatively significant band
230
broadening, especially for the amide 8 and 9. The lower column efficiency was observed for
231
these amides. Overall, methanol as mobile phase modifier provides the best separations and
232
reasonable enantiomeric resolution in the shortest time. Therefore, it was chosen for further
233
method development.
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For all eight of the nine amides whose enantiomeric pairs were resolved on the IC
Ac ce pt e
234
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221
235
column, and amide 4 which was resolved on the OD-H column, the retention and resolution
236
decrease with an increase in the percentage of methanol in the mobile phase as shown in Figures
237
6 and 7. In Figure 6，the retention plots for all but amide 9 are approximately linear in the
238
methanol composition range studied. As methanol was changed from 15% to 10%, the retention
239
time of the later eluting peak of amide 9 increased from 3.05 to 8.29 min. This dramatic retention
240
increase indicated stronger interactions between amide 9 and CSP. A relatively small decrease in
241
methanol content may allow for stronger hydrogen bonding between the solute and CSP, leading
242
to longer retention as observed.
11
Page 11 of 34
243
In Figure 7, the resolutions for all but amide 7 increase linearly as methanol composition decreases from 30% to 10%. As methanol composition decreased from 30% to 10%, the
245
resolution of amide 7 changed from 0.8 to 6.0 and the selectivity increased from 1.1 to 1.5.
246
Therefore, it might be an indication that hydrogen bonding is not only a dominant interaction for
247
retention but also a considerable contribution to chiral recognition of the amide 7 [28].
It is noted that with 10% methanol, the baseline separation was achieved for eight of the
cr
248
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244
nine enantiomeric pairs on the IC column and also for the amide 4 on the OD-H column.
250
However, 10% methanol causeed relatively long retentions for amides 8 and 9. Therefore, 20%
251
methanol was chosen for these two pairs and 10% methanol was used for the rest.
252
3.3. Effect of column temperature
an
Temperature is a potential factor affecting chiral separation by changing the mobile phase
M
253
us
249
viscosity and the diffusion coefficient of the solute as well as the thermodynamic parameters.
255
The temperature effect on the separation was studied for the nine racemates at 25, 30, 35 and 40
256
ºC. Except for amide 4, eight amides were studied on IC column using methanol as an organic
257
modifier with the flow rate of 4 mL/min. Amide 4 was studied on OD-H column with a similar
258
mobile phase and the same flow rate.
Ac ce pt e
259 260 261
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254
According to van’t Hoff equation
262 263 264 265
Where ΔH and ΔS are the standard molar enthalpy and molar entropy of transferring solute from
266
mobile phase to stationary phases, respectively.
267 268
Retention factor (lnk) as a function of temperature (1/T) is plotted as shown in Figure 8. As temperature increased, a linear relationship was demonstrated for all 9 amides as predicted by 12
Page 12 of 34
269
van’t Hoff equation. However, the retention increased for amides 1, 3, 4, 5, and 9 and decreased
270
for amide 8. For amides 2, 6, and 7, their retention stayed the same. As temperatures increases, the density and thus solvating power of CO2 decreases,
272
leading to an increase in retention. At the same time, the analyte solubility increases with
273
increasing temperature, resulting in a decrease in retention. For amides 1, 3, 4 ,5 and 9, the first
274
effect might be more pronounced and as a result retention increases with increasing temperature
275
[10, 21, 31]. For the amide 8, the second effect might be more dominating, leading to a decrease
276
in retention as temperature increases. For amides 2, 6 and 7, these two effects might be even out,
277
and their retention stays consistent with increasing temperature.
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In our experiments, temperature had negligible impact on selectivity (data not shown) except for the case of amide 3 in which its selectivity decreased from 1.18 to 1.15 as the
280
temperature increased from 25 to 40°C. The decreasing selectivity led to a decrease in the
281
resolution of amide 3 at higher temperature although better column efficiency was achieved at
282
higher temperature. However, except for amide 3, the resolution for the rest of the enantiomeric
283
pairs increased with temperature, mainly due to the increasing column efficiency with
284
temperature. For example, in the case of amide 9, as the temperature changes from 25 to 40ºC,
285
the selectivity stayed almost constant, the retention for the late-eluting peak increased from 2.07
286
to 2.14 min, and the average plate number for the pair increased from 3400 to 4200,
287
corresponding to a resolution increase from 2.0 to 2.5. For amide 8, as the temperature changed
288
from 25 to 40ºC, the selectivity stayed almost constant. Although the retention decreased from
289
3.54 to 3.41 and 4.64 to 4.46 for the pair, the resolution enhanced from 4.2 to 4.7 as a result of
290
increasing column efficiency from 3700 to 4900. Except for amide 3, higher temperature favors
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13
Page 13 of 34
291
higher resolution for this group of amides. Therefore, 40ºC was chosen for further method
292
development.
293
3.4. Effect of additive The effect of mobile phase additive on the separation was discussed in “column
ip t
294
selection” section. It was found that additives have more impact on amide 9 which bears a
296
carboxylic acid, and IPAm provides better resolution and peak shape. The impact of increasing
297
IPAm concentration in the mobile phase on the enantioselectivity of amide 9 was studied. As
298
shown in Figure 9, greater concentrations of IPAm provide for longer retention times and better
299
resolution. 0.2% IPAm was used for separation of amide 9 as it provided good resolution within
300
a reasonable separation time.
301
3.5. Applications
303
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Table 3 describes the final optimized method conditions, and Table 4 lists the retention, selectivity, and resolution using those conditions.
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For the application purpose, the method validation standards such as linearity,
305
reproducibility, and limit of quantitation were determined for amide 9 using the optimized
306
instrument conditions listed. Good linearity was achieved at both nominal (3.3 mg/mL) and low
307
level concentration ranges. Within the concentration range from 2.4 to 4.3 mg/mL (n = 5), the
308
linear equation y = 4.98 x 106 x – 2.78 x 105, (r2 = 0.998) was obtained, while within the
309
concentration range from 9.9 to 99 µg/mL (n = 5), the linear equation y = 4.79 x 106 x -1.34 x 105
310
(r2 = 0.998) was achieved for low level concentration range.
Ac ce pt e
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311
The system reproducibility was investigated using six injections of 3.3 mg/mL and 9.9
312
μg/mL of the compound 9 desired enantiomer, respectively. The RSD at 3.3 mg/mL was 0.46%,
313
while the RSD at 9.9 μg/mL was 3.2%. 14
Page 14 of 34
All validation was carried out using a Berger SFC system. The relatively poor signal to
315
noise in the SFC/UV detector only allowed quantitation to 9.9 µg/mL (0.3 area% of nominal
316
concentration) of the undesired enantiomer as shown in Figure 10 (a) with acceptable signal to
317
noise ratio of 10. With improved instrument design towards a less noisy baseline in an Aurora
318
SFC system, the LOQ was re-evaluated as shown in Figure 10 (b) using 9.9 μg/mL of amide 9.
319
The signal to noise ratio was improved from 10 to 25. The LOQ as low as 0.10% was achieved
320
using the new instrument.
321
4. Conclusions
cr
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Our study showed that the enantiomers of nine amide analogs bearing an α-stereocenter
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322
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314
and different substitutions on the amide nitrogen can be efficiently resolved using SFC. Of the
324
CSPs investigated, OD-H and IC columns showed the best results with baseline resolution of
325
eight of the nine enantiomeric pairs. However, amide 4 is separated by OD-H column but not by
326
IC column, whereas amide 2 is separated by the IC column, but not on OD-H column. The
327
elution order of some enantiomeric pairs was reversed on OD-H relative to IC column. These
328
results indicate that different chiral recognition mechanisms are involved in the separations on
329
the OD-H and IC columns based on the respective carbamate derivatives in the two CSPs. The
330
effects of substitution on the amide nitrogen on the enantiorecognition were also investigated in
331
this study. It was found that steric hindrance may play a critical role in the chiral separations on
332
both OD-H and IC columns. This effect may be more significant on IC column for this series of
333
amides since amides 3 and 4 with relatively bulky substitutions at the amide nitrogen were either
334
poorly resolved or not resolved. A proton on the amide nitrogen was found necessary for the
335
enantiorecognition on the OD-H column, indicating that the proton can hydrogen bond with the
336
stationary phase, serving as an important enantiorecognition force for the amides on the OD-H
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Page 15 of 34
column. In addition to CSPs, the organic modifier and organic modifier composition in the
338
mobile phase have dramatic impacts on the chiral separation of amides. Compared to acetonitrile
339
and IPA, methanol was found to provide the best separations as eight of nine amides were
340
resolved. Also, reasonable resolutions in the shortest time were achieved using methanol.
341
Retention, selectivity, and resolution increased as methanol content decreased, indicating that
342
hydrogen bonding is not only a dominating interaction for retention but also a contributing
343
attraction for chiral recognition, especially for amide 7. The effect of temperature on the
344
retention varied among the amides. Although the temperature had negligible impact on
345
selectivity, the resolution increased with temperature as a result of higher column efficiency at
346
higher temperature for most of the amides investigated.
cr
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Using optimized conditions, validation of a SFC chiral method for amide 9 was
M
347
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337
evaluated, confirming that the method can be used for quantitation. Finally, the substantial
349
increase in signal to noise due to SFC instrument improvement enabled a method with lower
350
detection and quantitation limits.
351
Acknowledgments:
353 354 355 356
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The authors thank Hongyao Zhu for the fruitful discussion on the chiral interactions and Frederick J. Antosz for review comments.
357 358 359 16
Page 16 of 34
References:
361
[1] H. Caner, E. Groner, L. Levy, I. Agranat, Drug Discov. Today 9 (2004) 105.
362
[2] L. Borgstrom, B. Kennedy, B. Nilsson, B. Angelin, Eur. J. Clin. Pharmacol.
363
38 (1990) 621.
364
[3] I. Wainer, (Ed) Drug Stereochemistry. Analytical Methods and Pharmacology, 2nd Ed.; 1993.
365
[4] D. B. Campbell, Eur. J. Drug Metab. Ph. 15 (1990) 109.
366
[5] W. H. De Camp, J. Pharm. Biomed. Anal. 11 (1993) 1167.
367
[6] Y. Xiang, G. W. Sluggett, J. Pharm. Biomed. Anal. 53 (2010) 878.
368
[7] V. Schurig, V. TrAC, 21 (2002) 647.
369
[8] O. Stephany, F. Dron, S. Tisse, A. Martinez, J.M. Nuzillard, V. Peulon-Agasse, J.
370
Chromatogr. A 1216 (2009) 4051.
371
[9] K. D. Klerck, D. Mangelings, D. Clicq, F. D. Boever, Y. V. Heyden, J. Chromatogr. A 1234
372
(2012) 72.
373
[10] Y. Zhao, W. A. Pritts, S. Zhang, J. Chromatogr. A 1189 (2008) 245.
374
[11] T. Q. Yan, C. Orihuela, J. P. Preston, Chirality 22 (2010) 922.
375
[12] L. Toribio, M. J. del Nozal, J. L. Bernal, J. Bernal, M. T. Martín, J. Chromatogr. A 1218
376
(2011) 4886.
377
[13] M.Ventura, B. Murphy, W. Goetzinger, J. Chromatogr. A 1220 (2012) 147.
378
[14] J. Qian-Cutrone, R. Hartz, V. T. Ahuja, V. M. Vrudhula, D. Wu, R.A. Dalterio, D. Wang-
379
Iverson, J. J. Bronson, J. Pharm. Biomed. Anal. 54 (2011) 602.
380
[15] A. Zhang, W. Gao, B. Ma, L. Jin, C. Lin, Anal Bioanal Chem 403 (2012) 2665.
381
[16] A. P. Kumar, J. H. Park, J. Chromatogr. A 1218 (2011) 1314.
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an
us
cr
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360
17
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[17] Y. Gong, Y. Xiang, B. Yue, G. Xue, J. S. Bradshaw, H. K. Lee, M. L. Lee, J. Chromatogr.
383
A 1002 (2003) 63.
384
[18] M. M. Warnke, C. R. Mitchell, R. V. Rozhkov, D. E. Emrich, R. C. Larock, D. W.
385
Armstrong, J. Liq. Chromatogr. R. T. 28 (2005) 823.
386
[19] H. Nelander, S. Andersson, K. Öhlén, J. Chromatogr. A 1218 (2011) 9397.
387
[20] L. Zhou, V. Antonucci, M. Biba, X. Gong, Z. Ge, J. Pharm. Biomed. Anal. 51 (2010) 153.
388
[21] N. Wu, Adv. Chromatogr. 46 (2008) 213.
389
[22] R. Helmy, M. Biba, J. Zang, B. Mao, K. Fogelman, V. Vlachos, P. Hosek, C. J. Welch,
390
Chirality 19 (2007) 787.
391
[23] C. White, J. Burnett, J. Chromatogr. A 1074 (2005) 175.
392
[24] A. K. Ghose, V. N. Viswanadhan, J. J. Wendoloski, J. Comb. Chem. 1 (1999) 55
393
[25] S. D. Roughley, A. M. Jordan, J. Med. Chem. 54 (2011), 3451.
394
[26] J. R. Dunetz, Y. Xiang, A. Baldwin, J. Ringling, Org. Lett. 13 (2011) 5048.
395
[27] J. R. Dunetz, M. A. Berliner, Y. Xiang, T. L. Houck, F. H. Salingue, C. Wang, Y. Chen, S.
396
Wang, Y. Huang, D. Farrand, S. J. Boucher, D. B. Damon, T. W. Makowski, M. T. Barrila, R.
397
Chen, I. Martínez, Org. Process Res. Dev., 16 (2012) 1635.
398
[28] M. Lämmerhofer, J. Chromatogr. A 1217 (2010) 814.
399
[29] G. Ferguson, C. Glidewell, I. L. J. Patterson, Acta Cryst. C52 (1996) 420.
400
[30] M. D. Altman, A. Ali, G. S. K. K. Reddy, M. N. L. Nalam, S. G. Anjum, H. Cao, S.
401
Chellappan, V. Kairys, M. X. Fernandes, M. K. Gilson, C. A. Schiffer, T. M. Rana, B. Tidor,
402
JACS 130 (2008) 6099.
403
[31] P. Mourier, P. Sassiat, M. Caude, R. Rosse, J. Chromatogr. A 353 (1986) 61.
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404 405 Captions:
407
Figure 1. Chemical structure of amides (single enantiomer shown)
408
Figure 2. Separation of amide 5 on IC column (a) methanol; (b) methanol with 0.1% IPAm; (c)
409
methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in Table
410
1.
411
Figure 3. Separation of amide 9 on OD-H column (a) methanol; (b) methanol with 0.1% IPAm;
412
(c) methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in
413
Table 1.
414
Figure 4. Separation of amide 9 on IC column (a) methanol; (b) methanol with 0.1% IPAm; (c)
415
methanol/ACN (75/25, v/v) with 0.1% TFA and 0.1%IPAm; other conditions are listed in Table
416
1.
417
Figure 5. The effect of organic modifiers on the separation of amide 7. Conditions: an IC
418
column, 40°C, 4 mL/min, back pressure of 150 bar and 10% organic modifier of (a) methanol;
419
(b) IPA; (c) ACN.
420
Figure 6. The effect of methanol composition on the retention. Conditions: IC and OD-H (for
421
amide 4 only) columns, 40°C, 4 mL/min, and back pressure of 150 bar.
422
Figure 7. The effect of methanol composition on the resolution. Same conditions as Figure 6.
423
Figure 8. The effect of temperature on retention. Conditions: IC and OD-H (for amide 4 only)
424
columns, 40°C, 4 mL/min, back pressure of 150 bar and 10% methanol (20% methanol for
425
amides 8 and 9).
Ac ce pt e
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406
19
Page 19 of 34
Figure 9. Separation of amide 9 at various isopropylamine additive concentrations (a) without
427
any additive; (b) with 0.1% IPAm; (c) with 0.2% IPAm.
428
Figure 10. Chromatograms of amide 9 at the concentration of 9.9 µg/mL (0.3% of nominal
429
concentration) using (a) Berger SFC system and (b) Aurora SFC, other conditions are listed in
430
Table 3.
Ac ce pt e
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426
20
Page 20 of 34
Ac ce p
te
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cr
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Figure 1
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ed
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cr
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Figure 2
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ed
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure 8
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Figure 9
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Figure 10
Page 30 of 34
Table 1
Table 1. Chiral SFC Screening Method Conditions. Description
Modifier Gradient: Detector: Flow Rate: Injection Volume: Column Temperature: Sample Concentration: Back Pressure: Total Run Time: Modifiers:
Modifier from 5 to 50% gradient at 6.5%/min, hold for 1 min 210 nm and 254 nm 4.0 mL/min 10 µL 40°C Approximately 3 mg/mL sample in ACN 150 bar 8 min MeOH, 0.1% IPAm in MeOH 0.3% formic acid and 0.3% IPAm in 75/25 MeOH/ACN OD-H, OJ-H, AD-H , AS-H and IC
cr
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Ac ce p
te
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M
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Columns:
ip t
Chromatographic parameters
Page 31 of 34
Table 2
Table 2. Selectivity and resolution results from method screening using methanol as a modifier AD-H Compound
Rs
1
AS-H
OD-H
OJ-H
IC
2
α
Rs
Α
Rs
α
Rs
α
Rs
α
3.03
1.11
1.65
1.07
0
1
7.36
1.29
0
1
2
1.28
1.06
0
1
0
1
0
1
3.16
1.11
3
1.65
1.06
0
1
6.95
1.25
1.7
1.06
1.5
1.05
4
2.03
1.11
0
1
2.15
1.09
1.3
1.07
0
1
5
1.40
1.06
1.61
1.08
3.60
1.12
6
0
1
0
1
1.42
1.07
7
0
1
0
1
10.7
1.60
0
8
2.80
1.14
0
1
4.45
1.18
9
1.60
1.08
0
1
1.95
1.07
Rs
1
2.11
1.08
0
1
1.71
1.05
us
cr 0
1
6.89
1.32
1.2
1.04
3.56
1.11
1.2
1.06
1.61
1.07
an
1
ip t
1
1.18(t 2 t1 ) , where t1 and t2 are the retention times for the less and more retained peaks, while W 0.5,1 W0.5,2 W0.5,1 W0.5, 2
Ac ce p
te
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M
are the half peak width of the less and more retained peaks, respectively; t t 2 2 0 , where t0 is the void time. t1 t 0
Page 32 of 34
Table 3
Table 3. Optimized chiral SFC method conditions.
ip t
Outlet Pressure: Injection Volume: Flow Rate: Detection: Mobile Phase A: Mobile Phase B:
Chiralpak IC for the compounds except for the compound 4 OD-H for the compound 4, 4.6 x 250 mm 40 °C on the inlet side of the column, and 46.5 °C on the outlet side of the column 150 bar 10 µL 4 mL/min 254 nm bandwidth 16nm (210 nm for the compound 7) CO2 MeOH for the amides 1-8
cr
Column Temperature:
Description
0.2% IPAm in MeOH the amide 9
an
ACN 8 minutes 90/10 Mobile Phase A / Mobile Phase B (v/v) for the compounds 1-7, while 80/20 Mobile Phase A / Mobile Phase B (v/v) for the compounds 8-9.
Ac ce p
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Dissolving solvent: Run Time: Isocratic Conditions:
us
Chromatographic parameters Column:
Page 33 of 34
Table 4
Table 4. Retention time, selectivity and resolution using the optimized conditions. Retention time
Retention time
Resolution1
Selectivity2
1
(t2, min) 2.4
(Rs) 3.8
(α) 1.3
2
(t1, min) 1.9 (t1) 2.6
3.3
3.8
1.3
3
3.0
3.5
2.3
1.2
4
3.0
3.6
1.9
1.2
5
2.2
2.6
2.4
6
3.7
4.3
1.5
7
1.3
2.0
6.0
8
3.4
4.4
4.7
9
2.9
4.0
cr 1.1
us
an
Rs
1.2
5.0
1.5 1.3 1.4
1.18(t 2 t1 ) , where t1 and t2 are the retention times for the less and more retained peaks, while W 0.5,1 W0.5,2 W0.5,1 W0.5, 2
M
1
ip t
Amide
Ac ce p
te
d
are the half peak width of the less and more retained peaks, respectively; t t 2 2 0 , where t0 is the void time. t1 t 0
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