Stereoselective high-performance liquid chromatography and analytical method characterization of evacetrapib using a brush-type chiral stationary phase: A challenging isomeric separation requiring a unique eluent system

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Stereoselective high-performance liquid chromatography and analytical method characterization of evacetrapib using a brush-type chiral stationary phase: A challenging isomeric separation requiring a unique eluent system V. Scott Sharp a,∗ , John D. Stafford b , Robert A. Forbes b , Megan A. Gokey a , Mary R. Cooper a a Product Design and Developability, Small Molecule Design and Development, Eli Lilly and Company, Lilly Technology Center North, Indianapolis, IN 46221, USA b Product and Process Performance, Small Molecule Design and Development, Eli Lilly and Company, Lilly Technology Center South, Indianapolis, IN 46221, USA

a r t i c l e

i n f o

Article history: Received 29 January 2014 Received in revised form 13 March 2014 Accepted 14 March 2014 Available online xxx Keywords: Chiral Evacetrapib Whelk O-1 Pirkle Brush Enantiomer

a b s t r a c t Using HPLC chiral separation screening, various columns representing the polysaccharide, macrocyclic antibiotic and brush classes were assessed in multiple chromatographic modes for the separation of evacetrapib, a potential cardiovascular drug, from its enantiomer, two diastereomers and several impurities. Screening data consistently pointed to the brush-type Whelk-O 1 chiral column as most promising for this task. A systematic separation optimization process is outlined using the (S,S) Whelk-O 1 chiral column, first for the resolution of the isomers, and later including six potential impurities. A relatively complex yet rugged separation system was eventually identified that effectively resolves all compounds within a reasonable analysis time, and should serve as an adequate tool for evacetrapib bulk drug enantiopurity measurement. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The systematic progression of a pharmaceutical candidate compound from its discovery, through development, and to commercialization is a lengthy and expensive endeavor. Along the entire process, analytical methodology designed to assess the quality and purity of the compound is necessary. Frequently, one of the most challenging requirements of this package for a chiral compound is the enantioseparation method. It is important to note that ICH guidelines require consideration of the control of the undesired enantiomer in the same manner as other impurities [1]. While various avenues exist for enantiopurity assessment, including supercritical and subcritical fluid chromatography, capillary electrophoresis, capillary electrochromatography and gas chromatography, HPLC remains the dominant technique for high quality chiral analysis. The HPLC chiral separation arena, as measured by the number of chiral stationary phases (CSPs) available, has grown

∗ Corresponding author. Tel.: +1 317 276 8140; fax: +1 317 655 2770. E-mail address: [email protected] (V.S. Sharp).

tremendously over the last 25 years, as well as the understanding of the science behind it. Chiral columns can be classified by selector type (polysaccharide, brush, macrocyclic antibiotic, cyclodextrin, crown ether, protein), bonding chemistry (coated or covalently bonded), or by functional chromatographic mode (normal phase, reversed phase, polar organic, polar ionic, hydrophilic interaction chromatography, extended solvent). This large assortment of CSPs and the various chromatographic modes in which they function must be taken into consideration when attempting to identify the best possible enantioseparation method. Despite a continually improving picture of how chiral selectors discriminate between enantiomers, the complexity of these interactions, as well as the unique microenvironment posed by each respective analyte at the atomic level, still limits the art of chiral separation development to a trial and error process. As a result, many laboratories initiate the search effort through the use of generalized multi-modal chiral separation screens employing multiple CSPs known for their broad enantioselectivity. A brief survey of chiral separation screens in the literature reveals a dominance of polysaccharide CSPs within the ranks of columns employed, including coated [2–11] and immobilized [5,7,12,13]. Literature reviews

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Fig. 1. Structure of Whelk-O 1 chiral selector. Source: Copied from Blum AM, Lynam KG, Nicolas EC. Use of a new Pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds. Chirality 1994;6:302–313.

by Akin et al. [14], Manglings and Vander Heyden [15] and Beesley [16] confirm this trend. On the occasion when a column of nonpolysaccharide origin is incorporated into a screening paradigm, it is most often a macrocyclic antibiotic CSP or a “brush-type” chiral column. A few published chiral separation screens, as well as some screens we employ, incorporate a specific brush-type ␲-electron acceptor/␲-electron donor CSP, the Whelk O-1 column, within their column repertoire [4,6,10,11,17]. Since the Whelk O-1 CSP was engineered specifically to resolve naproxen enantiomers, at least a measured understanding of this column’s chiral recognition mechanism has existed from the beginning. Pirkle and colleagues successfully employed a logical approach known as reciprocity to develop their many brush-type CSPs. In the case of the Whelk O-1 chiral selector, a CSP derived from a single enantiomer of naproxen was initially produced. This selector was tested for its resolving capability for several racemates. Those chiral molecules best resolved were then investigated for use as enantioselectors for the original naproxen racemate [18]. In this way, the most versatile brush-type chiral selector produced to date was synthesized [19–21]. The “brush” moniker refers to the appearance of the chiral selector. These low molecular weight structures are attached to silanol groups via a tether-like linkage, resulting in an ordered monomolecular chiral layer resembling a brush [22,23]. Enantiorecognition with brush-type CSPs is believed to occur from differences in the CSP–analyte relationship according to a threepoint interaction model. One interaction in this model must be stereochemically dependent. While some of the interactions must necessarily be attractive to allow for analyte retention, one or more stereospecific attractive or repulsive relationships must exist to allow for chiral discrimination. The resulting respective diastereomeric complexes formed between the analyte and CSP possess two distinct association energies, which results in different degrees of interaction, hence variable retention times, and therefore a chiral separation [24,25]. Significant effort has been expended toward the understanding of the three-point interaction model as it relates to a typical chiral separation using the Whelk-O 1 CSP. The selector consists of a 3,5-dinitrobenzamide group connected to a 1,2,3,4tetrahydrophenanthrene moiety via an amide linkage. The chirality of the selector is introduced at two chiral carbons, one linking the 1,2,3,4-tetrahydrophenanthrene group to the amide nitrogen, and the other linking the aryl moiety to an alkyl tether linked to silica (Fig. 1) [23]. The CSP structure is classified as semi-rigid, whereby a cleft is formed by the perpendicular arrangement of the dinitrobenzamide group to the phenanthryl moiety. The amide nitrogen rests at the base of this cleft [26]. The structure of the Whelk-O 1 CSP is ideal for compounds possessing an aromatic group and a hydrogen bond acceptor situated adjacent to it [27]. Using the aforementioned three-point

Fig. 2. Structure of evacetrapib.

interaction model idea, a ␲–␲ stack is formed between the electron deficient ␲-acidic dinitrobenzamide ligand of the CSP and the aryl moiety of the analyte. The second interaction is believed to occur between the amide nitrogen of the selector and a hydrogen bond acceptor in the analyte. While this ␲–␲ attraction and hydrogen bonding docking arrangement are fairly well understood as a necessary framework for retention and enantiorecognition with the Whelk-O 1, the third point of interaction that occurs is less understood [23]. Given the immobilizing constraints imposed upon both enantiomers when docked within the selector cleft, enantioselectivity is believed to be tied closely to this final point of interaction in the three-point model, which must necessarily be more sterically stable for one enantiomer. Consequently, the less stable diastereomeric complex terminates earlier, leading to its quicker elution [23,25]. A cursory literature review of the structures of the chiral analytes separated by the Whelk-O 1 column supports the above theory of enantiorecognition [19,21,23,26,28]. Compounds successfully separated possess an aromatic moiety along with a hydrogen bond accepting group nearby. Considering the very limited understanding of chiral separation theory across carbohydrate CSPs, this admittedly crude but substantiated understanding of the Whelk-O 1 chiral recognition mechanism is refreshing. At this point, we introduce a chiral molecule more structurally complex than most reported as enantiomerically discernable by the Whelk-O 1 CSP in the literature. The compound, known as evacetrapib, inhibits cholesterylester transfer protein (CETP) and has been shown to modify lipoprotein levels, an important factor in managing cardiovascular disease [29,30]. The molecule is relatively large, with a molecular weight of 639, and is quite bulky. It possesses three aromatic substituents, including a trifluoromethyl substituted phenyl moiety and a tetrazole linked via an aliphatic nitrogen to a benzazepin group. The molecule additionally contains multiple electronegative atoms and a carboxyl group (Fig. 2). In brief, evacetrapib is loaded with substituents that could potentially interact with the Whelk-O 1 CSP in an enantioselective manner. To complicate matters, evacetrapib contains not only a chiral center (the ring carbon in the benzazepin moiety attached to the aliphatic tertiary nitrogen), but cis–trans isomerism in the cyclohexyl ring. The initial task before us was to define HPLC chiral separation conditions for the resolution of evacetrapib (the trans-S isomer), from its trans-R enantiomer and the cis-S and cis-R diastereomers. Additionally, an ideal isomeric separation would elute evacetrapib last, allowing for maximal minor isomer sensitivity in the presence of a predominant amount of this primary analyte.

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2. Materials and methods While various instrumentation platforms and an array of columns and solvents were employed for the chiral separation screens of evacetrapib, a detailed listing of these constituents is not provided here since the focus of this report is upon method development using primarily the Whelk-O 1 column. Selectivity optimization with the (S,S) Whelk-O 1 column was completed using an Agilent (Santa Clara, CA) 1100 series HPLC equipped with an auto-sampler and photodiode array detector controlled by Empower® software (Waters, Milford, MA). Robustness experiments were conducted using an Agilent 1260 Infinity series HPLC with analogous components, a standard detector flow cell and Empower® software. Regis Technologies Inc. of Morton Grove, IL, USA provided the 4.6 mm × 250 mm 5 ␮m particle size Whelk-O 1 brush-type CSPs. The amylose-based Chiralpak® 4.6 mm × 150 mm 5 ␮m particle size CSPs were purchased from Chiral Technologies Inc., West Chester, PA, USA. The macrocyclic antibiotic ChirobioticTM columns of the same dimensions were obtained from Supelco Analytical, Bellefonte, PA, USA. HPLC grade solvents (purity of 99% or greater unless indicated otherwise) were employed for separation development. Acetonitrile (ACN) and methanol (MeOH) were obtained from EMD Millipore Corporation, Billerica, MA, USA. Ethanol (EtOH) (200 proof) was purchased from Decon Labs, Inc., King of Prussia, PA, USA. Isopropanol (IPA), dichloromethane (DCM), n-butanol (NBA) and n-hexane (95%) were purchased from Fisher Scientific, Inc., Fair Lawn, NJ, USA. n-Propanol was acquired from Macron Chemicals of Charlotte, NC, USA. The basic eluent additive triethylamine (TEA) was purchased from EMD Millipore Corporation, while the acidic eluent additives trifluoroacetic acid (TFA) and glacial acetic acid (HOAc) were acquired from Sigma–Aldrich Corporation, St. Louis, MO, USA. Ammonium acetate (NH4 OAc) and ammonium trifluoroacetate (ATFA) were also purchased from Sigma–Aldrich.

3. Results and discussion 3.1. Separation development As mentioned previously, most in the field of enantioseparations rely upon automated screening systems to obtain direction toward the identification of the ideal chiral separation. We likewise followed this approach, employing over 100 column-eluent combinations across the normal phase, reversed phase, polar organic and extended solvent chromatographic modes. The chiral separation screens revealed multiple polysaccharide CSPs that partially separated as many as three of the isomers. Greatest promise was observed with the normal phase chromatographic mode employing hexane as the primary eluent constituent with either EtOH or IPA as modifier. The additive TFA was included in all screens, which is frequently a requirement for use with analytes containing a carboxyl moiety. While selected carbohydrate columns, namely the Chiralpak® AD-H, Chiralpak® IA and Chiralpak® IC, provided limited isomer selectivity, subsequent optimization studies revealed that analyte retention was a significant issue with these normal phase systems. In short, all polysaccharide normal phase scenarios investigated required an eluent system with a fairly small polar solvent component (less than 3% in hexane) to affect the four-isomer separation within an acceptable analysis time. To complicate matters, the better isomer resolutions were achieved when using a mixture of two alcohols (usually MeOH and EtOH) to constitute the minimally present modifier component. An isomer separation displaying such sensitivity toward modifier content, to say the least, is not ideal for constituting a rugged and transferable

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enantioseparation method. Concerning other chromatographic modes, all polysaccharide CSPs, in addition to the macrocyclic antibiotic columns assessed (ChirobioticTM V2, ChirobioticTM T, ChirobioticTM TAG), proved largely unsuccessful in our reversedphase screens. Evacetrapib did not retain with any column tested when employing TFA-modified ACN or EtOH/MeOH eluent combinations in the polar organic chromatographic mode. The Chiralpak® IA, one of the columns that demonstrated some potential for separating evacetrapib isomers when using traditional hexane/alcohol eluents, showed some promise when we employed an extended solvent eluent composed of hexane, DCM and MeOH. Beyond the many CSPs assessed across multiple eluent modes, one column, the Whelk-O 1, distanced itself in subsequent isomer separation optimization. Compared to the carbohydrate columns, where the evacetrapib isomers typically eluted at between 4 and 6 min, the analytes retained beneficially longer with the Whelk-O 1 CSP. Isomer retention times ranged from 11 to 14 min when using EtOH in the gradient screen, and 12–16 min with IPA. While some of this increased retention can be explained due to the need to use a Whelk-O 1 column of 250 mm length (150 mm columns are not currently available), this fact alone does not fully account for the two to threefold increase in retention with the brush column. From screening results, this column frankly presented as the best hope toward achieving our separation goals. The task would be challenging, encompassing not only the development of a four-isomer separation, but also a system that demonstrates selectivity between the isomer group and six potential impurities of similar structure to the isomers. Additionally, the method would need to pass rigorous characterization and ruggedness challenges to prove acceptable for use in support of the late-stage pharmaceutical development of evacetrapib. Following a general approach toward enantioseparation method development in the normal phase mode, initial isocratic optimization experiments focused upon identifying the amount of alcohol modifier that would be necessary to appropriately elute the isomers in the desired retention window (6–20 min). This was determined to be roughly 10%, with EtOH proving more promising than IPA concerning isomer resolution. Since it was determined that evacetrapib eluted second, and its enantiomer fourth with the (R,R) version of the Whelk-O 1, development was continued with the (S,S) version of the CSP, which retains evacetrapib the longest. Since typical analysis samples would be composed of a large concentration of evacetrapib in the presence of much smaller amounts of the other isomers, having the major isomer elute last can improve minor isomer sensitivity If the minor isomers retained longer than evacetrapib, chances are that accurate isomer integration would be hampered due to potential tailing of the large evacetrapib peak. The capability to invert isomer elution order is a valuable asset when using CSPs available in opposing chiral forms [9,31]. The simplistic 10% EtOH in hexane isocratic eluent (with 0.1% TFA) well resolved the cis-R diastereomer (peak 1) from evacetrapib (peak 4), but the cis-S diastereomer and enantiomer (peaks 2 and 3, respectively) were not separated from each other. Using the additive HOAc in place of TFA provided similar results. Additional experiments revealed that employing a mixture of MeOH/EtOH in the mobile phase with hexane provided superior isomer resolution as compared to using single alcohol modifiers, as well as using other alcohols or dichloromethane mixed with equal parts of MeOH (Fig. 3) [32]. While the mixed-alcohol and acid-modified system effectively separated the four isomers, greater isomer resolution was still desired (for method ruggedness) and the observed peak fronting was concerning. Adding a basic additive (0.1% TEA) to the acidic hexane/MeOH/EtOH/TFA system shifted retention time but did not significantly affect the selectivity of all four isomer peaks. Removing the acidic additive and adding a small concentration of ammonium acetate (NH4 OAc) to the alcohol mixture, however,

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4 0.55

Column: (S,S) Whelk-O 1 (4.6 x 250 mm) Flow: 1.0 mL/min. Wavelength: 260 nm

Eluent:

0.50 0.45

90% Hexane with 0.1% TFA / 5% MeOH / 5% NBA

0.40 0.35

90% Hexane with 0.1% TFA / 5% MeOH / 5% NPA

AU

0.30 0.25 0.20

90% Hexane with 0.1% TFA / 5% MeOH / 5% IPA 0.15 0.10

Eluon Order: 1 - cis-R diastereomer 2 - cis-S diastereomer 3 - enanomer 4 - evacetrapib

0.05

90% Hexane with 0.1% TFA / 5% MeOH / 5% EtOH 0.00 0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

18.00

16.00

20.00

Minutes Fig. 3. Effect of mixed alcohol mobile phase modifiers upon the separation of evacetrapib from its isomers.

greatly increased isomer selectivity [21]. In addition, the introduction of the salt allowed for a return to the original 10% EtOH single alcohol modifier eluent. Increasing the column temperature to 40 ◦ C provided the peak sharpness lost by the removal of MeOH from the mobile phase (Fig. 4) [22]. With the optimized resolution and retention for quantitation of evacetrapib and the three isomers in view, this separation system would likely provide for an acceptable analytical enantioseparation analysis method. For many in the field of enantioseparation screening and optimization, the task is complete at this juncture. Likewise, many a publication would halt at this point with a claim of success for the resolution of evacetrapib and its isomers using the Whelk-O 1 CSP. For us, however, the chiral separation represents only a portion of the task at hand. At this point, six impurities and degradation products were added to the mix. Would the

above tailored CSP and eluent combination resolve these additional molecules? Using the conditions outlined in Fig. 4, samples of the six compounds were injected. While none interfered with the late-eluting evacetrapib, the evacetrapib penultimate and an adduct impurity co-eluted with the enantiomer. Additionally, a different impurity interfered with each of the respective cis diastereomers. Clearly, separation modification would be necessary. Potential improvements in the resolution of isomers and impurities were explored in experiments that utilized TFA, HOAc and NH4 OAc as additives to a hexane/EtOH mobile phase. The separations obtained using the acids by themselves or in combination with NH4 OAc resulted in poorer selectivity than achieved using NH4 OAc by itself. With the benefits of including NH4 OAc in the eluent established, optimization continued with an assessment of the effect of various

0.050

Column: (S,S) Whelk-O 1 (4.6 x 250 mm) Eluent: 5 mM NH4OAc in (90% Hexane / 10% EtOH) Flow: 1.0 mL/min. Wavelength: 260 nm Column Temperature 40°C

0.045 0.040 0.035

AU

0.030 0.025 0.020 0.015

Eluon Order: 1 - cis-R diastereomer 2 - cis-S diastereomer 3 - enanomer 4 - evacetrapib

0.010 0.005 0.000 -0.005 0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes Fig. 4. Effect of column temperature and NH4 OAc upon the separation of evacetrapib from its isomers.

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additional additives and alcohol modifier combinations upon the chromatography, all conducted while maintaining a constant 5 mM concentration of the buffer salt in the mobile phase. Initial experiments focused upon the effects of including a proportion of methanol (MeOH) in the mobile phase in combination with EtOH while keeping the total alcohol modifier constant at 10%. Both 3:1 and 1:3 ratios of MeOH:EtOH were tested. With a predominance of MeOH present as modifier, resolution of the four isomers was negatively affected. Isomeric resolution was maintained, however, when EtOH was the dominant modifier, with the added MeOH providing resolution of one impurity that previously interfered with the isomer group when EtOH was the lone alcohol in the eluent. It was also observed that the reintroduction of 0.1% TFA to the hexane component of the mobile phase (90%) appeared to stabilize the chromatography baseline with only a minimal reduction in analyte retention. Co-elution of many of the potential impurities with the isomers of evacetrapib remained an issue, however (Fig. 5). Further experimentation with TEA as an additive in the eluent cocktail provided enhanced resolution between the cis-S diastereomer and enantiomer, a key selectivity advantage that could not be ignored. Despite the improvements obtained to this point, selectivity was still lacking between the resolved isomeric group and the evacetrapib penultimate, an adduct and other impurities. A simple reduction in the overall alcohol content in the eluent from 10% to 7%, along with further modification of the EtOH to MeOH ratio, provided the desired results. Despite the successful resolution of all components, the complexity of this final eluent system (two alcohol modifiers, two additives and a salt in hexane) is admittedly not ideal. Realizing the ruggedness demands to be imposed upon this separation method, it behooved us to attempt to simplify the multi-component mobile phase. Various attempts were made. Chief of these was the substitution of ATFA in place of the current combination of NH4 OAc and TFA. Unfortunately, the addition of this salt, with the corresponding removal of the NH4 OAc/TFA additives, or of either constituent alone, significantly reduced compound retention and negatively affected resolution between the isomers. The removal of TEA from the original NH4 OAc/TFA eluent system resulted in a similar fate. After multiple tests, it was determined that all ingredients within the mobile phase were indeed necessary to meet the selectivity demands of the method. Later separation optimization was conducted employing a secondary UV absorbance maximum of 267 nm, as significant baseline interference from TEA was observed in the absorbance region below 230 nm, where evacetrapib and its structurally related impurities absorb most strongly. The final optimized chromatographic separation of evacetrapib from its enantiomer, and for the resolution of both components from their diastereomers and related impurities, are shown in Fig. 6. 3.2. Method characterization To finalize the development of the method, and to ensure that it would successfully pass ICH method validation in the Quality Control (QC) Laboratory, characterization experiments were completed to assess several key validation parameters [33]. With the significant hurdle of selectivity overcome, questions remained as to if this separation system would prove adequate with regard to sensitivity, linearity and robustness. Ideally, the method should be capable of accurately measuring levels of the undesired enantiomer (and potentially the other stereoisomers) down to, or below, the ICH qualification threshold of 0.15%. Generally, the sample concentration is maximized to provide an enantiomer peak as large as possible to provide maximal signal-to-noise. To enable accurate quantification of the enantiomer by an area percent approach, linearity at both the high and low concentrations must be demonstrated. Presuming the compound becomes a commercial product,

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Table 1 Evacetrapid linearity analysis. Sample concentration (mg/mL)

Percent of nominal (5.0 mg/mL)

High range 1.004 2.007 3.011 4.014 5.018 6.021

20.1 40.1 60.2 80.3 100.4 120.4

Sample Concentration (␮g/mL)

Percent of Nominal (5.0 mg/mL)

Low range 2.5 5.0 10.0 16.1 20.1 24.1 30.1

0.05 0.10 0.20 0.32 0.40 0.48 0.60

Peak area response (␮V s) 6,782,182 16,332,782 23,371,933 31,202,663 41,996,987 45,335,333 Peak Area Response (␮V s) 20,367 41,400 87,790 110,939 146,930 169,308 249,584

the method will be used in the QC laboratory throughout the product lifecycle, so there must be a degree of confidence that the method is robust enough to still perform appropriately when parameters vary slightly from instrument to instrument and analyst to analyst. The demands are high for an analytical isomeric purity method designed to support a chiral molecule in late phase clinical development, with an eye to commercialization. 3.2.1. Linearity Linearity of concentration versus absorbance was assessed over two ranges. The high level linearity ranged in concentration from 1.0 to 6.0 mg/mL (20–120% of a nominal 5.0 mg/mL anticipated evacetrapib sample concentration). The low-level linearity assessment covered the range of 2.5–30 ␮g/mL (0.05–0.60% of nominal). In all, 13 samples across the 2 concentration ranges (6 low and 7 high) were injected using the sample concentrations outlined in Table 1. Peak area values obtained in each range of study were fit by linear least squares regression to sample concentrations. The ratio of slopes (high range/low range) was 1.03. We concluded that the method provides the desired linearity for direct peak area percent measurement of a 5.0 mg/mL evacetrapib sample along with minor isomers present in concentrations as low as 0.05% of nominal. 3.2.2. Sensitivity For this method it was desired to report results down to 0.05%, the ICH limit of disregard. To support this limit of quantitation, a signal to noise ratio of 10 or greater must be demonstrated [33]. Sensitivity of the method was assessed using an evacetrapib sample preparation spiked at a level corresponding to 0.05% of nominal (5.0 mg/mL) with the enantiomer and each diastereomer. Signal-tonoise values (S/N) were calculated for the minor isomers using the European Pharmacopeia method where peak height is measured from the maximum of the peak to the extrapolated baseline and noise is calculated as half of the peak-to-peak baseline noise. The spiked sample chromatogram and S/N values ranged from 67 to 87. The method exhibits more than the required sensitivity for our needs. 3.2.3. Robustness As a final test of the separation, we evaluated the impact of small, deliberate variations in key method and instrumental parameters using a statistical screening experimental design. Seven factors (column temperature, detection wavelength, injection volume, total alcohol modifier content, EtOH/MeOH ratio, NH4 OAc concentration, and TFA/TEA ratio) were evaluated for significant effects

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6 0.60

Column: (S,S) Whelk-O 1 (4.6 x 250 mm) Eluent: 90% (0.1% TFA in Hexane) / 10% (50 mM NH4OAc in 3:1 EtOH:MeOH) Flow: 1.0 mL/min. Wavelength: 260 nm Column Temperature 40°C

0.55 0.50 0.45 0.40

AU

0.35 0.30

Impuries

0.25 0.20 0.15

Penulmate

0.10 1 - cis-R diastereomer

Eluon Order: 2 - cis-S diastereomer

0.05

Evacetrapib with isomers

3 - enanomer 4 - evacetrapib

0.00 0.00

2.00

6.00

4.00

Minutes

10.00

8.00

12.00

14.00

Fig. 5. Separation of evacetrapib from its isomers and selected impurities: incorporation of a mixed-alcohol modifier.

2.80

Column: (S,S) Whelk-O 1 (4.6 x 250 mm) Eluent: 93% (0.1% TFA and 0.1% TEA in Hexane) / 7% (50 mM NH4OAc in 2:1 EtOH:MeOH) Flow: 1.0 mL/min. Wavelength: 267 nm Column Temperature 40°C

2.60 2.40 2.20 2.00 1.80

AU

1.60

Impuries

1.40 1.20 1.00

Penulmate

0.80

evacetrapib

0.60

Enanomer

0.40

Cis-S Diastereomer

0.20

Cis-R Diastereomer

0.00 0.00

2.00

4.00

6.00

8.00

10.00

12.00

Minutes

14.00

16.00

18.00

20.00

22.00

Fig. 6. Separation of evacetrapib from its isomers and selected impurities: optimized conditions.

Table 2 Method robustness parameters. Run number

Column temperature (◦ C)

Wavelength (nm)

Injection volume (␮L)

Percent alcohol modifier

EtOH/MeOH ratio

NH4 OAc concentration (mM)

TFA/TEA ratio

1 2 3 4 5 6 7 8 9 10 11

40 37 43 43 37 40 43 37 37 43 40

267 270 264 264 270 267 270 264 264 270 267

10 12 8 12 8 10 8 12 8 12 10

7 6 8 6 8 7 6 8 6 8 7

2.0 2.2 2.2 1.8 1.8 2.0 1.8 1.8 2.2 2.2 2.0

50 45 45 55 55 50 45 45 55 55 50

0.10 0.08 0.08 0.08 0.08 0.10 0.12 0.12 0.12 0.12 0.10

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7

Fig. 7. Variability chart demonstrating the impact of method parameters on the resolution of the S-cis diastereomer from the enantiomer.

on the chromatography (Table 2). Particular attention was given to the effects of changes in the seven experimental parameters upon resolution between evacetrapib and its enantiomer (≥1.5), evacetrapib tailing (≤2.0) and the signal to noise ratio of the enantiomer (≥20). While separation quality was certainly affected by varying method conditions, the changes explored did not result in any trials missing these performance limits. The separation between the enantiomer and its nearest neighboring impurity was informative toward understanding which mobile phase components were most critical to achieving the necessary specificity for quantitation of the enantiomer. All of the method parameters were found to have statistically significant effects on the resolution of said impurity from the enantiomer, although the magnitude of the effect was small for most (see Fig. 7). The largest effect was observed for the TFA/TEA ratio in mobile phase A. A decrease in resolution of 0.75 was observed when the TFA/TEA ratio was changed from 0.08/0.12 to 0.12/0.08. The lowest resolution (1.4) was observed for run number 10, which had most of the influential parameters at their worst settings, but the enantiomer peak could still be accurately integrated such that there was no impact on the reported results. 4. Conclusions The HPLC separation of evacetrapib from its enantiomer, two diastereomers and multiple impurities was achieved using normal phase HPLC and the (S,S) Whelk-O 1 chiral column. Beginning with an extensive multi-modal chiral separation screening process employing several CSP and eluent combinations, systematic optimization trials led to the identification of this brush-type column as the superior selector for the complex separation. Utilizing a tailored eluent system consisting of hexane, a mixed-alcohol modifier, a buffer salt and acidic and basic additives, an impressive separation of 10 compounds was achieved within a 20 min analysis time. Characterization tests verified that the method possessed the required sensitivity and response linearity. Additionally, the separation withstood the challenges of a robustness study which assessed method response to various changes in instrument and eluent parameters.

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