The Chiral Notebook Multiple Solutions for Chiral Applications

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• Chiral Resources © 2014 Phenomenex, Inc. All rights reserved. 2 Phenomenex l WEB: www.phenomenex.com Table of Contents Method Development Strategies Alternative Selectivity of Lux® Cellulose-1 and Cellulose-2 [TN-1047]...... 4 Method Development for RP LC/MS/MS on all 5 Lux Phases [TN-1079]...... 12 Lux Cellulose-1 Versus Immobilized CHIRALPAK® IB® [TN-1128]...... 20 Chiral Separation of FMOC Amino Acids by RP Mode [TN-1148]...... 26 Novel Screening Approach for the Separation of Pharmaceutical Compounds in SFC Mode [TN-9003]...... 30

Purification Techniques Purification of Chiral APIs using Axial Compressed Columns [TN-1056]...... 34 SFC and HPLC Chiral Purification on Lux Axia™ [TN-9001]...... 39 Axia Technology vs. Standard Hardware by HPLC and SFC [TN-9002]...... 43

Pharmaceutical Drugs Applications Separation of Generic PPIs in RP Mode [TN-1102]...... 51 Beta Blockers in NP, RP, and PO Modes [TN-1142]...... 54 Anti-Allergic Agents in NP, RP, and PO Modes [TN-1143]...... 58 Pain Relievers in NP, RP, and PO Modes [TN-1144]...... 62 Vasodilator Drugs in NP, RP, and PO Modes [TN-1145]...... 66 Anti-Anxiety and Antidepressive Drugs in NP, RP, and PO Modes [TN-1146]...... 70 Antifungal Drugs in NP, RP, and PO Modes [TN-1147]...... 74

Clinical Drugs Applications Synthetic Cannabinoids Metabolites [TN-1167]...... 78

Pesticide Applications Enantioseparation of Racemic Herbicides [TN-1162]...... 82 Enantiomeric and Diastereoisomeric Resolutions of Chiral Triazole Fungicides [TN-1164]...... 86

Preparative Columns Axia: Award-Winning Column Packing Technology...... 92

Guard Columns SecurityGuard™ Extends Column Lifetime...... 94

Free Chiral Resources Easily Search Over 2,000 Chiral Applications, Method Development Posters, and More...... 95

Ordering Information...... 96

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Alternative Selectivity of Chiral Stationary Phases Based on Cellulose tris(3-chloro-4-methylphenylcarbamate) and Cellulose tris(3,5-dimethylphenylcarbamate Liming Peng, Tivadar Farkas and Swapna Jayapalan Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Introduction HPLC Conditions Polysaccharide-based chiral stationary phases (CSP) are widely Instrumentation used due to their wide chiral recognition ability. Several cellulose Agilent 1100 series (www.agilent.com) and amylose derivatives are extremely effective in the separation HPLC System: of a wide range of compounds of interest in the pharmaceutical Pump: G1311A Quaternary Pump industry1. This work demonstrates the different chiral recognition Autosampler: G1313A ALS capabilities of CSPs based on cellulose tris(3-chloro-4-methyl- Detector: G1315A Diode Array Detector phenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate). Over 180 racemates of pharmaceutical interest were an- HPLC Conditions alyzed on these two phases in normal (NP), polar-organic (PO) Flow Rate: 1.0 mL/min and reversed phase (RP) separation modes. Numerous examples Injection Volume: 5 - 20 μL (depending on analyte response) including important classes of drug compounds as well as statis- Sample tical data prove that cellulose tris(3-chloro-4 methylphenylcarba- 500 μg/mL racemate dissolved in mobile phase mate) offers a good alternative to the commonly used cellulose Concentration: tris(3,5-dimethylphenylcarbamate) in the separation of difficult Columns: Lux® 5 μm Cellulose-1; 250 x 4.6 mm Lux 5 μm Cellulose-2; 250 x 4.6 mm racemic mixtures. CHIRALCEL® 5 μm OD-H®; 250 x 4.6 mm Temperature: Ambient Figure 1. Detector: UV @ 220 nm Structures of Chiral Selective Phases

® Lux Cellulose-1 Table 1. Cellulose tris(3,5-dimethylphenylcarbamate) Mobile Phase Compositions

Mobile Phase NP PO Basic and 0.1 % DEA 0.1 % DEA 0.1 % DEA

Neutral in Hexane:IPA in MeOH:IPA in CH3CN:IPA Compounds Acidic and 0.1 % HAC 0.1 % HAC 0.1 % HAC Neutral (or FA) in (or FA) in (or FA) in

Compounds Hexane:IPA MeOH:IPA CH3CN:IPA IPA: Iso-propanol; DEA: Diethylamine; HAC: Acetic Acid; FA: Formic acid; MeOH: Metha-

nol; CH3CN: Acetonitrile

Lux® Cellulose-2 Cellulose tris(3-chloro-4-methyl-dimethylphenylcarbamate)

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Figure 2. Oxprenolol on Lux Cellulose-1 Enantioseparations of ß-Blockers in Normal Phase 0.1 % DEA in Hexane / 0.1 % DEA in IPA (90:10) mAU Toliprolol on Lux Cellulose-1 0.1 % DEA in Hexane / 0.1 % DEA in IPA (80:20) 400 mAU

400 200 App ID 17481 200 0 0 4 8 min

App ID 17478 Oxprenolol on Lux Cellulose-2 0 0.1 % DEA in Hexane / 0.1 % DEA in IPA (90:10) 0 10 20 min mAU

400 Toliprolol on Lux Cellulose-2 0.1 % DEA in Hexane / 0.1 % DEA in IPA (80:20) 200 mAU

400 App ID 17482 0 0 4 8 min 200 Column: Lux 5 µm Cellulose-1 Lux 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm ID

App ID 17479 Part No.: 00G-4459-E0 N 0 O NH 00G-4457-E0 H 0 10 20 min Flow Rate: 1 mL/min OH Detection: UV @ 220 nm Temperature: Ambient Oxprenolol Toliprolol on Lux Cellulose-2 0.1 % DEA in Hexane / 0.1 % DEA in IPA (90:10) mAU Bopindolol on Lux Cellulose-1

400 0.1 % DEA in Hexane / 0.1 % DEA in IPA (90:10) mAU

800 200

400

0 App ID 17480

0 10 20 min App ID 17483 0 0 4 8 min Column: Lux 5 µm Cellulose-1 Lux 5 µm Cellulose-2 Bopindolol on Lux Cellulose-2 Dimensions: 250 x 4.6 mm ID 0.1 % DEA in Hexane / 0.1 % DEA in IPA (90:10) Part No.: 00G-4459-E0 HN 00G-4457-E0 mAU Flow Rate: 1 mL/min Detection: UV @ 220 nm O 800 Temperature: Ambient

OH 400 Toliprolol App ID 17484 0 0 4 8 min

Column: Lux 5 µm Cellulose-1 Lux 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm ID HN Part No.: 00G-4459-E0 00G-4457-E0 O NH O Flow Rate: 1 mL/min Detection: UV @ 220 nm Temperature: Ambient O

Bopindolol

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Figure 3. Enantioseparations in Normal Phase

Warfarin on Lux Cellulose-1 Sulconazole on Lux Cellulose-1 0.1 % Formic Acid in Hexane / 0.1 % Formic Acid in IPA (60:40) 0.1 % DEA in Hexane / 0.1 % DEA in IPA (60:40) mAU Rs: 7.41 mAU Rs: 2.37 400 40

200 20 App ID 17324 App ID 17311 0 0

0 4 8 min 0 10 20 min

Warfarin on Lux Cellulose-2 Sulconazole on Lux Cellulose-2 0.1 % Formic Acid in Hexane / 0.1 % Formic Acid in IPA (60:40) 0.1 % DEA in Hexane / 0.1 % DEA in IPA (60:40)

mAU Rs: 3.12 mAU 400 Rs: 5.90 40

200 20 App ID 17312 App ID 17325 0 0

0 4 8 min 0 10 20 min Column: Lux 5 µm Cellulose-1 Lux 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm ID Column: Lux 5 µm Cellulose-1 Part No.: 00G-4459-E0 Lux 5 µm Cellulose-2 00G-4457-E0 Dimensions: 250 x 4.6 mm ID Flow Rate: 1 mL/min Part No.: 00G-4459-E0 Detection: UV @ 220 nm 00G-4457-E0 Temperature: Ambient Flow Rate: 1 mL/min Detection: UV @ 220 nm Temperature: Ambient

Warfarin on CHIRALCEL®† OD-H® Sulconazole on CHIRALCEL®† OD-H® 0.1 % Formic Acid in Hexane / 0.1 % Formic Acid in IPA (60:40) 0.1 % DEA in Hexane / 0.1 % DEA in IPA (60:40)

mAU Rs: 4.40 mAU Rs: 2.19

400 40

200 20 App ID 17313 App ID 17326 0 0

0 4 8 min 0 10 20 min

Cl Cl Column: CHIRALCEL® 5 µm OD-H® Column: CHIRALCEL® 5 µm OD-H® Dimensions: 250 x 4.6 mm ID Dimensions: 250 x 4.6 mm ID Flow Rate: 1 mL/min N Flow Rate: 1 mL/min O N Detection: UV @ 220 nm S Detection: UV @ 220 nm Temperature: Ambient Temperature: Ambient O O

Warfarin Cl Sulconazole

*with 0.1 % Formic Acid †CHIRALCEL and OD-H are registered trademarks of DAICEL Chemical Industries, Ltd. Comparative separations may not be representative of all applications.

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Figure 4. Complementary Enantioselectivity in Normal Phase and Polar-Organic

Sulconazole on Lux Cellulose-2 Milnacipran on Lux Cellulose-2 0.1 % DEA in Hexane / 0.1 % DEA in IPA (60:40) 0.1 % DEA in Hexane / 0.1 % DEA in IPA (80:20 ) mAU Rs: 5.80 mAU Rs: 1.12 40 1200 1000 800 20 600 400 App ID 17325

200 App ID 17489 0 0

0 10 20 min 0 4 8 12 min

Sulconazole on Lux Cellulose-2 Milnacipran on Lux Cellulose-2 0.1 % DEA in MeOH / 0.1 % DEA in IPA (95:5) 0.1 % DEA in MeOH / 0.1 % DEA in IPA (90:10) mAU mAU Rs: 0.00 1000 Rs: 4.28 600 800

400 600

400 200 200 App ID 17488 App ID 17485 0 0

0 4 8 min 0 4 8 12 min

Sulconazole on Lux Cellulose-2 Milnacipran on Lux Cellulose-2

0.1 % DEA in CH3CN / 0.1 % DEA in IPA (95:5) 0.1 % DEA in CH3CN / 0.1 % DEA in IPA (95:5) mAU Rs: 2.54 mAU No Elution 400

200 200 App ID 17513 App ID 17492 0 0

0 4 8 12 min 0 4 8 12 min

Column: Lux 5 µm Cellulose-2 Cl Cl Column: Lux 5 µm Cellulose-2 H Dimensions: 250 x 4.6 mm ID Dimensions: 250 x 4.6 mm ID Part No.: 00G-4457-E0 Part No.: 00G-4457-E0 N Flow Rate: 1 mL/min Flow Rate: 1 mL/min H N Detection: UV @ 220 nm N Detection: UV @ 220 nm O Temperature: Ambient S Temperature: Ambient N

Cl Milnacipran Sulconazole

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Figure 4. (cont’d) Figure 5. Complementary Enantioselectivity in Normal Phase and Polar-Organic Enantioseparations in Reversed Phase

Chlormezanone on Lux Cellulose-1 Clenbuterol on Lux Cellulose-2

0.1 % DEA in CH3CN / 0.1 % DEA in IPA (60:40) 0.1 % DEA in MeOH / 0.1 % DEA in Water (80:20) 1 mAU Rs: 0.00 Rs: 0.51

1200

800

400 App ID 17728 App ID 17504 0

0 4 8 12 min

0 10 20 min Clenbuterol on Lux Cellulose-2

Chlormezanone on Lux Cellulose-1 0.1 % DEA in CH3CN / 0.1 % DEA in Water (60:40) 0.1 % DEA in CH CN / 0.1 % DEA in IPA (95:5) mAU 3 Rs: 0.97 1 Rs: 0.00 1200

800

400 App ID 17505 0

0 4 8 12 min App ID 17729

0 4 8 min Clenbuterol on Lux Cellulose-2

0.1 % DEA in CH3CN / 0.1 % DEA in Water (40:60) Chlormezanone on Lux Cellulose-1 mAU Rs: 1.46 0.1 % DEA in MeOH / 0.1 % DEA in IPA (90:10) 800 1 Rs: 3.21 600 2 400

200

0 App ID 17506

0 4 8 12 min

App ID 17730 Column: Lux 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm ID Cl Part No.: 00G-4457-E0 NH 0 4 8 min Flow Rate: 1 mL/min

Detection: UV @ 220 nm H2N CI Temperature: Ambient Column: Lux 5 µm Cellulose-1 OH Dimensions: 250 x 4.6 mm ID O O Cl Clenbuterol Part No.: 00G-4459-E0 S Flow Rate: 1 mL/min Detection: UV @ 220 nm Temperature: Ambient N

CH3 O Mobile Phase Rs Chlormezanone Hexane:IPA 0.00 MeOH:IPA 0.00

CH3CN 0.00

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Figure 6. Success Rates for over 180 Racemates on Lux® Cellulose-1 and -2

80 75 73 71 67 68

26 60 55

31 26 31 11

25 26 18 11 25 18

20 11 18 13 9 12 18

9 9 11 6 5 0 Lux Lux Lux Lux Lux Lux Cellulose-1 Cellulose-2 Cellulose-1 Cellulose-2 Cellulose-1 Cellulose-2

(MeOH) (MeOH) (CH3CN) (CH3CN) (NP) (NP)

Total number of separations Partial separation on both Lux Cellulose-1 and Cellulose-2 Baseline separation on both Lux Cellulose-1 and Cellulose-2 Partial separation Baseline separation

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Results and Discussion or polar organic separation modes. The number of uniquely base- Lux, a new line of polysaccharide-based chiral selective phases, line resolved racemates are given at the bottom of the bar graph. has recently been introduced into the market. One phase, Lux® The same selection criteria was applied to partially separated ra- Cellulose-1 is based on cellulose tris (3,5- dimethylphenyl carba- cemates. For example, Lux Cellulose-2 shows good chiral recog- mate) similar to other chiral phases on the market (e.g. CHIRACEL® nition in acetonitrile mobile phase with 9 baseline separations of OD-H®). The other phase, Lux Cellulose-2, is a new member to the racemates that could not be separated on Lux Cellulose-1. This family of polysaccharide based chiral selective phases and uses complementary enantioselectivity of Lux Cellulose-2 over Lux Cel- cellulose tris (3-chloro-4-methylphenyl carbamate) (Figure 1) as lulose-1 is most evident in acetonitrile: IPA mobile phase mixtures, a chiral selector; this new chemistry delivers a unique selectivity and is less pronounced in standard normal phase mixtures (hexane versus other phases 2,3. In this study over 180 diverse compounds / IPA) and methanol mixtures. of pharmaceutical interest were screened on the Lux line of chiral selective phases as well as other comparative medias to better characterize the selectivity delivered by each Lux phase.

Table 1 summarizes the screening conditions used for each column; different types of mobile phases (NP, PO, and RP) as well as additives used (0.1 formic acid or acetic acid for acidic analytes or 0.1 % diethylamine for basic analytes).

Figures 2-5 show several representative examples of the differ- References ent selectivities provided by Lux Cellulose-1 and Lux Cellulose-2 in chiral separations across normal phase, polar-organic and reversed 1. Y. Okamoto, Y. Kaida J. Chromatography A 666 (1994), phase separation modes. Representative compounds such as var- 403-4192. ious ß-blockers, warfarin, sulconazole, milnacipran, and clenbuter- 2. N. Matthijs, M. Maftouh, Y. Vander Heyden J. Chromatography A 1111 ol demonstrate the complementary behavior of Lux Cellulose-2 to (2006), 48-613. the commonly used cellulose tris (3.5-dimethylphenylcarbamate) 3. T. Huybrechts, G. Torrok, T. Vennekens, R. Sneyers, S. Vrielynck, I. Somers based CSPs (CHIRACEL® OD-H® and Lux Cellulose-1) in the sepa- LCGC Europe June 1, 2007 ration of difficult racemates. 4. N. Maier, P. Franco, W. Lindner J. Chromatography A 906 (2001), 3-33 Figure 2 demonstrates the behavior of the two Lux phases in normal phase separations. While Lux Cellulose-1 generally demonstrates slightly better resolution and increased retention versus CHIRACEL® OD-H®, there are several cases where Lux Cellulose-2 is a better separation choice when using normal phase. While Bopindolol is equally well separated on the two Lux phases, Ox- prenolol enantiomers are better resolved on Lux Cellulose-2. Tolip- rolol enantomers are separated on Lux Celluose-1 with spectacular resolution but at the expense of extensive retention for one of the enantomers. Lux Cellulose-2 barely separates racemic Oxprenolol under similar mobile phase conditions, but with minimal optimization (i.e. a reduction of IPA in the mobile phase) a better separation is achieved with Lux Cellulose-2 with significantly shorter analysis time. Figure 3 shows additional normal phase enantomeric separations using Lux Cellulose-1, Lux Cellulose-2 and CHIRACEL® OD-H®. Such separations further demonstrate the complementary selectivity offered by Lux Cellulose-2 versus the cellulose tris (3,5-dimethylphenyl carbamate) phases Lux Cellulose-1 and CHI- RACEL® OD-H®.

Figures 4 and 5 demonstrate the effect of mobile phase composition on chiral resolution. Figure 4 demonstrates the dramatic changes in selectivity for each phase when traditional normal phase separation is substituted with polar organic separation mode using either acetonitrile or methanol as mobile phase. Figure 5 shows changes in selectivity are observed in reversed phase mode. Dif- ferent solvents can alter the steric structure of the polysaccharide backbone and the arrangement of binding sites4, providing alternative selectivity for separating difficult chiral compounds. Such mobile phase alteration offers a powerful tool in developing and Trademarks Lux is a registered trademark of Phenomenex, Inc. CHIRALCEL and OD-H are registered optimizing chiral separations. trademarks of DAICEL Chemical Industries, Ltd. of Japan. Figure 6 compares the success rates of Lux Cellulose-1 and Lux Disclaimer Comparative separations may not be representative of all applications. Subject to Phe- Cellulose-2 in the analysis of over 180 racemates in normal phase nomenex Standard Terms & Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions. © 2008 Phenomenex, Inc. All rights reserved.

10 Phenomenex l WEB: www.phenomenex.com FIVE Complementary Chiral Phases for Greater Selectivity

Cellulose-1

Cellulose-2

Cellulose-3

Cellulose-4

Amylose-2

Resolve 92 % of all your Enantiomers using Lux® Screening Kits FREE Chiral Screening and Separation Services

Phenomenex l WEB: www.phenomenex.com 11 TN-1079 APPLICATIONS Method Development for Reversed Phase Chiral LC/MS/MS Analysis of Stereoisomeric Pharmaceutical Compounds with Polysaccharide-based Stationary Phases Philip J. Koerner, Kari Carlson, Liming Peng, Tivadar Farkas et al. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Five different Lux® polysaccharide-based chiral stationary phases Results and Discussion were explored in the reversed phase elution mode using mobile Chiral LC/MS/MS Experiments Five different polysaccha- phases consisting of 0.1 % formic acid in acetonitrile or methanol ride-based chiral stationary phases (Lux Cellulose-1, Lux Cel- to demonstrate the feasibility of LC/MS/MS analysis of a variety of lulose-2, Lux Cellulose-3, Lux Cellulose-4, and Lux Amylose-2 acidic pharmaceutical racemates. (Figure 1) were explored in the reversed phase elution mode for the separation of a variety of acidic compounds of pharmaceu- Introduction tical interest in mobile phases consisting of 0.1 % formic acid in Developing simple and straightforward reversed phase chiral LC acetonitrile or methanol and MS/MS detection. separations coupled with highly sensitive MS detection is a chal- lenging requirement for conducting drug metabolism and pharma- Figure 1. cokinetic studies of stereoisomers. Polysaccharide derivatives are Structures of Polysaccharide-based Chiral Stationary (CSPs) Phases the most widely used chiral stationary phases (CSP) due to their wide chiral recognition and high loading capacity. As normal phase Lux Cellulose-1 is favorable for their principal mechanism of chiral recognition – hy- Cellulose tris (3,5-dimethylphenylcarbamate) drogen bonding interaction – the majority of chiral separations with CH polysaccharide phases are performed in normal phase using hex- 3 ane and alcohol modifiers as mobile phase components. However, OCONH CH these mobile phases are highly flammable and are not compatible O 3 O CH with atmospheric pressure ionization (API) MS ion sources. The 3 CH3

current research describes the effectiveness of an acidic mobile HNOCO OCONH phase for the separation and detection of using acidic stereoiso- CH CH mers by ESI or APCI LC/MS/MS and for method development of 3 3 these applications. Lux Cellulose-2 Cellulose tris (3-chloro-4-methylphenylcarbamate) Experimental with R1=Cl and R2=CH3 Analytes: 15 acidic compounds of pharmaceutical interest were or analyzed. The structures are shown in Figure 2 and the MS ion- Lux Cellulose-4 ization and mass reaction monitoring (MRM) transitions monitored Cellulose tris (4-chloro-3-methylphenylcarbamate)

are listed in Table 1. with R1=CH3 and R2=Cl.

OCONH R2 Columns: Lux 3 μm Cellulose-1, 150 x 2.0 mm Lux 3 μm Cellulose-2, 150 x 2.0 mm O Lux 3 μm Amylose-2, 150 x 2.0 mm O Rl Rl Lux 5 μm Cellulose-1, 250 x 4.6 mm Lux 5 μm Cellulose-4, 250 x 4.6 mm HNOCO R2 OCONH R2 Lux 5 μm Cellulose-3, 250 x 4.6 mm Lux 5 μm Amylose-2, 250 x 4.6 mm Kinetex® 2.6 μm C18, 50 x 2.1 mm (used for achiral analysis) Flow Rate: 0.2 mL/min (3 μm, 150 x 2.0 mm) or 1.0 mL/min (5 μm, 250 x 4.6 mm) – flow split to 0.25 mL/ Lux Cellulose-3 min into MS/MS Cellulose tris (4-methylbenzoate) Temperature: 25 °C O Detection: UV @ 220 nm C CH O 3 Injection Volume: 5 μL (150 x 2.0 mm) or 10-20 μL (250 x 4.6 mm) Mobile Phases: 1. 0.1 % Formic acid in Acetonitrile or Methanol O 2. 5 mM Ammonium bicarbonate in Acetonitrile or Methanol (achiral analysis) O 3. 5 mM Ammonium formate in Acetonitrile or Methanol (achiral analysis) O O

4. 5 mM Ammonium acetate in Acetonitrile or Methanol (achiral analysis) C CH3 CH3 C Instrument: HPLC System: Agilent® 1200 series equipped with binary pumpand autosampler O O (Agilent, Palo Alto, CA)MS Detector: AB SCIEX™ 4000 LC/MS/MS Turbo V™ source with ESI or APCI probe MS Detection: TurbolonSpray® – ESI or APCI in Positive or NegativeIon Mode; MRM Lux Amylose-2 Amylose tris (5-chloro-2-methylphenylcarbamate) Cl

OCONH O Cl CH3 Cl O HNOCO OCONH CH3

CH3 Cl

12 OCONH Phenomenex l WEB: www.phenomenex.com MeCH3 O O Cl Cl

Me HNOCO OCONH MeCH3 TN-1079 APPLICATIONS

Selection of Mobile Phase Additives Table 1. As acidic analytes are present as anions in mobile phases of neu- MRM Transitions and Concentrations of Acidic Racemates tral pH their retention is not favorable on polysaccharide-based CSPs. Early elution and poor or no enantioseparation can result Compound IS and MRM Conc.* Compounds IS and MRM Conc.* under these conditions. Acidic mobile phase additives are often Ibuprofen ESI-205.1/160.1 100 Abscisic acid APCI-263.0/152.7 50 required to suppress the dissociation of acidic analytes, resulting in increased retention and improved enantioselectivity. Flurbiprofen ESI-243.0/198.7 50 Mecoprop ESI-214.1/141.7 50

Suprofen ESI+261.1/111.0 50 Ketorolac ESI-254.0/209.8 50 Three volatile organic acids, (trifluoroacetic acid (TFA, pKa 0.59),

ESI+256.2/105.0 formic acid (FA, pKa 3.75), and acetic acid (HAc, pKa 4.76) were evaluated on Lux® Cellulose-1 and Lux Amylose-2 CSPs as acidic Fenoprofen APCI-241.0/196.8 100 Etodolac ESI-286.1/242.0 50 additives. In general, these additives provide similar enantiore- Carprofen APCI-272.8/228.8 100 Warfarin ESI-307.1/160.9 50 solution for weakly acidic racemates (Figures 3A-3C) while the ESI+309.2/163.1 stronger acidic additive (TFA) performs better for the stronger Indoprofen ESI+282.1/236.1 50 Bendroflumethiaze ESI-420.1/77.9 50 acidic racemates (Figures 3D–3F). Formic acid provides comparable enantioseparations and peak shapes to TFA. Considering Proglumide ESI-333.1/120.9 50 Trichlormethiazide ESI-377.8/241.6 50 the “memory effect” of TFA commonly experienced on polysac- 1-(Phenylsulfonyl)- ESI-300.0/209.8 100 charide-based CSPs and its ion suppressing tendency in MS de- 3-indoleboronic tection, formic acid was selected in preference over TFA in re- acid * Conc. (ng/mL) versed phase mobile phase for the chiral separation and MS/MS detection of acidic racemates.

Figure 2. Effect of Mobile Phase Additives Molecular Structures of Acidic Racemates The LC/MS/MS responses of acidic racemates with ESI negative (ESI-) mode in 5 mM ammonium formate, 5 mM ammonium ace-

OH OH OH Cl tate, and 5 mM ammonium bicarbonate containing mobile phases O with either acetonitrile or methanol as organic modifier using an O O S O ® F achiral column (Kinetex 2.6 µm C18) were compared to responses N H

O N in 0.1 % formic acid (Figures 4 and 5). The results show that MS/ O

O O O MS responses using 0.1 % formic acid in acetonitrile or methanol

OH OH HO are comparable to the responses using the other acid additives in Ibuprofen Flurbiprofen Suprofen Fenoprofen Carprofen Indoprofen ESI- mode, with the exception of carprofen which showed poor response with all of the acidic mobile phase additives. This shows that using 0.1 % formic acid as acidic mobile phase additive is fully OH HO O OH OH compatible with MS/MS detection and can be implemented as the O O N O B O OH first choice for mobile phase additive. O O S N HO O N NH O O Effect of Organic Modifier on Chiral Resolution

OH Acetonitrile or methanol was the organic modifier used in chiral re- O Cl versed phase HPLC. Decreasing the eluting strength of the mobile Proglumide Abscisic Acid Mecoprop Ketorolac 1-(Phenylsulfonyl)-3- indoleboronic acid phase by decreasing the percentage of acetonitrile or methanol in the mobile phase will increase retention and resolution (Figure 6). OHOOO However, once enantiomers elute later than about 10 minutes with CH3 Cl H H C H H 3 OH O N Cl only partial resolution, baseline separation can be rarely achieved N Cl N CF3 O O O O O O N by further decreasing the % organic modifier in the mobile phase. N H S S H S S In our study, acetonitrile was more successful than methanol in HO NH O NH2 O O O 2 O O Etodolac Warfarin Bendroflumethiaze Trichlormethiazide providing chiral resolution on Lux CSPs in reversed phase (RP) mode (Figure 8), typically yielding sharper and narrower peaks.

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Figure 3. Effect of Acidic Additives on the Enantioseparation of Acidic Racemates in Reversed Phase Bendroflumethiaze Flurbiprofen Lux® 5 µm Cellulose-1 Lux 5 µm Amylose-2 Part Number: 00G-4458-EO Part Number: 00G-4471-EO

A. D. Acetonitrile / Acetic acid (40:60) Acetonitrile / 0.1 % Acetic acid (60:40)

Column: As noted Dimensions: 250 x 4.6 mm Flow Rate: 1 mL/min Temperature: 25 °C B. E. Detection: UV @ 220 nm Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Injection Volume: 10 µL acid (40:60) Formic acid (60:40) Mobile Phase: As noted

C. F. Acetonitrile / 0.1 % Trifluoroacetic Acetonitrile / 0.1 % acid (TFA) (40:60) Trifluoroacetic acid (60:40) App ID 19327 App ID 19326

Figure 4. LC/MS/MS Responses of Acidic Racemates in ESI- with Different Additives and Methanol

1500000

Enlargement

1250000 250000

200000 1000000

150000 0.1 % Formic acid / Methanol 750000 100000 5 mM Ammonium formate / Methanol 5 mM Ammonium bicarbonate / Methanol 50000 5 mM Ammonium acetate / Methanol 500000

250000 FenoprofenFlurbiprofenProglumide KetorolacMecoprop Bendroflumethiaze Carprofen 1-(PS)-3-indoleboronic

WarfarinEtodolacIbuprofen FenoprofenFlurbiprofen Proglumide KetorolacMecopropCarprofen Bendroflumethiaze 1-(PS)-3-indoleboronic

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Figure 5. LC/MS/MS Responses of Acidic Racemates in ESI- with Different Additives and Methanol

1250000

1000000 5 mM Ammonium bicarbonate / Acetonitrile 5 mM Ammonium formate / Methanol 750000 0.1 % Formic acid / Acetonitrile 5 mM Ammonium acetate / Acetonitrile

500000 Ibuprofen Proglumide Ketorolac MecopropCarprofen Bendroflumethiaze 1-(PS)-3-indoleboronic

250000

WarfarinEtodolac Fenoprofen Ibuprofen Flurbiprofen Proglumide c KetorolacMecopropCarprofen Bendroflumethiaze 1-(PS)-3-indoleboroni Figure 6. Effect of Acidic Additives on the Enantioseparation of Acidic Racemates in Reversed Phase Proglumide Ibuprofen Lux® 3 µm Cellulose-1 Lux 5 µm Cellulose-3 Flow Rate: 0.2 mL/min Flow Rate: 1 mL/min Dimensions: 150 x 2.0 mm Dimensions: 250 x 4.6 mm

Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Formic acid (60:40) acid (60:40)

Column: As noted Dimensions: As noted Flow Rate: As noted 5 mM Ammonium bicarbonate / Methanol Temperature: 25 °C

Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Formic Detection: Mass Spectrometer (MS) acid (40:60) acid (50:50) Injection Volume: 5-20 µL Mobile Phase: As noted Sample: As noted

Acetonitrile / 0.1 % Formic acid (30:70) Acetonitrile / 0.1 % Formic acid (40:60) App ID 19328 App ID 19329 Phenomenex l WEB: www.phenomenex.com 15 TN-1079 APPLICATIONS

Chiral LC/MS/MS Applications Figures 7-11 demonstrate 15 chiral separations on several dif- improved signal intensity for warfarin and ketorolac versus ESI- (Fig- ferent Lux chiral stationary phases. APCI negative (APCI-) mode ure 10). Most compounds evaluated here eluted in less than 10 min was employed for the MS/MS detection of carprofen, abscisic with baseline resolution in mobile phases of various elution strength. acid, and fenoprofen as it provides much better MS/MS signals The results show that Lux® Cellulose-3 was most successful in sep- than ESI- for these acidic racemates. ESI positive (ESI+) mode arating acidic racemates (ten racemates), especially nonsteroidal was used for the MS/MS detection of suprofen and gave slightly anti-inflammatory drugs.

Figure 7. Enantioseparations in Reversed Phase on Lux 3 µm Cellulose-1 with Acetonitrile

Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Formic acid (60:40) acid (40:60) Sample: Etodolac Sample: Proglumide App ID 19334 App ID 19330 Acetonitrile / 0.1 % Formic acid (60:40) Acetonitrile / 0.1 % Formic acid (60:40) Sample: Warfarin Sample: Carprofen Column: Lux 3 µm Cellulose-1 Dimensions: 150 x 2.0 mm Part No.: 00F-4458-B0 Flow Rate: 0.2 mL/min App ID 19331 App ID 19335 Temperature: 25 °C

Acetonitrile / 0.1 % Formic acid / (30:70) Acetonitrile / 0.1 % Formic acid (40:60) Detection: Mass Spectrometer (MS) Sample: Trichlormethiazide Sample: Bendroflumethiaze Injection Volume: 5 µL Mobile Phase: As noted Sample: As noted App ID 19332 App ID 19336 Acetonitrile / 0.1 % Formic acid (30:70 Sample: 1-(Phenylsulfonyl)- Acetonitrile / 0.1 % Formic acid (30:70) 3-indoleboronic acid Sample: Ketorolac App ID 19333 App ID 19337

Figure 8. Enantioseparations on Lux 3 µm Cellulose-2 with Acetonitrile or Methanol as Modifier

Acetonitrile / Formic Methanol / 0.1 % Formic acid (40:60) acid (70:30) Sample: Proglumide Sample: Proglumide App ID 19338

Acetonitrile / Formic acid (40:60) Methanol / 0.1 % Formic acid (70:30) Sample: Ketorolac Sample: Ketorolac App ID 19339 Column: Lux 3 µm Cellulose-2 Acetonitrile / Formic Dimensions: 150 x 2.0 mm acid (40:60) Part No.: 00F-4456-B0 Sample: Methanol / 0.1 % Formic acid (70:30) Bendroflumethiaze Sample: Bendroflumethiaze Flow Rate: 0.2 mL/min Temperature: 25 °C

App ID 19340 Detection: Mass Spectrometer (MS) Injection Volume: 5 µL Acetonitrile / Formic acid (40:60) Methanol / 0.1 % Formic acid (70:30) Sample: 1-(Phenylsulfonyl)- Sample: 1-(Phenylsulfonyl)- Mobile Phase: As noted 3-indoleboronic acid 3-indoleboronic acid Sample: As noted App ID 19341

Methanol / 0.1 % Formic Acetonitrile / Formic acid (40:60) acid (70:30) Sample: Etodolac Sample: Etodolac App ID 19342 16 Phenomenex l WEB: www.phenomenex.com TN-1079 APPLICATIONS

Figure 9. Enantioseparations on Lux® 5 µm Cellulose-4 with Acetonitrile

X I C o f -M R M ( 6 p a i rs ): 2 8 6 .1 0 1 /2 4 2 .0 0 0 D a fro m S a m plep l... M a x . 1 .3 e 6 cp s . X IC o f - M R M (6 p a ir s ) : 3 0 7 .1 3 4 /1 6 0 .9 0 0 D a fr o m S a m plep l ... M a x. 1 .8 e 5 c p s.

7 .1 2 6 .4 9 1 .3 0 e 6 7 .5 6 6 .9 1 1 .5 e 5 E t o d o la c W a r f a r in 1 .0 0 e 6 Acetonitrile / 0.1 % Formic acid (50:50) Acetonitrile / 0.1 % Formic acid(60:40) M P : C H 3 C N : 0 . 1 % F A / 6 0 : 4 0 Sample:M P : C HEtodolac3 C N : 0 . 1 % F A /5 0 : 5 0 1 .0 e 5 Sample: Warfarin I 5 .0 e 4 5 .0 0 e 5 n t Column: Lux 5 µm Cellulose-4 0 .0 2 4 6 e8 1 0 2 4 6 8 1 0 Dimensions: 250 x 4.6 mm

T im e , m i n n T im e , m i n App ID 19347 App ID 19343 X I C o f -M R M ( 6 p a i rs ): 2 9 9 .9 9 4 /2 0 9 .8 0 0 D a fro m S a m ple... s M a x . 1 5 7 7 .1 cp s . X IC o f - M R M (6 p a ir s ) : 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a fr o m S a m ple... M a x. 4 5 5 6 .1 c p s. Part No.: 00G-4491-E0 i Flow Rate: 1 mL/min t 1 2 .7 7 1 2 .7 5 1 5 0 0 1 3 .5 3 1 3 .5 4 1 - ( P h e n y ls u lf o n y l ) - 3 - in d o l e b o r n i c a c id y 4 0 0 0 AcetonitrileK e t o r o la c / 0.1 % Formic acid (40:60) Temperature: 25 °C AcetonitrileM P : C H 3 C N/ :0.10 .1 % %F AFormic/4 0 : 6 0 acid (40:60) , M P : C H 3 C N : 0 . 1 % F A / 4 0 : 6 0 1 0 0 0 Sample: Ketorolac Detection: Mass Spectrometer (MS) Sample: 1-(Phenylsulfonyl)- I 2 0 0 0 5 0 0 c 3-indoleboronic acid n p Injection Volume: 20 µL t 0 s 0 2 4 6 8 1 0 e1 2 1 4 2 4 6 8 1 0 1 2 1 4 Mobile Phase: As noted T im e , m i n n T im e , m i n App ID 19348 App ID 19344 X I C o f -M R M ( 6 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a fro m S a m plep l... s M a x . 2 .7 e 4 cp s . X IC o f - M R M (2 p a ir s ) : 3 7 7 .7 7 4 /2 4 1 .6 0 0 D a fr o m S a m plep l ... M a x. 9 .3 e 4 c p s. Sample: As noted i 6 .8 4 t 1 0 .7 0 2 .7 e 4 9 .3 e 4 P r o g lu m id e 7 .6 1 y T r ic h l o r m e t h ia z id e 1 3 .6 0 2 .0 e 4 AcetonitrileM P : C H 3 C N/ : 0.10 .1 % %F AFormic/4 0 : 6 0 acid (40:60) , AcetonitrileM P : C H 3 C/N 0.1: 0 .1 %% F FormicA / 3 0 : 7 0 acid Sample: Proglumide 5 .0 e 4 (30:70) 1 .0 e 4 cI Sample: Trichlormethiazide pn t 0 .0 s 0 .0 2 4 6 8e 1 0 2 4 6 8 1 0 1 2 1 4 T im e , m i n T im e , m i n n App ID 19349 App ID 19345 X I C o f -M R M ( 6 p a i rs ): 4 2 0 .0 4 8 /7 7 .9 0 0 D a fro m S a m p le ... s M a x . 1 .2 e 4 cp s . X IC o f - M R M (6 p a ir s ) : 4 2 0 .0 4 8 /7 7 .9 0 0 D a fr o m S a m p l e ... M a x. 1 .5 e 4 c p s. i 5 .1 6 t 3 .7 9 5 .8 0 1 .5 e 4 4 .1 4 B e n d ro f lu m e t h ia z e y 1 .0 0 e 4 B e n d r o f lu m e t h i a z e Acetonitrile / 0.1 % Formic AcetonitrileM P : C H 3 C N/ :0.10 .1 % %F A Formic/5 0 : 5 0 , 1 .0 e 4 M P : M e O H : 0 .1 % F A /8 : 2 acid (50:50) acid (80:20) 5 0 0 0 .0 0 I c 5 0 0 0 .0 Sample: Bendroflumethiaze Sample: Bendroflumethiaze n p t 0 .0 0 s 0 .0 2 4 6 e8 1 0 2 4 6 8 1 0 T im e , m i n n T im e , m i n App ID 19350 App ID 19346 s i t y , Figure 10. Enantioseparations in Reversed Phase on Lux 3 µmc Amylose-2 with Acetonitrile p Negative Ion Mode s Positive Ion Mode

Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Formic acid (60:40) acid (60:40) Sample: Ketorolac Sample: Ketorolac Column: Lux 3 µm Amylose-2 Dimensions: 150 x 2.0 mm

App ID 19351 Part No.: 00F-4471-B0 Flow Rate: 0.2 mL/min Temperature: 25 °C Detection: Mass Spectrometer (MS) Acetonitrile / 0.1 % Formic Acetonitrile / 0.1 % Formic Injection Volume: 5 µL acid (60:40) acid (60:40) Mobile Phase: As noted Sample: Warfarin Sample: Warfarin Sample: As noted App ID 19352

Acetonitrile / 0.1 % Acetonitrile / 0.1 % Formic acid (60:40) Formic acid (60:40) Sample: Suprofen Sample: Flurbiprofen App ID 19353 App ID 19354

Phenomenex l WEB: www.phenomenex.com 17 TN-1079 APPLICATIONS

Figure 11. Enantioseparations in Reversed Phase on Lux® 5 µm Cellulose-3 with Acetonitrile or Methanol as Modifier

Acetonitrile / 0.1 % Formic acid (40:60) Acetonitrile / 0.1 % Formic acid (40:60) Sample: Mecoprop Sample: Flurbiprofen App ID 19356 App ID 19355

Column: Lux 5 µm Cellulose-3 Dimensions: 250 x 4.6 mm Acetonitrile / 0.1 % Formic acid (40:60) Acetonitrile / 0.1 % Formic acid (40:60) Sample: Suprofen (ESI+) Sample: Ketorolac Part No.: 00G-4493-E0 Flow Rate: 1 mL/min App ID 19358 Temperature: 25 °C App ID 19357 Detection: Mass Spectrometer (MS) Acetonitrile / 0.1 % Formic acid (40:60) Methanol / 0.1 % Formic acid (80:20) Injection Volume: 20 µL Sample: Etodolac Sample: Indoprofen Mobile Phase: As noted Sample: As noted App ID 19363 App ID 19359

Methanol / 0.1 % Formic Methanol / 0.1 % Formic acid (95:5) acid (80:20) Sample: Ibuprofen Sample: Proglumide App ID 19364 App ID 19362

Methanol / 0.1 % Formic Acetonitrile / 0.1 % Formic acid (40:60) acid (60:40) Sample: Fenoprofen (APCI-) Sample: Abscisic acid (APCI-) App ID 19361 App ID 19360

Conclusions Free Chiral Screening Services The chiral LC/MS/MS analysis of fifteen different acidic racemates are successfully demonstrated on the polysaccharide-based CSPs Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cel- lulose-4, and Lux Amylose-2 in reversed phase (RP) elution mode.

Formic acid is a good first choice for an acidic RP mobile phase Visit us online at: www.phenomenex.com/Phenologix additive as it leads to increased retention and improved enantioselectivity for acidic enantiomers and is also compatible with MS/ MS detection.

Increasing the percentage of organic modifier (acetonitrile or methanol) in the RP mobile phase has the expected effect of decreasing retention and enantioselectivity. Adjusting the organic If Lux analytical columns (≤ 4.6 mm ID) do modifier content of the mobile phase is therefore essential to op- not provide at least an equivalent or better timizing chiral resolution. separation as compared to a competing column of the same particle size, similar phase and dimensions, return the column with comparative data within 45 days for a FULL REFUND.

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux and Kinetex are registered trademarks, and Axia and SecurityGuard are trademarks of Phenomenex. TurboIonSpray is a registered trademark and AB SCIEX, API 3000, and Turbo V are trademarks of AB Sciex Pte. Ltd. TN76320312_W Disclaimer Comparative separations may not be representative of all applications. © 2012 Phenomenex, Inc. All rights reserved.

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Phenomenex l WEB: www.phenomenex.com 19 TN-1128 APPLICATIONS Performance Evaluation of Immobilized and Coated Polysaccharide Chiral HPLC Columns Using Generic Screening Mobile Phase Systems Zdravko Milanov, Liming Peng, Jeff Layne, Marc Jacob et al. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Two chiral stationary phases (Lux® Cellulose-1 and CHIRALPAK® Figure 1. IB®) that consist of the same chiral selector, cellulose tris(3,5-di- Structures of cellulose based chiral stationary phase used in this study methylphenylcarbamate), were evaluated. The major difference between the two phases is that the first phase (Lux Cellulose-1) is Lux Cellulose-1 prepared by coating the underlying silica with the modified polysac- Cellulose tris(3,5-dimethylphenylcarbamate) charide, while the second phase is an immobilized phase in which Me the polysaccharide is covalently bonded to the underlying silica. OCONH This covalent linkage allows for the use of an extended range of solvents (e.g., THF, DMF, acetone, ethyl acetate, methylene chloride) O Me O that are not compatible with coated chiral stationary phases (CSPs). Me Me In this study, we sought to determine if this expanded solvent range HNOCO OCONH increased the success rate of chiral separations using the immobilized CSP. Me Me

Introduction CHIRALPAK IB Modified polysaccharide-based stationary phases are the most Cellulose tris(3,5-dimethylphenylcarbamate) widely used CSPs due to their broad-spectrum chiral selectivity Me and high loading capacity. Most separations performed using poly- OCONH saccharide CSPs are performed in normal phase using solvents such as hexane and alcohol, and these conditions have been prov- O Me O en to be very favorable for chiral recognition mechanisms. Me Me

The majority of polysaccharide-based CSPs are coated phases, in HNOCO OCONH which the stationary phase is not covalently bonded to the under- Me Me lying silica. Recently, immobilized polysaccharide CSPs, in which the polysaccharides are covalently linked to the silica, have also become available. Immobilized CSPs allow for the use of more aggressive solvents, such as chlorinated solvents (e.g., methylene Material and Methods chloride) or ethyl acetate, which cannot be used with conventional All analyses were performed using an HPLC Agilent® 1100 series coated phases due to solubility issues. (Agilent Technologies, Palo Alto, CA, USA) equipped with an autosampler and a quaternary pump. Chiral chromatographic sepa- There has been speculation that the expanded solvent range of im- rations followed by UV detection were performed using Lux Cel- mobilized CSPs might increase the selectivity options and, hence, lulose-1 (coated phase) and CHIRALPAK IB (immobilized phase) lead to enhanced enantiorecognition relative to coated CSPs. HPLC columns with dimensions 250 x 4.6 mm ID packed with However, there is a lack of extensive comparative studies to deter- 5 µm particles. The system flow rate was set to 1 mL/min and the mine if this is indeed the case. Thus, in this study, we have sought column temperature was ambient unless noted otherwise Mobile to determine if an immobilized CSP (CHIRALPAK IB) exhibits sig- Phase Conditions used for each column are described in Table 1. nificantly increased chiral separation success rates as compared to a coated CSP (Lux Cellulose-1), when evaluated under generic Table 1. normal phase screening conditions (including the use of chlorinat- Mobile phase conditions used in this study. ed solvents). The two chiral stationary phases evaluated in this CSP Mobile Phase (MP) study consist of the same chiral selector, cellulose tris(3,5-dimeth- Coated Phase 80:20:0.1 Hex:IPA:DEA; and 85:15:0.1 Hex:EtOH:DEA ylphenylcarbamate) as depicted in Figure 1. Lux Cellulose-1 Generic screens in Normal Phase (NP) are common in the industry, Immobilized 80:20:0.1 Hex:IPA:DEA; 85:15:0.1 Hex:EtOH:DEA; as NP is favorable for the principal mechanisms of chiral recogni- Phase 65:35:0.1 Hex:EtOAc:Ethanolamine; and ® tion. The majority of chiral separations with polysaccharide phases CHIRALPAK IB 65:35:0.1 Hex:CHCl3:Ethanolamine are performed using hexane and alcohol modifiers. Previous work DEA = Diethyl amine; IPA = Isopropyl alcohol; CHCl3 = Chloroform; Hex = Hexane; EtOH = Ethanol; EtOAc = has identified the different selectivities offered between Isopropyl Ethyl acetate alcohol (IPA) and Ethanol (EtOH), and we have used these solvents in our generic screen of both columns. Chlorinated solvents and ethyl acetate may also be used as NP modifiers to offer a different analyte solvent selectivity; however, these cannot be used on coated polysaccharide columns. Our generic screen incorporated the addition of the above solvents only for the immobilized phase.

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Results and Discussion The same 51 compounds were analyzed under different mobile To evaluate the enantioresolution between coated and immobilized phase conditions using EtOH as organic modifier with DEA as ad- CSP, 51 chemical compounds of pharmaceutical interest were an- ditive. Out of those 51 compounds, 28 analytes were resolved on alyzed under various mobile phase conditions. In the first set of either CSP using 85:15:0.1 Hexane:EtOH:DEA as mobile phase. experiments, IPA was used as a modifier with DEA as additive. Out Table 3 summarizes the difference in enantioselectivity using those of those 51 compounds, 22 were resolved on either CSPs using conditions. 80:20:0.1 Hexane:IPA:DEA as mobile phase. Table 2 summarizes the difference in enantioselectivity using those conditions. Table 3. Comparison of enantioresolution of 28 racemates between coated and Im- Table 2. mobilized CSP using 85:15:0.1 Hex EtOH:DEA as organic modifier Comparison of enantioresolution of 22 racemates between coated and immobilized CSP using 80:70:0.1 Hex:IPA:DEA as organic modifier Compounds Lux CHIRALPAK Compounds Lux CHIRALPAK Cellulose-1 IB Cellulose-1 IB Compounds Lux® CHIRALPAK® Compounds Lux CHIRALPAK® DL-B- x Partial Carazolol √ Partial Cellulose-1 IB® Cellulose-1 IB Hydroxyphenethylamine Tetrahydrozoline √ x Toliprolol √ √ Miconazole √ Partial Toliprolol √ √ Metoprolol √ x Bisoprolol √ Partial Tetrahydrozoline √ x Bisoprolol √ Partial Tetramisole √ x Sulfconazole x Partial Metoprolol x √ Sulfconazole √ √ Halofantrine √ √ Orphenadrine √ √ Acebutolol Partial x Orphenadrine √ Partial Bopindolol √ Partial Mianserin √ x Tetramisole √ √ Mianserin √ Partial Bupranolol √ Partial 1,1-Dihydroxy-6,6- √ √ Halofantrine √ √ 1,1-Dihydroxy-6,6- Partial √ Dimethylbiphenyl Dimethylbiphenyl Carazolol Partial x Methoxy-p-tolyl √ x Bopindolol √ √ Methoxy-p-tolyl √ x sulfoxide sulfoxide Metomidate √ x Prilocaine √ Partial Bupranolol √ x 5-Methyl-5- Partial x Mephenesin √ x Nifedpine x √ phenyl-hydantoin Oxazapam √ √ Bupivacaine √ x Metomidate √ Partial Nifedpine x √ Oxprenolol x √ Disopyramide Partial x Mephenesin √ Partial Bupivacaine Partial x Oxazapam √ √ Omeprazole √ √ Lux CHIRALPAK Cellulose-1 IB Oxprenolol √ √ Indapamide x √ Prilocaine Partial Partial Bendroflumethiazide √ Baseline Resolution Rs > 1.5 17 7 x √ x No resolution R < 0.8 s 3 10 Lux CHIRALPAK Cellulose-1 IB Partial Resolution 0.8 < Rs < 1.5 2 5

√ Baseline Resolution Rs > 1.5 18 13 The percentage of compounds that showed a resolution > 2 on x No resolution R < 0.8 5 6 coated Lux Cellulose-1 is over twice that observed on immobilized s Partial Resolution 0.8 < R < 1.5 CHIRALPAK IB using Hexane/IPA as mobile phase as represented s 5 9 in Figure 2.

Figure 2. Percentage of compounds showing resolution > 2 using IPA as modifier.

Lux Cellulose-1 vs. CHIRALPAK IB 1 .0 0

Lux Cellulose-1 0 .9 0 CHIRALPAK® IB

0 .8 0 77.22 %

0 .7 0 63.75 %

0 .6 0

0 .5 0

0 .4 0

Fraction of Compounds 0 .3 0 27.50 % Fraction of Compounds

0 .2 0 Flow Rate: 1.0 mL/min 11.39 % 11.39 % 8.75 % Temperature: 25 ˚C 0 .1 0 Detection: UV @ 220 nm Mass Loaded: 10 µg 0 .0 0 <1 1-2 >2 Resolution

Phenomenex l WEB: www.phenomenex.com 21 TN-1128 APPLICATIONS The percentage of compounds that showed a resolution > 2 on Interestingly, although both columns shared similar chiral selector, coated Lux® Cellulose-1 is over twice that observed on immobi- cellulose tris(3,5-dimethylphenylcarbamate), there was very little lized CHIRALPAK® IB® using Hexane/EtOH as mobile phase as correlation between the compounds that were resolved on the represented in Figure 3. coated phase and those that were resolved on the immobilized phase (< 25 % of the racemates were resolved on both columns Figure 3. under identical running conditions) as depicted in Figure 4. Percentage of compounds showing resolution > 2 using EtOH as modifier

® ® ® Figure 4. Lux Cellulose-1 vs. CHIRALPAK IB ® Extent of Complementary Selectivity between Coated and Immobilized Columns: Lux 5 µm Cellulose-1 CSPs in Hex:IPA and Hex:EtOH Dimensions: 250 x 4.6 mm 1 .0 0 Mobile Phase: Hex/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min 0 .9 0 1.00 Detection: UV @ 220 nm

0.90 0 .8 0 80:20 HEX:IPA Cellulose-1 vs. IB Lux Cellulose-1 85:15 HEX:EtOH Cellulose-1 vs. IB 0.80 ® 0 .7 0 CHIRALPAK IB 63.29 % 0.70 67.2 % 68.4 % 58.23 % 0 .6 0 Columns: Lux 5 µm Cellulose-1 0.60 Dimensions: 250 x 4.6 mm 0 .5 0 0.50 Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min 0 .4 0 0.40 Detection: UV @ 220 nm 31.65 % Fraction of Compounds 0.30 0 .3 0 Fraction of Compounds 24.05 % 23.1 % 0.20 14.8 % 12.9 % 0 .2 0 FractionFraction of Comps. that Overlap of Comps. that Overlap 12.66 % 0.10 8.0 % 10.13 % 0 .1 0 0.00 <1 1-2 >2 0 .0 0 Resolution Columns: Lux 5 µm Cellulose-1 <1 1-2 >2 Resolution Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Flow Rate: 1.0 mL/min % Match of Compounds Resolved Flow Rate: 1 mL/min Detection: UV @ 220 nm Temperature: 25 ˚C on both Lux Cellulose-1 and CHIRALPAK® IB® for Different Resolution UV @ 220 nm Detection: Ranges Mass Loaded: 10 µg

Columns: Lux 5 µm Cellulose-1 Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Detection: UV @ 220 nm

Columns: Lux 5 µm Cellulose-1 Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min Detection: UV @ 220 nm

Columns: Lux 5 µm Cellulose-1 Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Detection: UV @ 220 nm

Columns: Lux 5 µm Cellulose-1 Dimensions: 250 x 4.6 mm Free Mobile Phase: Heptane/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min Chiral Screening Detection: UV @ 220 nm Services, provided by PhenoLogixsm www.phenomenex.com/PhenoLogix

Columns: Lux 5 µm Cellulose-1 Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Detection: UV @ 220 nm

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A few representative examples of the effect on the mobile phase composition on the chiral separation on coated and immobilized CSP are presented on Figure 5. The data clearly indicates that under the same mobile phase conditions, coated CSP Lux Cellulose-1 has a greater separation properties over immobilized CSP. Figure 5. Effect of Organic Modifier on Lux Cellulose-1 (coated) and CHIRALPAK® IB® (immobilized) CSPs 19196 19195 Mephenesin RS= 1.92 RS= 1.92 Columns: Lux® 5 µm Cellulose-1 Columns: CHIRALPAK® IB® 5 µm Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19195 App ID 19196 0 10 20 min 0 10 20 min 19198 19197

RS= 3.04 Columns: Lux 5 µm Cellulose-1 Columns: CHIRALPAK IB 5 µm RS= 1.32 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19198 min App ID 19197 0 10 20 0 10 20 min

19199 19200

Orphenedrine® R = 1.53 RS= 4.58 S

Columns: Lux 5 µm Cellulose-1 Columns: CHIRALPAK IB 5 µm Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm 0 2 4 6 8 10 min App ID 19199 0 2 4 6 8 10 min App ID 19200 19202 19201

Columns: Lux 5 µm Cellulose-1 Columns: CHIRALPAK IB 5 µm R = 1.15 R = 2.63 S Dimensions: 250 x 4.6 mm S Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm

0 2 4 6 8 10 min 0 2 4 6 8 10 min App ID 19202 App ID 19201 19204 19203 Prilocaine RS= 1.88 R = 1.88 Columns: Lux 5 µm Cellulose-1 S Columns: CHIRALPAK IB 5 µm Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/IPA/DEA (80:20:0.1)

Flow Rate: 1 mL/min Flow Rate: 1 mL/min 0 2 4 6 8 10 min App ID 19204

Detection: UV @ 220 nm min App ID 19203 Detection: UV @ 220 nm 0 2 4 6 8 10 19206 19205

Columns: Lux 5 µm Cellulose-1 Columns: CHIRALPAK IB 5 µm R = 1.12 R = 1.28 S Dimensions: 250 x 4.6 mm S Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19206

0 2 4 6 8 10 min App ID 19205 0 19208 2 4 6 8 10 min

19207 Tetramisole

R = 2.95 S Columns: CHIRALPAK IB 5 µm Columns: Lux 5 µm Cellulose-1 RS= 1.17 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Heptane/IPA/DEA (80:20:0.1) Mobile Phase: Heptane/IPA/DEA (80:20:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min

Detection: UV @ 220 nm 0 10 20 min App ID 19208 Detection: UV @ 220 nm App ID 19207 0 10 20 min

19210 19209

Columns: CHIRALPAK IB 5 µm R =1.92 Columns: Lux 5 µm Cellulose-1 S R = 3.02 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm S Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19209 App ID 19210 0 10 20 min 0 10 20 min

Phenomenex l WEB: www.phenomenex.com 23 TN-1128 APPLICATIONS Immobilized CSP can be used with mobile phases of various natures, ranging from the so called “standard solvents “ such as acetonitrile, alcohols, and alkanes recommended for coated CSPs to mobile phase containing “non-standard” solvents such as chlorinated solvents, ethyl acetate, tetrahydrofuran (THF) and methyl tertiary butyl ether (MTBE). Figures 6-8 show separation of three chiral analytes using “standard” and Me “non-standard” solvents indicating that using chloroform (CHCl3) or ethyl acetate (EtOAc) in the mobile phase does not necessarily improve the OCchiralONH separation.

Me O 19216 O 19215 Figure 6. Me Me Bisoprolol on CHIRALPAK® IB® HNOCO OCONH R = 0.85 S Columns: CHIRALPAK IB 5 µm Columns: CHIRALPAK IB 5 µm R = 1.45 Me Me S Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) App ID 19215

Flow Rate: 1 mL/min Flow Rate: 1 mL/min App ID 19216 OH Detection: UV @ 220 nm Detection: UV @ 220 nm H 19217 19218 N 0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 min

Columns: CHIRALPAK IB 5 µm Columns: CHIRALPAK IB 5 µm R = 5.90 R = 2.66 S Dimensions: 250 x 4.6 mm S Dimensions: 250 x 4.6 mm

Mobile Phase: Hex/EtOAc/Ethanolamine (65:35:0.1) Mobile Phase: Hex/CHCl3/Ethanolamine (65:35:0.1) 1 2 1 2 O Flow Rate: 1 mL/min Flow Rate: 1 mL/min UV @ 220 nm UV @ 220 nm App ID 19218 O Detection: App ID 19217 Detection:

0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 min

Figure 7. ® 19219 Bupranolol on CHIRALPAK IB 19220 2 1

® R = 0.66 Columns: CHIRALPAK 5 µm IB RS= 0.82 Columns: CHIRALPAK IB 5 µm S 2 Dimensions: 250 x 4.6 mm 1 Dimensions: 250 x 4.6 mm Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min App ID 19219 CI OH Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19220 CH H 3 0 2 4 6 8 10 min O N CH 0 2 4 6 8 10 min 3 19221

CH Columns: CHIRALPAK IB 5 µm Columns: CHIRALPAK IB 5 µm 3 19222 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm R = 0.00 0.00 S 1 Mobile Phase: Hex/EtOAc/Ethanolamine (65:35:0.1) Mobile Phase: Hex/CHCl3/Ethanolamine (65:35:0.1) RS = 0.00 CH3 Flow Rate: 1 mL/min Flow Rate: 1 mL/min Detection: UV @ 220 nm Detection: UV @ 220 nm App ID 19222 App ID 19221 0 2 4 6 8 10 min 0 2 4 6 8 10 min

Figure 8. 19223 Metomidate on CHIRALPAK® IB 19224 1 2

2 1

RS= 1.37 Columns: CHIRALPAK IB 5 µm RS= 0.77 Columns: CHIRALPAK IB 5 µm O Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm

Mobile Phase: Hex/IPA/DEA (80:20:0.1) Mobile Phase: Hex/EtOH/DEA (85:15:0.1) App ID 19224

Flow Rate: 1 mL/min App ID 19223 Flow Rate: 1 mL/min O Detection: UV @ 220 nm Detection: UV @ 220 nm 0 2 4 6 8 10 min 0 2 4 6 8 10 min 19226 19225 1 2 N N 1 2

Columns: CHIRALPAK IB 5 µm Columns: CHIRALPAK IB 5 µm RS= 1.19 R = 3.11 Dimensions: 250 x 4.6 mm S Dimensions: 250 x 4.6 mm Mobile Phase: Hex/EtOAc/Ethanolamine (65:35:0.1) Mobile Phase: Hex/CHCl3/Ethanolamine (65:35:0.1) Flow Rate: 1 mL/min Flow Rate: 1 mL/min

Detection: UV @ 220 nm UV @ 220 nm App ID 19226

App ID 19225 Detection: 0 2 4 6 8 10 min 0 2 4 6 8 10 min

24 Phenomenex l WEB: www.phenomenex.com TN-1128 APPLICATIONS Of the 51 unique compounds investigated using various NP solvent Conclusions systems only 18 were partially or fully resolved using CHIRALPAK® In this Technical Note, we compared the chiral separation success IB® as summarized in Table 4. The option of using non-standard rate of an immobilized CSP (CHIRALPAK IB) to a conventional coat- solvents on immobilized CSPs clearly did not offer any advantages ed CSP (Lux® Cellulose-1) under generic normal phase screening over coated CSP for those 51 compounds. conditions using 51 different racemates of pharmaceutical interest.

Table 4. Using the conventional mobile phase of Hexane/IPA/DEA, the coated Lux Cellulose-1 column was able to resolve 17 racemates, while Compounds 80:20:0.1 85:15:0.1 65:35:0.1 65:35:0.1 ® ® Hex:IPA:DEA Hex:EtOH:DEA Hex:EtOAc:Ethanolamine Hex:CHCl :Ethanolamine the immobilized CHIRALPAK IB column was only able to resolve 3 7 racemates. Using another conventional mobile phase (Hexane/ 1,1-Dihydroxy-6,6- √ √ x Partial Dimethylbiphenyl Ethanol/DEA), the coated phase column was able to resolve 18 racemates, while the immobilized phase column was only able to Bisoprolol Partial Partial √ √ resolve 13 racemates with baseline or greater resolution. Bopindolol Partial √ x √ The overall resolution success rate of the coated Lux Cellulose-1 Bupranolol Partial x x √ column was 45 % (Rs > 2) using two mobile phases (Hexane/IPA/ Disopyramide x x √ x DEA and Hexane/EtOH/DEA) compared to the overall success rate ® ® Mephenesin Partial Partial √ x of 37 % for the immobilized CHIRALPAK IB columns using four different mobile phases (Hexane/IPA/DEA, Hexane/EtOH/DEA, Methoxy-p-tolyl x x Partial x sulfoxide Hexane/Chloroform/Ethanolamine, and Hexane/Ethyl acetate/Eth- anolamine). Metomidate x Partial √ Partial Metoprolol x √ √ x Overall, the data indicates that, under generic normal phase screening conditions, traditional coated CSPs display greater en- Mianserin x Partial Partial Partial antioselectivity in terms of % of compounds resolved with greater Miconazole x Partial Partial x than baseline resolution than do immobilized CSPs. Orphenadrine √ Partial √ √ Oxprenolol √ √ √ √ Prilocaine Partial Partial √ √ Sulfconazole √ √ √ √ Tetramisole Partial √ Partial x Toliprolol √ √ √ x Zopiclone x x √ √

√ Baseline resolution: Rs > 1.5

x No resolution Rs < 0.8

Partial resolution 0.8 < Rs < 1.5

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux is a registered trademark and Axia is a trademark of Phenomenex. CHIRALPAK and IB are registered trademarks of DAICEL Corporation. Agilent is a registered trademark of Agilent Technol- ogies, Inc. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with DAICEL Chemical Industries, Ltd. or Agilent. © 2012 Phenomenex, Inc. All rights reserved.

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HPLC Enantioseparation of N-FMOC a-Amino Acids Using Lux® Polysaccharide- Based Chiral Stationary Phases Under Reversed Phase Conditions Marc Jacob, Michael Klein, Liming Peng, and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technical note, we report the chiral separation of the most es, the final purity and overall yield of the peptide produced common 19 FMOC protected a-amino acids derivatives under re- is directly affected by the chemical and chiral purity of the pro- versed phase separation mode using Lux polysaccharide-based tected amino acids used. Currently, for the most common com- chiral stationary phases. All FMOC a-amino acids analyzed in this mercially available FMOC protected a-amino acids (19 natural study are baseline resolved with an analysis time below 25 min in amino acids), the expected enantiomeric purity is > 99.0 % en- isocratic conditions. The order of elution as well as the enantiomer antiomeric excess (ee) for the L form and sometimes the purity identification are also reported. required must be >= 99.8 % ee. This level of precision can only be achieved by very few analytical techniques, chiral HPLC be- Introduction ing one of them. The main advantages of chiral HPLC analysis N-Fluorenylmethoxycarbonyl (FMOC) a-amino acids are import- over other techniques are speed, detection level, and ease of use. 1 ant building blocks for the solid phase synthesis of peptides. HPLC is also used on a regular basis by the peptide chemists to 2 After the development of FMOC/tBu strategy for solid phase analyze purified fractions as well as peptide purity. In this applica- peptide syntheses, FMOC a-amino acids have become the raw tion, we report for the first time, the chiral separation of the most materials of choice for the preparation of synthetic peptides. common commercially available FMOC protected a-amino acids Using this methodology, long peptides (up to 100 residues) can be under reversed phase conditions using polysaccharide-based prepared in a few days with high yield from micro molar (g) up to chiral stationary phases (CSPs) depicted in Figure 1.3 molar scale (kg). As the number of amino acids residues increas-

Figure 1. Structures of Polysaccharide-Based CSPs

Amylose Backbone Cellulose Backbone R R O O O O O * * n * O O R O R O O R n R

Lux Cellulose-1 (Cell-1) Lux Cellulose-3 (Cell-3) Lux Amylose-2 (Amy-2) H O H N O N O H O N O Lux Cellulose-2 (Cell-2) Lux Cellulose-4 (Cell-4) Cl Cl Cl H H H N H N Cl N N Cl O O O O

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Materials and Methods Table 1 summarizes all the separations and chiral recognition ob- All analyses were performed using an Agilent® 1100 series LC sys- served after performing RP screening using the protocol described tem (Agilent Technologies, Inc., Palo Alto, CA, USA) equipped with above. As shown in Table 1, all the amino acids tested were success- quaternary pump, in-line degasser, multi-wavelength UV detector, fully resolved on at least one of the five Lux polysaccharide-based and autosampler. Lux® columns used for analysis were obtained CSPs. In the case of Ile, Leu, Met, Phe, and Val FMOC derivatives, from Phenomenex (Torrance, CA, USA). The HPLC column dimen- baseline resolution was achieved on the five CSPs. sions were 250 x 4.6 mm ID and all columns were packed with 5 μm particles. FMOC protected L and D amino acids used in this study Table 1. were provided by Bachem® (Bubendorf, Switzerland). All solvents Chiral Recognition of the 19 Most Common FMOC Protected were purchased from EMD (San Diego, CA, USA). a-Amino Acids HPLC Conditions: Baseline resolution Chiral separation No resolution Columns: Lux 5 μm Cellulose-1 250 x 4.6 mm OOG-4458-EO Lux 5 μm Cellulose-2 250 x 4.6 mm OOG-4456-EO Lux 5 μm Cellulose-3 250 x 4.6 mm OOG-4492-EO FMOC-AA-OH Cell-1 Cell-2 Cell-3 Cell-4 Amy-2 Lux 5 μm Cellulose-4 250 x 4.6 mm OOG-4490-EO FMOC-Ala-OH Lux 5 μm Amylose-2 250 x 4.6 mm OOG-4471-EO Flow Rate: 1 mL/min FMOC-Arg(Pbf)-OH Temperature: Ambient Detection: UV @ 220 nm FMOC-Asn(Trt)-OH Injection Volume: 5 µL FMOC-Asp(OtBu)-OH Sample concentration: 2 mg/mL in Methanol (MeOH) or Acetonitrile (ACN) (pure FMOC amino acids enantiomer L and D were mixed in a ratio of 2:1 (L:D)) FMOC-Cys-(Trt)-OH

FMOC-Gln(Trt)-OH Results and Discussion Five different polysaccharide-based chiral stationary phases (CSPs) FMOC-Glu(OtBu)-OH Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, FMOC-His(Trt)-OH and Lux Amylose-2 (Figure 1) were explored in the reversed phase FMOC-Ile-OH

(RP) HPLC enantioseparation of the 19 most common FMOC pro- FMOC-Leu-OH tected a-amino acids. FMOC-Lys-(Boc)-OH

Due to the acidic nature of FMOC amino acid derivatives and based FMOC-Met-OH on our previous extensive screening work in RP mode,4 it was decid- FMOC-Phe-OH ed to use trifluoroacetic acid (TFA) or formic acid (FA) as acidic additives with acetonitrile (ACN) or methanol (MeOH) as organic modifier FMOC-Pro-OH (see experimental conditions). Those mobile phases are arguably the FMOC-Ser(tBu)-OH most used in RP mode. All the analysis were performed in isocratic FMOC-Thr(tBu)-OH mode with run time below 25 min. FMOC-Trp(Boc)-OH Initial screening of the Lux CSPs was performed with 0.1 % TFA/ FMOC-Tyr(tBu)-OH ACN in a volume ratio of 40:60. For retention time (Rt) < 6 min and FMOC-Val-OH resolution (Rs) < 1.5 (no baseline resolution), the amount of ACN was decreased in order to improve retention and chiral recognition. If no chiral separation was obtained with ACN as modifier, columns were Under our RP screening protocol, Cellulose-2 was the most suc- screened with 0.1 % FA/MeOH in a volume ratio of 20:80. In general, cessful phase with 18 chiral recognitions followed by Cellulose-3 as we observed more retention with TFA as an additive than with FA represented in Figure 2. when using ACN as modifier and as expected ACN elution power is Figure 2. stronger than MeOH. Quite a few FMOC amino acids can be sepa- Enantioselectivity Comparison Between Polysaccharide-Based CSPs rated with either ACN or MeOH as modifier. Baseline resolution Partial resolution No resolution

References 1. Merrifield R. B. J. Am. Chem. Soc., 1963, 85, 2149. 1 3 2. Carpino L.A. and Han G.Y. J. Org. Chem., , 37, 3404. 1972 3 5 3. Chankvetadze B. J. Chromatogr. A, 2012, 1269, 26. 6 8 4. Peng L., Jayapalan S., Chankvetadze B. and Farkas T. 3 J. Chromatogr. A 2010, 1217, 6942. 1 3 Acknowledgements Bachem AG, Switzerland for generously providing the 38 FMOC 1 protected L and D amino acids used in this study.

Julissa Fernandez and Michael McCoy (PhenoLogix group, Cali- 15 13 fornia, USA) for additional work performed on FMOC-Asn(Trt)-OH 12 11 separation. 10

Cell-1 Cell-2 Cell-3 Cell-4 Amy-2

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Table 2 describes some of the best separation observed for each amino acids such as His, Asn, and Cys derivatives are more chal- FMOC amino acid screened. Retention time for both enantiomers, lenging to separate and baseline resolution is only achieved using alpha value, resolution achieved, and order of elution are provided. Cellulose-2, Cellulose-3, and Cellulose-1, respectively. Selected chi- All the separation reported are baseline resolved and the run time is ral separation of FMOC-Asp(OtBu)-OH and FMOC-Tyr(tBu)-OH are less than 25 min. Interestingly, Trityl (Trt) side chain protected FMOC shown in Figure 3.

Table 2. Optimal RP HPLC Enantioseparation of the 19 Most Common FMOC Protected a-Amino Acids

a a App IDb FMOC-AA-OH CSP Mobile Phase Rt1 Rt2 Alpha Rs FMOC-Ala-OH Cell-3 MeOH / 0.1 % TFA (80:20) 7.165 9.551 1.55 5.63 21550

FMOC-Arg(Pbf)-OH Cell-1 ACN / 0.1 % TFA (70:30) 8.547 9.991 1.24 2.71 21580

FMOC-Asn(Trt)-OH Cell-2 ACN / 0.1 % TFA (55:45) 20.825 23.124 1.10 1.60 21873

FMOC-Asp(OtBu)-OH Cell-1 ACN / 0.1 % TFA (60:40) 12.577 15.426 1.28 4.18 21589

FMOC-Cys-(Trt)-OH Cell-4 MeOH / 0.1 % TFA (90:10) 9.969 11.375 1.20 1.79 21641

FMOC-Gln(Trt)-OH Cell-4 ACN / 0.1 % TFA (70:30) 7.184 8.866 1.39 4.47 21631

FMOC-Glu(OtBu)-OH Cell-1 ACN / 0.1 % TFA (60:40) 13.979 16.652 1.23 3.55 21590

FMOC-His(Trt)-OH Cell-1 ACN/ 0.1 % FA (60:40) 4.865 5.783 1.39 2.33 21582

FMOC-Ile-OH Cell-3 ACN / 0.1 % TFA (40:60) 12.22 13.64 1.15 2.86 21553

FMOC-Leu-OH Cell-3 MeOH / 0.1 % TFA (90:10) 4.56 5.654 1.64 3.60 21647

FMOC-Lys-(Boc)-OH Cell-3 ACN / 0.1 % TFA (50:50) 5.615 6.52 1.33 3.59 21546

FMOC-Met-OH Cell-1 ACN / 0.1 % TFA (60:40) 11.423 13.064 1.18 2.96 21559

FMOC-Phe-OH Cell-1 ACN / 0.1 % TFA (60:40) 18.965 21.963 1.18 2.80 21585

FMOC-Pro-OH Cell-4 ACN / 0.1 % TFA (60:40) 5.865 6.818 1.32 3.31 21643

FMOC-Ser(tBu)-OH Cell-3 ACN / 0.1 % TFA (40:60) 8.654 9.599 1.16 2.87 21549

FMOC-Thr(tBu)-OH Cell-4 ACN / 0.1 % TFA (60:40) 7.69 8.92 1.26 3.78 21629

FMOC-Trp(Boc)-OH Cell-1 ACN / 0.1 % TFA (80:20) 8.179 9.576 1.25 3.28 21586

FMOC-Tyr(tBu)-OH Cell-3 ACN / 0.1 % TFA (60:40) 5.973 6.773 1.26 2.89 21570

FMOC-Val-OH Cell-1 ACN / 0.1 % TFA (60:40) 11.669 15.052 1.37 3.90 21579

a Highlighted in blue is the retention time for the D enantiomer b To view the full application enter the App ID into the search field on our website at www.phenomenex.com/ChiralAppSearch

Figure 3. RP HPLC Enantioseparations of FMOC-Asp(OtBu)-OH and FMOC-Tyr(tBu)-OH

FMOC-Asp(OtBu)-OH on Lux 5 μm Cellulose-1 FMOC-Tyr(tBu)-OH on Lux 5 μm Cellulose-3 21570 21589 ACN / 0.1 % TFA (60:40) ACN / 0.1 % TFA (60:40) mAU mAU (L) 120 6.773 (L) 100 15.426 400 (D) 80 (D) 5.973 12.577 60

200 40

20 App ID 21589 App ID 21570 0 0

0 2 4 6 8 10 12 14 16 18 20 min 0 2 4 6 8 min

Conclusion Five different polysaccharide-based chiral stationary phases were acidic additive and Acetonitrile as organic modifier are the best explored in reversed phase HPLC for the separation of the 19 choice combination for successful chiral separation of FMOC most common FMOC protected a-amino acids. Under our RP a-amino acids derivatives. screening protocol, Lux® Cellulose-2 was the most successful phase with 18 chiral recognitions (15 baseline resolved) followed Based on this study, we feel confident that with a proper screen- by Lux Cellulose-3. ing protocol most of the FMOC protected amino acids can be resolved with the five polysaccharide-based chiral stationary phase All FMOC amino acids evaluated were fully resolved (Rs > 1.5) in used in this study. less than 25 min analysis time by RP separation mode. TFA as

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Novel Screening Approach for the Separation of Pharmaceutical Compounds using Lux® Polysaccharide-Based Chiral Stationary Phases in SFC Mode Richard Hodgson1, Michael Klein2, Tom Cleveland2, and Marc Jacob2 1Phenomenex LTD, Queens Avenue, Hurdsfield Industrial Estate, Macclesfield, Cheshire, SK10BN, United Kingdom 2Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technical note, we report a novel screening approach for the chiral chromatographic separation, derived from a 56-pharmaceuti- Table 1. cal compound test set, using five Lux polysaccharide-based chiral Fifty-six racemic pharmaceutical compounds screened in this study stationary phases in supercritical fluid chromatography mode within an analysis time of 30 min or less. Compounds Compounds Compounds Introduction Of the many techniques available for the separation of enantiomers, Acebutolol Flurbiprofen Oxazepam high performance liquid chromatography (HPLC) using polysaccha- Acenocoumarol Hexobarbital Oxprenolol ride-based chiral stationary phases (CSP) is currently the most popular.1,2 Some of the reasons for this include ease of use, high success Alprenolol Ibuprofen Pindolol rate, and ability to scale to preparative separations.3 Ambucetamide Isothipendyl Praziquantel

However, over the past few years supercritical fluid chromatography Atenolol Ketoprofen Procyclidine (SFC) has regained interest as a valuable alternative chromatograph- Atropine Labetalol Promethazine ic technique for chiral separations. The supercritical mobile phase, Betaxolol Mandelic acid Propiomazine which typically is constituted of a large percentage of carbon dioxide (>60 %), has a higher diffusivity and lower viscosity than liquid chro- Bisoprolol Mebeverine Propranolol matography mobile phases. As a result, it is possible to run instru- Bopindolol Mepindolol Salbutamol ments at higher flow rates, which enables higher throughput by a reduction in column equilibration and analysis times. In addition, SFC Bupranolol Meptazinol Salmeterol results in lowering consumption of organic solvent, decreasing costs, Carazolol Methadon Sotalol and reducing environmental impact.4 Carbinoxamine Metoprolol Sulpiride With increasing workloads and decreasing resources, fast and effi- Carvedilol Mianserine Suprofen cient chiral method development screening strategies are required to save development time. In this technical note, we wish to report the Clorphenamine Nadolol Terbutaline screening strategy, derived from a representative group of 56 chi- Chlorthalidone Naringenin Tertatolol ral pharmaceutical compounds (Table 1) using five Lux polysaccharide-based CSPs under SFC conditions. Dimethindene Nicardipine Tetramisol Ephedrine Nimodipine Verapamil The results summarized in this application are extracted from an extended study performed by De Klerck et al 5. For all results and expla- Esmolol Nisoldipine Warfarin nations, we recommend the reader to consult the recent published Fenoprofen Nitrendipine article from this group as well as the references cited therein. Acidic compounds are written in italic

Material and Methods The analyses shown in this technote were performed using an analytical SFC method station from Thar Instruments (Pittsburgh, PA, USA, a Waters® company) equipped with a Waters 2998-DAD detector (Milford, MA, USA). Data acquisition and processing were performed using ChromScope™ V1.10 software (2011) from Waters. The columns used for analysis: Lux Cellulose-1 (Cell-1), Cellulose-2 (Cell-2), Cellulose-3 (Cell-3), Cellulose-4 (Cell-4), and Amylose-2 (Amy-2) were obtained from Phenomenex (Torrance, CA, USA). All columns had dimensions 250 x 4.6 mm I.D. and 5 μm particle size. SFC conditions unless noted otherwise were the following: flow rate: 3 mL/min, temperature: 30 °C, detection: UV @ 220 nm, backpressure: 150 bar, injection volume: 5 µL, run time: 30 min. Compounds that did not elute (entirely) within the set time frame of 30 minutes are considered as non-eluted. All solutions were prepared at sample concentration of 0.5 mg/mL in methanol (MeOH). Pharmaceutical compounds and materials were purchased from various suppliers (see reference 5 for further details).

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Results and Discussion The increase in the number of baseline separations for mobile phases The test group of 56 racemic pharmaceutical compounds listed in Ta- D and H is related to a decrease in retention time, resulting in elution ble 1 was screened on five Lux® polysaccharide-based CSPs (Cel- (and separation) of a number of analytes that were eluting outside the lulose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) with 30 minute time window with weaker mobile phases. In addition, peaks eight mobile phases under SFC conditions. The SFC mobile phases were sharper when using higher modifier concentrations, resulting in tested in this study are described in Table 2. slightly higher resolutions.

Table 2. The cumulative baseline separations for the five Lux polysaccha- SFC mobile phases used in this study ride-based chiral columns using a mobile phase of CO2/ (MeOH with MP Description 0.1 % IPA and 0.1 % TFA), 80/20, v/v (D) is shown in Figure 2. Using A CO /(MeOH with 0.5 % additive) 90/10 this mobile phase, the baseline separations from the most successful 2 CSP are recorded first. Then the second CSP is selected based on the B CO /(MeOH with 0.5 % additive) 80/20 2 highest number of added unique baseline separations compared with C CO2/(MeOH with 0.25 % IPA and 0.25 % TFA) 90/10 the first, followed by the third, fourth, and then fifth. Using this strategy, D CO /(MeOH with 0.1 % IPA and 0.1 % TFA) 80/20 46 of the 56 pharmaceutical compounds are baseline separated using 2 mobile phase D and all five Lux CSPs. E CO2/(2PrOH with 0.5 % additive) 90/10 F CO2/(2PrOH with 0.5 % additive) 90/10 Figure 2. G CO2/(2PrOH with 0.25 % IPA and 0.25 % TFA) 90/10 Cumulative baseline separations across five Lux phases using mobile phase of CO2 / (MeOH with 0.1% IPA and 0.1% TFA), 80/20 v/v H CO2/(2PrOH with 0.1 % IPA and 0.1 % TFA) 80/20 56 100 % MP = mobile phase, MeOH = methanol, 2PrOH = isopropanol/ 2-propanol, TFA = trifluoroacetic acid, IPA = isopropylamine. For acidic compounds, additive was TFA and for all other compounds (neutral, amphoteric, basic) IPA was used as additive. 51 91 %

80 % 82 % 46 82 % 77 % 46 73 % 45 41 43 73 % 41 The number of baseline separations (Rs > 1.5) with the five commercially available Lux CSPs are summarized in Figure 1 for each mobile 36 64 % phase condition. For this set of 56 pharmaceutical compounds, Lux 54 % Cellulose-1, Cellulose-2, and Cellulose-4 returned the highest number 31 55 % of baseline separations for mobile phases B, D, F, and H. Lux Cellu- 30 lose-1 showed the largest number of baseline separations for five of 26 46 % the eight mobile phases tested. The mobile phases showing the high- Cell-1 Cell-4 Cell-2 Amy-2 Cell-3 est number of baseline separations were D and H. Both of these mobile phases contain a high concentration of organic solvent (20 %) and a combination of acidic (0.1 % TFA) and basic (0.1 % IPA) additives.

Figure 1. Number of baseline separations for mobile phase A through H.

Cell-1 Cell-2 Cell-2 Cell-4 Amy-2 30

0 A B C D E F G H Mobile Phase

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The cumulative baseline separations of the five Lux polysaccha- In Figures 5a and 5b, some typical SFC separations are represented.

ride-based chiral columns using a mobile phase of CO2/(2PrOH with All chiral separations with resolutions greater than 1.5 can be found on 0.1 % IPA and 0.1 % TFA), 80/20, v/v (H) are shown in Figure 3. In our application search web page www.phenomenex. com/Application/ the same way as before, 43 of the 56 pharmaceutical compounds are Search. baseline resolved. In fact, only four CSPs are needed to obtain the maximal success rate. Figure 5a. Chiral separation of Alprenolol by SFC Figure 3. mAU Cumulative baseline separations across five Lux® phases using mobile

phase of CO2/(2PrOH with 0.1% IPA and 0.1% TFA), 80/20 v/v (H) 200 56 100 %

51 91 % 100

46 82 % 77 % 77 % App ID 21009 0 41 71 % 43 43 73 % 0 2 4 min 66 % 40 Column: Lux 5 µm Cellulose-1 36 64 % Dimensions: 250 x 4.6 mm 37 Mobile Phase: CO2 / (Methanol + 0.5 % IPA) 80:20 (v/v) (B) 31 55 % Flow Rate 3 mL/min 50 % Temperature: 30 °C

26 28 46 % Figure 5b. Cell-1 Cell-4 Cell-2 Cell-3 Amy-2 Chiral separation of Mianserine by SFC

By using 7 chromatographic systems, which require three mobile phases mAU (C, D, and F) and four Lux CSPs (Cellulose-1, Cellulose-3, Cellulose-4, and Amylose-2), 55 of the 56 test group compounds are baseline separated 200 (Figure 4).

Figure 4. 100 Cumulative baseline separations across seven chromatographic systems made up of four Lux phases and three mobile phases. 98 % App ID 21234 56 100 % 0 95 % 96 % 55 0 2 4 6 8 min 89 % 53 54 51 91 % Column: Lux 5 µm Cellulose-4 84 % 50 Dimensions: 250 x 4.6 mm Mobile Phase: CO / (Methanol + 0.1 % IPA + 0.1 % 46 82 % 2 47 TFA) 80:20 (v/v) (H) 73 % Flow Rate: 3 mL/min 41 73 % Temperature: 30 °C 41

36 64 % Conclusion The results from this study clearly suggest the complexity of chiral 54 % 31 55 % screening under SFC conditions and the differences which can occur with relatively small changes in mobile phase composition. In particu- 30 26 46 % lar, the influence of additives on the polysaccharide-based chiral sta- Cell-1 Cell-4 Cell-3 Cell-4 Cell-1 Cell-3 Amy-2 tionary phases is yet to be fully understood. For the selected mixture MP D MP D MP F MP C MP C MP C MP C of 56 racemic pharmaceutical compounds, we have demonstrated that screening with a single, well-selected mobile phase and four or five Lux polysaccharide based CSPs can give a high probability of baseline separation.

References 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at 4. Miller L. J. Chromatogr. A 2012, 1250, 250. http://www.phenomenex.com/TermsAndConditions. 5. De Klerck K.; Mangelings D.; Vander Heyden Y. J. of Supercritical Fluids 2013, Trademarks 80, 50-59. Lux is a registered trademark of Phenomenex. Waters is a registered trademark and ChromScope is a trademark of Waters Corporation. Acknowledgements Disclaimer The authors would like to acknowledge K. De Klerck, D. Mange- Phenomenex is not affiliated with Waters. lings and Y. Vander Heyden (Department of Analytical Chemistry and Pharmaceutical Technology of the Vrije Universiteit Brussel (Bel- © 2013 Phenomenex, Inc. All rights reserved. gium)) who allowed us to use data from reference 5. 32 Phenomenex l WEB: www.phenomenex.com Purification Techniques

Purification Techniques page

Purification of Chiral APIs using Axial Compressed Columns [TN-1056]...... 34 SFC and HPLC Chiral Purification on Lux® Axia™ [TN-9001]...... 39 Axia Technology vs. Standard Hardware by HPLC and SFC [TN-9002]...... 43

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Purification of Optically Active Pharmaceutical Compounds using Axial Compressed Columns Peter Rahn and William Cash Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

A major improvement in preparative chiral column performance has How Axia packed columns perform better been achieved by adapting axial compression to manufacture Lux® A computerized mechanical process packs the column bed. The chiral preparative columns. This paper demonstrates the advan- force applied to the column is carefully controlled during the packing tages of combining the Lux media and Axia™ packing technology process to prevent crushing or cracking of the media. The Lux me- to produce high performance stable preparative chiral columns. dia is engineered to be mechanically stronger than previous chiral This process produces preparative columns packed with 5 µm me- media allowing higher packing pressures to be applied (Figure 3). dia with the same efficiency (plates per meter) in prep columns as Once the column bed forms, the media is never allowed to expand found in analytical columns and peak symmetry independent of col- or extrude from the column and the internal packing force is main- umn length and internal diameter. tained on the column during final hardware assembly.

Introduction Creating efficient, more productive chiral purification methods Previous limitations with preparative chiral columns This paper demonstrates the advantages of combining the Historically, it has been a limitation of chiral columns that perfor- new Lux media and Axia packing technology to produce high mance and lifetime decrease as the column’s internal diameter in- performance stable preparative chiral columns. This pro- creases from analytical to preparative dimensions, despite being cess produces preparative columns packed with 5 µm media packed with the same particle size media. Column stability was with the same efficiency (plates per meter) and peak symme- also inherently less for the preparative chiral columns compared try independent of column length and internal diameter (Fig- to analytical columns. This lower initial performance and/or loss ure 4). Axia packed preparative columns are manufactured with of performance is inherent in all slurry packed chiral preparative 5 µm media in 100, 150, and 250 mm lengths with 21.2, 30, and columns, and is caused by: 50 mm internal diameters (Figure 5).

1. Packed bed structure being disturbed after the media is packed Figure 2. Axia Packing Process Integrates Axial Compression Technology into Pre- 2. Media fracture, and or fines, created by packing media in large packed Chiral Preparative Columns diameter columns 3. Packing density not uniform throughout the column 4. Media extrudes from the packed bed during final hardware assembly (Figure 1)

Figure 1. Conventional Slurry Packed Preparative Chiral Column Media Hydraulic Piston Column Bed Piston Retainer End-Fitting High pressure solvent forces After sedimentation, column is disassembled from Added Compresses Bed Formed Added Sleeve Attached sedimentation of the slurry slurry chamber and capped (as quickly as possible) The Axia process uses highly controlled pneumatic mechanical pressure to drive the piston into the column to produce a uniformly packed bed. Once the bed is formed the pressure on the piston and bed is not released, the During disassembly bed is not disturbed and the piston is locked in place leaving the chiral the bed “relaxes” and media under pressure. This packing process won the 2006 R&D 100 Award extrudes from column for its innovation. Figure 3. Controlled Axia Packing Process for Lux Cellulose-2 Prevents Crushing the Inherent in all slurry packed columns Media

SEM of Virgin Media SEM After Axia Packing

With slurry packed columns, the packing hardware must be disassembled before the end fitting is placed on the column. During this procedure the pressure on the media must be released, the packed bed is disturbed and the media begins to extrude from the column creating non-uniform density.

Recent improvements in chiral purification technology A major improvement in preparative chiral column performance has been achieved by adapting axial compression to manufacture Lux preparative columns. For the last six years the Axia packing technology (explained in Figure 2) has been utilized to produce Lux media is mechanically stronger allowing higher packing pressures than high performance stable achiral preparative columns. This same previous chiral media. technology is now employed to produce preparative chiral columns The SEM of virgin media and after Axia packing proves that Axia’s comput- packed with 5 µm chiral stationary phases (CSP). er controlled process does not crush the Lux high porosity media that is engineered to be mechanically strong. 34 Phenomenex l WEB: www.phenomenex.com TN-1056 APPLICATIONS

Figure 4. Experimental Axia™ Packed Lux® Preparative Columns Provide the Same High Perfor- Lux is a media engineered to provide a straightforward approach mance Independent of Column Diameter to enantiomeric recognition and separation by HPLC and Super- critical Fluid Chromatography (SFC). Two Lux phases have been Dimensions: 250 x 21.2 mm N = 77,180 developed using a coated derivatized cellulose material as the a = 1.22 chiral selector (Figure 6). Lux Cellulose-1 features the classical SN 455733-2 tris(3,5-dimethylphenylcarbamate) cellulose derivative used industry-wide for many enantiomer separations. This particular chiral selector has well-established enantiomeric abilities to resolve a wide

Absorbance range of racemates.

Figure 6. Structures of Lux Cellulose-1 and Lux Cellulose-2 Chiral Phases 0 18 min

Dimensions: 250 x 50 mm Lux 5 µm Cellulose-1 N = 75,156 Cellulose tris(3,5-dimethylphenylcarbamate) Me a = 1.19 SN 456633-1 OCONH O Me O Me Me

Absorbance HNOCO OCONH

Me Me

0 18 min Lux 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm Cellulose tris(3-chloro-4-methylphenylcarbamate) Cl N = 70,664 OCONH a = 1.19 Me SN 461603-13 O O Cl Cl

Me HNOCO OCONH Me Absorbance

Lux Cellulose-1 and Lux Cellulose-2 are derivatized phenyl carbamates with different functional groups substituted on the aromatic rings. The substitu- 0 18 min tion of the chlorine molecule with Lux Cellulose-2 provides unique selectivity Conditions for all columns: compared to the traditional Lux Cellulose-1 structure. Column: Lux 5 µm Cellulose-2 Mobile Phase: Hexane:IPA (90:10) Detection: UV @ 220 nm Lux Cellulose-2 incorporates an advanced halogenated derivative, Sample: Trans-Stilbene oxide leading to unique enantioselectivity compared to previously com- mercialized cellulose phases. The unique selectivity of Lux Cellu- Axia technology has the highest process control and produces reproducible, stable, high efficiency columns with the same plates per meter and lose-2 makes it an ideal CSP providing excellent complementary peak asymmetry independent of column length and ID. selectivity to Lux Cellulose-1, and any improvement in the alpha value is extremely important for preparative separations. The two Lux phases are compatible with a wide range of solvent systems Figure 5. including normal phase, polar organic, reversed phase, and SFC. Axia packed Lux Product Family Available in Three Diameters and Three Lengths Methocarbamol represents an important class of compounds 30 mm routinely separated and purified by HPLC with CSP columns. The initial separation was developed on a 100 x 4.6 mm column using MeOH:IPA (90:10) and the response monitored at 220 and 254 nm. Increasing the column diameter from 4.6 to 21.2 mm provides higher throughput (32 mg) for each run without increasing the overall purification time Figure 7( ). By increasing the column length to 250 mm the load can be further increased to 80 mg per run. The methocarbamol separation demonstrates that the separa- 21.2 mm 50 mm tion scales up linearly based on the column length with no loss of resolution. An important factor to consider when performing these higher mass loading separations is the UV detector response is not The Axia packing process is utilized to produce the Lux preparative col- linear making it difficult to determine where the major mass is lo- umns in 100, 150 and 250 mm lengths and in the three diameters 21.2, 30 and 50 mm. cated based solely on the UV signal. Although de-tuning a detector will keep the peaks on scale, the UV signal is not the best indicator for purity and resolution when column overloading occurs. It is cru- cial to first evaluate the purity and yield for the collected fractions to determine the maximum load per run that could be achieved.

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Figure 7. 17780 Figure 8b. ® Direct Scale Up of Methocarbamol Purification on Lux 5 µm Cellulose-1 Comparison of Yield and Purity for Different Loads on Lux® Cellulose-1 1 H N 2 Chiral Column O 2 Dimensions: 100 x 4.6 mm O Mobile Phase: Methanol/Isopropanol (90:10) O Flow Rate: 1 mL/min α = 1.93 Detection: UV @ 220 nm & 254 nm O OH Sample: 5 µg in 2 µL

17781 80 mg Load 0 1 2 min App ID 17780 10 20 min 10 20 min 10 20 min

1 Isomer 1 Mixed Fractions Isomer 2 Yield 97 % @ 100 % pure 0.7 % of Isomer 1 Yield 95 % @ 100 % pure Dimensions: 100 x 21.2 mm 4 % of Isomer 2 99 % @ 99 % pure Mobile Phase: Methanol/Isopropanol (90:10) 99 % @ 99 % pure = 1.84 Flow Rate: 20 mL/min 2 α Detection: UV @ 220 & 254 nm Sample: 32 mg in 640 µL

17782 App ID 17781

0 1 2 min mg180 Load

10 20 min 10 20 min 10 20 min 1 Isomer 1 Mixed Fractions Isomer 2 Yield 96 % @ 100 % pure 2.5 % of Isomer 1 Yield 81 % @ 100 % pure Dimensions: 250 x 21.2 mm 97.6 % @ 99 % pure 16.2 % of Isomer 2 84 % @ 99 % pure Mobile Phase: Methanol/Isopropanol (90:10) α = 1.80 Flow Rate: 20 mL/min 2 Detection: UV @ 220 & 254 nm Sample: 80 mg in 1600 µL App ID 17782

0 1 2 3 4 5 6 7 min 240 mg Load Separation scales up directly based on column length. With the 100 mm 10 20 min min 10 20 min length column a 32 mg/load separation was achieved and a higher sample Isomer 1 Mixed Fractions Isomer 2 load required the longer 250 mm length column. As expected when increas- Yield 89 % @ 100 % pure 10 % of Isomer 1 Yield 20 % @ 99 % pure ing the load, the peak width and tailing increased but there was no loss 95 % @ 99 % pure 55 % of Isomer 2 32 % @ 98 % pure of resolution. For the higher sample loads, the detector sensitivity was de- Each fraction was individually evaluated and then pooled providing three creased by monitoring at 254 nm. fractions that were then evaluated for the overall purity and yield. At 80 mg load both enantiomer 1 and 2 were obtained in high yield and purity. When To further improve sample throughput and productivity, the sample 180 mg load was purified, the yield for pure enantiomer 1 was not signifi- load on the column was increased from 80 mg to 180 mg and then cantly affected but enantiomer 2’s yield decreased to 81 %. With the 240 mg finally to 240 mg (Figure 8a). The UV signal was used to determine load, the column overload was too great and the capability to collect pure the starting and ending collection points, but time based fractions enantiomer 2 was lost. Even if a 2 % enantiomer impurity was acceptable were collected across the peaks including the valley area. After the only 32 % of the load was recovered at this purity level. fractions were collected, each fraction was first analyzed using a The dramatic effect increasing sample load has on throughput, pu- 100 x 4.6 mm Lux Cellulose-1 column on an analytical HPLC and rity, and productivity is illustrated in Figure 9. then the fractions were pooled together and the overall yield and purity were assessed (Figure 8b). Figure 9. Effect on Purity and Yield When Increasing Methocarbamol Load to Figure 8a. Improve Throughput Resolution Change with Increased Load for Chiral Separations 120

80 mg Load 80

1 2 3 4 5 6 7 min 4 5 min

Dimensions: 250 x 21.2 mm 180 mg

Flow Rate: 20 mL/min mg Pure Load Detection: UV @ 220 and 254 nm 40 Sample: Load as shown 1 2 3 4 5 6 7 min 4 5 min

0 240 mg 80 100 120 140 160 180 200 220 240 Load mg Load

Theoretical Yield 100 % Pure Enanatiomer 1 100 % Pure Enantiomer 2

1 2 3 4 5 6 7 min 4 5 min 99 % Pure Enantiomer 1 99 % Pure Enantiomer 2 Fractions were collected across the peak and evaluated for purity at 220 nm The quantity of 100 % pure enantiomer 1 collected increases with larger sam- using a 100 x 4.6 mm Lux Cellulose-1 column. The purity for each pooled ple loads with a slight sacrifice in yield. The later eluting enantiomer’s purity fraction was determined. The 254 nm detector trace for the preparative sep- and yield dramatically drops when the load is above 180 mg on the 250 x aration shows the presence of each material but cannot be used to predict 21.2 mm column. A larger diameter column with the same load would provide

the purity of the fraction. increased throughput for the second enantiomer without sacrificing purity. 36 Phenomenex l WEB: www.phenomenex.com TN-1056 APPLICATIONS

Enantiomer 1 – Even when 240 mg was loaded onto the column, Conclusions the purity of the first enantiomer was not greatly affected and Axia technology is the industry standard for consistency and robust- 107 mg (89 % of this enantiomer) was determined to be 100 % pure. ness in preparative columns with the same performance achieved Increasing the mass loading per run is a tremendous advantage from 4.6 mm analytical columns to 50 mm ID preparative columns. when larger quantities of this enantiomer are required. If the enan- Over the last several years the Axia technology with its high level tiomer purity requirement was 99 %, then additional fractions could of process control has been proven to produce columns with the be pooled and a total of 114 mg (95 %) of the first enantiomer mass same performance (plates per meter) independent of length and was collected. diameter. The 5 µm Lux preparative columns are available in 100, 150, and 250 mm lengths with 21.2, 30, and 50 mm diameters. Enantiomer 2 – When 180 mg was loaded onto the column, 73 mg There has been a significant improvement in the asymmetry and ef- (81 %) of the enantiomer was obtained at 100 % purity level which ficiency across all lengths and IDs for the Axia packed preparative is only a slight change from enantiomer 1 results where 86 mg was columns allowing chemists more flexibility to achieve their goals for collected for the same purification. Whereas when the mass loading increased purity and yield for their preparative purifications. was increased to 240 mg the purity for enantiomer 2 dramatically decreased. In fact, at the 240 mg load, the highest purity achieved Since these preparative Lux columns packed with 5 µm have the was 99 % for enantiomer 2, but only 24 mg was recovered repre- same plates/meter (efficiency and asymmetry factors) independent senting only about 20 % of the initial load. With the 240 mg load, the of ID and length, the chemist has more options to quickly scale first enantiomer contaminates the second enantiomer and 0 % was up a separation to obtain higher quantities of purified enantiomer obtained with a 100 % purity level. The extent of the peak overlap without sacrificing purity or yield. Many times the shorter column is very evident in the preparative chromatograms in Figure 8a. Al- provides sufficient resolution for the required compounds resulting though the UV trace indicates there is still resolution between the in faster turn-around times and higher productivity. two compounds the amount of overlap between enantiomer 1 and enantiomer 2 is very significant. If the desired enantiomer is the Selectivity is still the most critical factor for chiral separations. later eluting compound on the chiral column, the sample load and Screening and having a choice of multiple chiral phases is import- throughput must be carefully monitored by evaluating the fractions ant to increase resolution prior to performing the preparative sep- to ensure an acceptable enantiomer purity is achieved. arations. This work has shown that sample load scales up directly based on column length, but the separation time increases as the column length increases. Whereas, sample load increases exponentially with column diameter without increasing the separation time. The judicious choice of overall column length and column diameter of the Axia packed Lux columns will have a major affect on a laboratory’s overall throughput.

Phenomenex l WEB: www.phenomenex.com 37 Innovative Preparative Chromatography Hardware

The packing piston head is integrated into the column and locked by the piston retainers, so the pressure is never released. Packing Piston

Media is never allowed Media packed under to relax, eliminating ideal pressure voids and dramatically improving reproducibility, column to column.

Long Column Lifetime. Guaranteed.

38 Phenomenex l WEB: www.phenomenex.com TN-9001 APPLICATIONS

Direct Comparison of HPLC and SFC for the Milligram to Gram Scale Purification of Enantiomers Philip J. Koerner1, Peter C. Rahn1, Gary W. Yanik2, and Irene Tranquil2 1 Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA 2 PDR-Chiral Inc. 1331A South Killian Drive, Lake Park, Florida 33403 USA

High efficiency Axia™ preparative columns packed with Lux® polysac- Background charide-based chiral stationary phases are utilized to compare chiral In the past, the same particle size chiral media packed in 4.6 mm SFC and HPLC purification of several pharmaceutical compounds. ID columns was packed into preparative columns but the column performance and lifetime decreased as the column internal diame- Introduction ter increased. Column stability was also inherently less for the pre- High-throughput analysis and purification of enantiomers are im- parative chiral columns compared to analytical columns. The lower portant in drug discovery. With today’s regulations to improve initial performance and/or loss of performance are inherent in all safety and efficacy of drugs, the pharmaceutical industry needs slurry packed chiral preparative columns and caused by: to provide high quality pure enantiomers for pharmacological testing. Historically, chiral purification has been achieved with normal 1. Packed bed structure being disturbed after the media is phase HPLC, and more recently, with reversed phase separations. packed In recent years, SFC has gained acceptance as a very effective 2. Media fracture, and or fines, created by packing media in large complementary tool for other chiral separation modes to produce diameter columns pure enantiomeric compounds. 3. Non-uniform packing density throughout the column This technical note compares the throughput (grams per hour) for 4. Media extrusion from the packed bed during final hardware the same chiral preparative separations performed under HPLC assembly (Figure 2). conditions and SFC conditions. In this work, three different polysaccharide-based chiral stationary phases (CSPs) are utilized (Figure 1): Lux Cellulose-1, Lux Cellulose-2, and Lux Amylose-2. Figure 2. The same 5 μm media was used to produce both the analytical col- Conventional Slurry Packed Preparative Chiral Column umns and the Axia packed preparative columns. This work also demonstrates the capability to directly scale analytical chiral sep- High pressure solvent forces sedimentation of the slurry arations to preparative chiral separations when both column sizes are packed with 5 μm media. After sedimentation, column is disassembled from slurry chamber and capped (as quickly as possible). Figure 1. Structures of Polysaccharide-based Chiral Phases

Lux® Cellulose-2 Lux® Amylose-2 Cellulose tris Amylose tris (3-chloro-4-methylphenylcarbamate) (5-chloro-2-methylphenylcarbamate) During disassembly, the bed “relaxes” and extrudes from column. Cl Cl CH OCONH 3 OCONH This problem is inherent in all slurry CH O 3 Cl O packed columns Cl CH Cl 3 O Cl A major improvement in preparative chiral column performance HNOCO CH O OCONH 3 HNOCO OCONH has been achieved by adapting Axia packing technology to CH 3 manufactured Lux chiral preparative columns. Axia packing CH3 technology (Figure 3) has been utilized to produce stable, high Lux® Cellulose-1 performance achiral preparative columns. This same technology Cellulose tris is now employed to produce preparative chiral columns packed (3,5-dimethylphenylcarbamate) with 5 μm chiral stationary phases. The Lux media is engineered CH 3 to be mechanically stronger than previous chiral media, allowing OCONH higher packing pressures to be applied. A computerized

O CH3 O mechanical process packs the column bed. The force applied CH CH 3 3 to the column is carefully controlled during the packing process HNOCO OCONH to prevent crushing or cracking of the media. Once the column CH CH 3 3 bed forms, the media is never allowed to expand or extrude from the column and the internal packing force is maintained on the column during final hardware assembly. The advantages of combining the new Lux media and Axia packing technology to produce high performance stable preparative chiral columns are

Phenomenex l WEB: www.phenomenex.com 39 TN-9001 APPLICATIONS illustrated in this technical note. The Axia packing technology pro- Experimental Conditions ® duces preparative columns packed with 5 μm media having the Analytical HPLC separations were developed using an Agilent same efficiency and peak symmetry as analytical columns Fig( - 1100 system with diode array detector (Agilent, Palo Alto, CA). ™ ure 4). Axia packed preparative columns are manufactured with The Gilson 845ZPREP HPLC system (Gilson, Middleton, WI) Lux 5 μm chiral media in 100, 150 and 250 mm lengths with 21.2, was used for the preparative HPLC separations and fraction 30 and 50 mm internal diameters. collection. For SFC separations, a Berger preparative SFC system was utilized consisting of the pumping system, variable Figure 3. UV and PDR-Chiral detectors, and a 6-port fraction collector Axia™ Packing Process Integrates Axial Compression Technology into Pre- capable of collecting hundreds of milliliters of eluent. The packed Chiral Preparative Columns advanced laser polarimeter (ALP) detector (PDR-Chiral, Lake Park, FL) measures the rotation of plane-polarized 660 nm laser beam passing through the flow cell and indicates the optical rotation of each enantiomer. Using a mobile phase consisting of 25 % polar modifier (methanol or ethanol) added to the carbon

dioxide (CO2), a flow rate of 50 mL/min through the 21.2 mm diameter columns was easily achieved without exceeding the 200 bar pressure limits of the SFC instrument.

Results and Discussion Media Added Hydraulic Piston Column Bed Piston Retainer End Fitting Sleeve Compresses Bed Formed Added Attached Figures 5A – 5D contain the SFC and HPLC chromatograms, purification conditions, and results for each sample. Figure 5A com- Figure 4. pares the Atenolol separation for SFC and HPLC. The SFC condi- Axia™ Packed Lux® Preparative Columns Provide the Same High Perfor- tions required 25 % methanol while the HPLC conditions yielded mance18283 Independent of Column Diameter the best separation with 20 % ethanol. The PDR-Chiral detector (ALP) indicates the optical rotation of each enantiomer. The SFC 450 Dimensions: 250 x 4.6 mm Conditions same for all separations: Flow Rate: 0.5 mL/min Column: Lux 5 µm Cellulose-2 cycle time was 4 minutes and the HPLC cycle time was 6 minutes. 400 N = 70,664 1 Mobile Phase: Hexane / IPA (90:10) In addition to a faster cycle time the total load on the column was 350 s = 1.19 Temperature: Ambient SN 461603-13 also 1.8 times higher for SFC (102 mg for SFC vs. 60). 300 Detection: UV @ 220 nm Sample: Trans-Stillbene oxide 250 2 Figure 5B compares the Terfenedine HPLC and SFC separations. 200 The Lux Cellulose-1 column using 25 % methanol as the polar 150 modifier provided the best SFC separation. The Lux Cellulose-1

Absorbance 100 column with the polar organic mobile phase of 3 % isopropanol and 50 App ID 18283 97 % acetonitrile provided the best HPLC separation. SFC sample 0 load per cycle was 105 mg compared to 12 mg/cycle for the HPLC runs in the same 7 minute cycle time for SFC and for HPLC. This 0 2 4 6 8 10 12 14 16 18 20 22 24 min results in a significantly higher throughput for SFC (840 mg/hour) 18284 compared with HPLC (102 mg/hour) and a significantly smaller vol- Dimensions: 250 x 21.2 mm ume collected per gram of product. The overall purity and recovery Flow Rate: 20 mL/min 480 for HPLC and SFC were the same. N = 77,180 1 420 s = 1.22

320 SN 455733-2 Figure 5C compares the two Propranolol chiral separations. For

300 SFC, the Lux Cellulose-1 column with 25 % methanol polar mod-

240 ifier provided the best separation with a sample load of 120 mg 2

180 per cycle. The HPLC conditions also utilized the Lux Cellulose-1 column with 20 % isopropanol as the polar solvent (hexane as the 120 Absorbance non-polar solvent) and a sample load of 60 mg per cycle. Since the 60 App ID 18284 load per cycle was higher for SFC, the throughput for SFC was 0 450 mg/hour compared to 553 mg/hour for HPLC and the total vol- 0 2 4 6 8 10 12 14 16 18 min ume collected for 1 gram of product was 508 mL for SFC compared

18285 to 799 mL for HPLC.

Dimensions: 250 x 50 mm The Propafenone separations using Lux Amylose-2 are shown in Flow Rate: 50 mL/min 280 Figure 5D. Lux Cellulose-1 and Lux Cellulose-2 could not resolve N = 75,156 1 these enantiomers in HPLC or SFC but a 50 mm long Lux Amy- 240 s = 1.19 SN 456633-1 lose-2 provided the best resolution possible although the resolution 200 is on the low end for a preparative purification. The SFC conditions 180 2 utilized 20 % ethanol as the polar solvent with a load of 15 mg per 120 7 minute cycle. Under HPLC conditions, 50 % isopropanol was re-

80 Absorbance quired to achieve the separation on the Lux Amylose-2 column with

40 a load of 18 mg per 4 minute cycle. The throughput is limited by App ID 18285 the resolution between the two compounds, which is minimal for a 0 preparative separation. A longer column could be used to improve 0 2 4 6 8 10 12 14 16 18 20 22 24 min resolution, but the overall throughput would remain the same. A larger diameter column is required for higher throughput.

40 Phenomenex l WEB: www.phenomenex.com TN-9001 APPLICATIONS Figure 5A. Figure 6 graphically compares the SFC and HPLC results to ob- Purification of Atenolol tain 1 gram of purified product. The total volume collected was al- ways less for SFC, in some cases as much as one-half the volume collected for HPLC. However, since significant amounts of polar solvent modifiers were required for SFC, the difference in volume was not as dramatic as expected. However, the lower volume of solvent collected for SFC would result in less time to remove the App ID 19266 App ID 19269 solvent and recover the desired product. With the exception of the propafenone separation, the SFC yields (mg/hour) were significant- Column: Lux 5 µm Cellulose-1 Column: Lux 5 µm Cellulose-1 ly greater than the HPLC yields. Dimensions: 250 x 21.2 mm Dimensions: 250 x 21.2 mm Part No.: 00G-4459-P0-AX Part No.: 00G-4459-P0-AX Figure 6. Mobile Phase: 0.1 % Diethylamine in Methanol / Mobile Phase: 0.1 % Diethylamine in Ethanol / 0.1 % Diethylamine in Carbon 0.1 % Diethylamine in Comparison of Yield and Total Collected Volume Dioxide (25:75) Hexane (20:80) Throughput (mg/hour) Flow Rate: 50 mL/min Flow Rate: 50 mL/min Collected Volume Per Gram

1 6 0 0 Temperature: 37 ˚C Temperature: 20 ˚C 3 5 0 0

Detection: UV @ 254 nm Detection: UV @ 254 nm 1 4 0 0 Injection Volume: 5.1 mL Injection Volume: 2 mL 3 0 0 0 1 2 0 0 Injection Injection 2 5 0 0 1 0 0 0 Concentration: 20 mg/mL in Ethanol Concentration: 30 mg/mL in Methanol m m 2 0 0 0 8 0 0 S F C S F C g L H P L C 1 5 0 0 H P L C Figure 5B. 6 0 0 1 0 0 0 4 0 0 Purification of Terfenedine 2 0 0 5 0 0

SFC SFC HPL C HPL C 0 0 4 4 A t e n o lo l T e r fe n e d in e P r o p r a n o lo l P r o p a f e n o n e A t e n o lo l T e r f e n e d in e P r o p r a n o lo l P r o p af e n o n e UV UV 3.5 3.5 UV UV 3 3 2.5 2.5 2 2 mV Conclusions mV 1.5 1.5 1 1 Axia preparative columns packed with 5 μm Lux polysaccha- 0.5 0.5 0 ride-based media are compatible with SFC and HPLC conditions 0 -0.5 App ID 19273 -0.5 0 2 4 6 8 10 12 14 16 18 20 22 24 and gave the same high performance as the analytical columns,

0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 App ID 19272 Minutes Mi n ut e 1 Minutes 0 2 Mi n ut e allowing for scale up under both SFC and HPLC separation condi- 1 Column: Lux 5 µm Cellulose-1 Column: Lux 5 µm Cellulose 2 tions. Larger diameter Axia packed preparative columns are avail- Dimensions: 250 x 21.2 mm Dimensions: 250 x 21.2 mm able if higher flow rate SFC systems are utilized. Part No.: 00G-4457-P0-AX Part No.: 00G-4457-P0-AX Mobile Phase: 0.1 % Diethylamine in Methanol / Mobile Phase: 0.1 % Diethylamine in Isopropanol / Sample solubility is a major problem for both SFC and HPLC. The 0.1 % Diethylamine in Carbon 0.1 % Diethylamine Acetonitrile (3:97) Dioxide (25:75) Flow Rate: 20 mL/min use of polar organic solvents such as methanol, ethanol and iso- Flow Rate: 50 mL/min (135 bar) Temperature: 20 ˚C propanol to improve solubility can have an adverse effect on peak Temperature: 37 ˚C Detection: UV @ 220 nm Detection: UV @ 220 nm shape, retention time, and resolution. If the polar solvent in the sample is too high, the strong polar solvent will cause sample break- Figure 5C. through and reduce resolution. For these examples the column ca- Purification of Propranolol pacity was higher than the actual load achieved because the polar solvent limited the total volume that could be injected. The use of polar solvents also directly impacts the column operating pressure and limits the overall flow rate on the SFC system. The polar solvents also create higher backpressure on the HPLC systems but generally these systems have higher pressure limits. App ID 19267 App ID 19271 Advantages of SFC relative to HPLC are dependent on the analyte,

Column: Lux 5 µm Cellulose-1 Column: Lux 5 µm Cellulose-1 the CSP, and the amount of polar solvent required. Generally, higher Dimensions: 250 x 21.2 mm Dimensions: 250 x 21.2 mm load and higher flow rates were achieved with SFC but the volume Part No.: 00G-4459-P0-AX Part No.: 00G-4459-P0-AX collected per gram of product was not as different as other achiral Mobile Phase: 0.1 % Diethylamine in Methanol / Mobile Phase: 0.1 % Diethylamine in Isopropanol / 0.1 % Diethylamine in Carbon 0.1 % Diethylamine in Hexane (20:80) separations previously reported. This is a result of the higher per- Dioxide (25:75) Flow Rate: 20 mL/min (135 bar) centage of polar mobile phase solvent used in the SFC separations. Flow Rate: 50 mL/min (135 bar) Temperature: 20 ˚C Temperature: 37 ˚C Detection: UV @ 254 nm Detection: UV @ 254 nm

Figure 5D. Purification of Propafenone

Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Gilson 845ZPREP is a trademark of Gilson, Inc. Agilent is a registered trademark of Agilent Technol-

App ID 19268 App ID 19270 ogies, Inc. Disclaimer Column: Lux 5 µm Amylose-2 Column: Lux 5 µm Amylose-2 The research presented in this technical note was a joint effort between Phenomenex, Inc. Dimensions: 250 x 21.2 mm Dimensions: 250 x 21.2 mm and PDR-Chiral Inc. Comparative separations may not be representative of all applications. Part No.: 00G-4472-P0-AX Part No.: 00G-4472-P0-AX Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 Mobile Phase: 0.1 % Diethylamine in Ethanol / Mobile Phase: 0.1 % Diethylamine in Isopropanol / 0.1 % Diethylamine in Carbon Dioxide 0.1 % Diethylamine in Hexane (50:50) Copying or re-use of this information is not allowed without written permission from (20:80) Flow Rate: 20 mL/min Phenomenex. Flow Rate: 20 mL/min Temperature: 20 ˚C © 2010 Phenomenex, Inc. All rights reserved. Temperature: 37 ˚C Detection: UV @ 220 nm Detection: UV @ 254 nm Phenomenex l WEB: www.phenomenex.com 41

ChiralFREE Screening Services

Polysaccharide Chiral Columns Dependable. Scalable. A ordable. Column Screening for Optimal Chiral Resolution 19146 α = 1.45 Use different chiral column selectivities to develop more efficient methods.19147 α = 1.06

1 1 Lux 5 µm Amylose-2 2 Lux 5 µm Cellulose-1 a = 1.45 a = 1.06 2 19151 App ID 19146

α = 1.35 App ID 19147 19148 α = 1.48

0 2 4 6 8 10 12 14 16 18 min 0 2 4 6 8 10 12 14 16 18 min

1 1 Lux 5 µm Cellulose-2 Lux 5 µm Cellulose-4 a = 1.48 2 a = 1.35 2 App ID 19148 App ID 19151

0 2 4 6 8 10 12 14 16 18 min 0 2 4 6 8 10 12 14 16 18 min

Optimal Separation Etozolin 1 O

N O N Lux 5 µm Cellulose-3 a = 1.70 S O

2 Conditions for all columns: Column: As noted Dimension: 250 x 4.6 mm Mobile Phase: Acetonitrile / 20 mM Ammonium bicarbonate with 0.1 % App ID 19152 Diethylamine (60:40) Flow Rate: 1 mL/min Temperature: Ambient Detection: UV @ 220 nm 0 2 4 6 8 10 12 14 16 18 min

Based on a five phase screen under reversed phase conditions, the optimal chiral stationary phase for resolving Etozolin is Lux Cellulose-3.

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Scaling from Analytical to Preparative Chiral Chromatography While Balancing Purity, Yield, and Throughput under HPLC and SFC Conditions J Preston, J.T. Presley III, Michael McCoy, Michael Klein, Marc Jacob et al. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Axia™ preparative column technology along with Axia specilized in the last several years. In contrast to traditional liquid chroma- hardware shows higher performance than traditionally packed tography, the SFC mobile phase consists of a mixture of liquid standard hardware preparative columns. The Axia packing tech- carbon dioxide and organic solvent, such as Methanol. The prin- nology is compatible with both SFC and HPLC conditions. In this ciple advantages of SFC over conventional HPLC techniques are application, we will demonstrate how the Axia packed columns increased speed, reduced waste generation and for preparative with the Lux® Cellulose-1 polysaccharide-based chiral stationary purifications, minimized post-chromatography sample manipu- phase can be a tool to increase throughput for purification of chi- lation. For chiral separations in particular, SFC is increasing in ral compounds. popularity because it is often very simple to convert an existing normal phase HPLC method into an SFC method. The use of pre- Introduction parative chiral chromatography has increased significantly over HPLC has been extensively studied since the late 1960’s and there the past 5-10 years, and SFC has been a significant driver for this have been numerous theoretical models developed to describe, increase. explain, and predict the results of chromatographic experiments. The typical goal of chromatography is to separate compounds It is well known that chromatography can be directly scaled from from each other, and the most straight forward way to evaluate very small columns to very large columns when the eluent compo- a separation is to calculate the resolution between two peaks of sition remains consistent. The work presented in this application interest. Resolution of two peaks will be a function of numerous will address the relationship between both normal phase and SFC factors, including mobile phase composition, stationary phase se- chiral methodologies at the analytical and preparative scale. The lectivity, and running conditions. In practical terms, the resolution impact on resolution at both scales due to flow rates will be eval- is predicted by how far apart the two peaks are separated in time uated and compared between SFC and normal phase. The effect and how broad the peaks are shaped. Thus, optimal resolution is of preparative column hardware technology along with resulting provided by obtaining narrower peaks, as this allows them to be purity and throughput from related SFC and normal phase purifi- more easily resolved from one another in any given time frame. cation methodologies will also be evaluated. One particular method of chromatography known as supercritical fluid chromatography (SFC) has become increasingly popular

Lux Cellulose-1 Chiral Stationary Phase Cellulose tris (3,5-dimethylphenylcarbamate) Me

OCONH

[ O Me O Me Me [ n HNOCO OCONH

Me Me

Cellulose backbone

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Material and Methods Analytical HPLC separations were developed using an Agilent® attributed to the packing quality with these larger I.D. columns. 1100 system with diode array detector (Agilent, Palo Alto, CA). The reasons for this change in performance are complex but in- SFC analytical was performed on a Waters® ACQUITY® UPC2® clude non-uniform packing density throughout the column, the system (Waters, Milford, MA USA) consisting of a convergence bed structure being disturbed after the media is packed, media manager, sample manager, binary solvent manager, PDA detector, fracture and/or fines created during the packing process, and column manager with 6 positions, and a Waters 3100 mass spec- media extrusion from the packed bed during final hardware as- trometer. Data analysis was performed using MassLynx® software sembly. (Version 4.1). The standard hardware column packing process is complicated Normal phase preparative scale separations were performed on and there are many opportunities for a loss in column perfor- a Shimadzu® LC20 Prep HPLC system, with an LC-10 autosam- mance. To address this issue, Phenomenex developed a unique pler and fraction collector. SFC purifications were performed on column packing technology and hardware, AXIA™, to maintain a Berger Automated PrepSFC™ system (Mettler-Toledo, USA) analytical-like column performance in preparative column di- consisting of a Bohdan automated injection/collection robot, mensions. The Axia technology, patented by Phenomenex, is an Berger SCM-250 (separator control module), Berger ECM-2500 advanced column packing and column hardware design that in- (electronic control module), KNAUER K-2500 UV variable detec- corporates Hydraulic Piston Compression technology that mim- ® ® tor, Varian SD-1 methanol and CO2 delivery systems, JULABO ics axial compression columns. This results in Axia preparative chiller, and SFC ProNTo™ control software (Version 1.5.305.15) columns outperforming column packed using traditional packing with SFC Automation Controller add-on (Version 1.5.92.3). methods.

Compounds were evaluated using a Phenomenex Lux® 5 µm Cel- Axia packing technology uses a computerized mechanical pro- lulose-1 column, dimensions are as noted in each Figure. HPLC cess to pack the column bed (Figure 1). The force applied to the conditions and injection amounts are as noted in each Figure. column is carefully controlled during the packing process to pre- Warfarin test solutions were prepared at 20 mg/mL in ethanol and vent crushing or cracking of the media. Once the column bed used for all testing. forms, the media is never allowed to expand or extrude from the column and the internal packing force is maintained on the col- Results and Discussion umn packing during final hardware assembly and into the final When scaling up from analytical sized columns (4.6 mm I.D.) to product. traditionally packed larger I.D. columns (>10 mm I.D.), there has historically been some loss in efficiency and performance that is

Figure 1. Axia Patented Packing Technology

Packing piston locked. No Media maintains initial optimal media or pressure escape packing density and shape

Silica media packed under piston pressure Column

Specialized computer control for ideal pressure

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Previous work by Jan Priess et. al. demonstrated increased col- The 150 x 21.2 mm traditionally packed standard hardware pre- umn efficiency and resolution for polysaccharide-based chiral parative column and Axia packed preparative column were first stationary phase (CSP) media packed using Axia™ columns.1 To evaluated by generating Van Deemter curves for trans-Stilbene better understand how much this hardware technology improves Oxide (TSO) to find out if there was any difference in column effi- column performance we packed the same 5 µm Lux® Cellulose-1 ciency versus linear velocity. The normal phase data indicated the chiral media into two different 150 x 21.2 mm I.D. columns. The Axia packing technology had a substantial 91.6 % increase in col- Lux media is engineered to be mechanically stronger than previ- umn efficiency over traditionally packed columns at a 0.1 cm/sec ous chiral media, allowing higher packing pressures to be applied; linear flow as depicted inFigure 2 . The difference in performance thus increasing the column plate count and column performance. was less pronounced in SFC, but still showed a 26.8 % increase One column was packed using a traditional HPLC column pack- in efficiency for the Axia packed column at 0.4 cm/sec Figure 3( ). ing process with standard hardware and the other column was As expected, the decrease in column efficiency as linear velocity packed using Axia technology with Axia hardware. The QC data increased was less under SFC conditions. for the Axia column showed 73,000 plates per meter, which was a > 22 % increase over the standard hardware column.

Figure 2. Van Deemter Plots - Normal Phase Mode Increase Performance in Axia > 90 % 0.040 Standard 0.035 Increase performance Hardware in Axia > 90 % 0.030

0.025

0.020 Axia Technology

0.015 H = L/N (mm) 0.010

0.005

0.000 0.05 0.1 0.15 0.2 0.25 0.3 Linear Velocity (cm/sec)

Figure 3. Van Deemter Plots - SFC Mode Increase Performance in Axia > 25 % 0.075 Increase performance in Axia > 25 %

0.07 Standard Hardware 0.065

0.06 H = L/N (mm)

0.055 Axia Technology

0.05 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Linear Velocity (cm/sec)

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To understand what advantage this would provide for a twenty fold for both of the 150 x 21.2 mm preparative columns. high-throughput purification laboratory, we performed a scale up The resolution and efficiency for the second peak were measured. experiment using Warfarin. Analytical separations were first de- Again, the preparative column packed using Axia™ technology veloped in normal phase on a 150 x 4.6 mm column and loading showed roughly a 30 % increase in resolution and 42 % increase was increased until a reasonable loading capacity was achieved. in efficiency over the traditionally packed standard hardware col- The injection volume was then directly scaled up (geometrically) umn. (Figure 4).

Figure 4. Standard Hardware Warfarin Purification in Normal Phase Mode

Column Analytical Standard Axia

(mm) 150 x 4.6 150 x 21.2 150 x 21.2 mV 2750

Mass Loaded (mg) 2 40 40 2500 Resolution* 1.5 2.85 3.72 2250 2000 Rs = 2.85

Plates (N) 117 535 760 1750

1500

* Resolution calculated with peak width at baseline and center retention time due to the 1250 overloaded peaks being off-scale 1000

750

500

Analytical 250

0 App ID 21920

0 1 2 3 4 5 6 7 min

mAU

4000

Axia Technology 3000 30 % Increase in Resolution 2000 mV 2750 1000 2500 Rs = 3.72 2250

0 2000

1750 App ID 21922

-1000 1500 0 1 2 3 4 5 6 7 min 1250

1000 Column: Lux® 5 µm Cellulose-1 750 Dimensions: 150 x 4.6 mm 500 Mobile Phase: Hexane/Ethanol (75:25) 250

Flow Rate: 1 mL/ min 0 App ID 21921 Temperature: Ambient min Inj. Volume: 100 µL 0 1 2 3 4 5 6 7 8

Conditions for both columns: Column: Lux 5 µm Cellulose-1 Dimensions: 150 x 21.2 mm Mobile Phase: Hexane / Ethanol (75:25) Flow Rate: 20 mL/ min Temperature: Ambient Inj. Volume: 2 mL

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The separation was then adapted to SFC to provide a reduced in efficiency for the second peak Figure 5( ) when compared with run time, while maintaining suitable resolution. Due to the SFC the traditionally packed standard hardware column. systems injection volume limitation, direct loading comparisons were not possible. We did attempt to make a more concentrat- Fractions were collected for both normal phase and SFC runs and ed solution of Warfarin, but reached a saturation point. However, yielded similar masses collected with similar purity profiles. This when comparing loads of 36 mg on-column, the Axia™ columns was to be expected since the peaks were still well resolved at again showed a 25 % increase in resolution and a 14 % increase this load.

Figure 5. Standard Hardware Warfarin Purification in SFC Mode

Column Analytical Standard Axia 2200 Rs = 1.87 (mm) 150 x 4.6 150 x 21.2 150 x 21.2 2000 Mass Loaded (mg) 1.5 36 36 1800 Resolution* 1.39 1.87 2.33 1600 1400 Plates (N) 206 441 503 1200 1000 * Resolution calculated with peak width at baseline and center retention time due to the mAU overloaded peaks being off-scale 800 600 Analytical 400 200 App ID 21919

4.0e+2 0 3.8e+2 -200 3.6e+2 0 1 2 min 3.4e+2 3.2e+2 25 % Increase in 3.0e+2 Axia Technology Resolution 2.8e+2 2.6e+2 2.4e+2 2200 2.2e+2 Rs = 2.33

A U 2.0e+2 2000 1.8e+2 1800 1.6e+2 1600 1.4e+2 1.2e+2 1400 1.0e+2 1200 8.0e+1

mAU 1000 6.0e+1 800 4.0e+1 2.0e+1 600 App ID 21917 0.0 400 0 1 2 min 200 App ID 21918 0 Column: Lux® 5 µm Cellulose-1 -200 Dimensions: 150 x 4.6 mm 0 1 2 min CO /Methanol (65:35) Mobile Phase: 2 Conditions for both columns: Flow Rate: 3.5 mL/min Column: Lux 5 µm Cellulose-1 55 °C Temperature: Dimensions: 150 x 21.2 mm Inj. Volume: 75 µL Mobile Phase: CO2/Methanol (65:35) Flow Rate: 70 mL/min Temperature: 55 ˚C Inj. Volume: 1.8 mL

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Conclusion Axia™ preparative columns packed with 5 μm Lux® polysaccha- References ride-based media gives higher performance than traditionally 1. Evaluation of Chiral Stationary Phase Packed Axia HPLC Column, J. Priess, packed standard hardware columns. The Axia technology is com- C. Valente, G. Diehl and E. Francotte, Novartis Institutes for Biomedical Re- patible with both SFC and HPLC separation conditions and can search, Basel, Switzerland, Poster 2137, SPICA 2008 be a tool to increase throughput for purification. Acknowledgements The authors would like to acknowledge Bill Ferrell at Pfizer, La Jolla, CA for his contribution to this work.

CH CI CH CH 3 O O 3 O 3 CH R = 3 R = R = O CH N CI N R = N 3 R = H H H N CH3 H O CI

Cellulose-O-R Cellulose-O-R Cellulose-O-R Cellulose-O-R Amylose-O-R Lux Cellulose-1 Lux Cellulose-2 Lux Cellulose-3 Lux Cellulose-4 Lux Amylose-2 Cellulose tris Cellulose tris Cellulose tris Cellulose tris Amylose tris (3.5-dimethylphenylcarbamate) (3-chloro-4-methylphenylcarbamate) (4-methylbenzoate) (4-chloro-3-methylphenylcarbamate) (5-chloro-2-methylphenylcarbamate)

Free Chiral Screening Services, provided by PhenoLogix www.phenomenex.com/PhenoLogix

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Phenomenex and Lux are registered trademarks, and Axia is a trademark of Phenomenex. Waters ACQUITY, UPC2 and MassLynx are registered trademarks of Waters Corp. SFC ProNTo™ is a trademark of Waters Corp. Agilent and Varian are registered trademarks of Agilent Technologies, Inc. Berger Automated PrepSFC™ is a trademark of Mettler-Toledo Co. JULABO is a registered trademark of Julabo. Shimadzu is a registered trademark of Shimadzu Corporation. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 © 2013 Phenomenex, Inc. All rights reserved. 48 Phenomenex l WEB: www.phenomenex.com Why Aren’t You Using Phenex Syringe Filters?

Phenex™ Yours Excellent Performance 1 Effective particulate removal, with low hold-up volumes High Quality 2 100 % integrity and performance tested Fast Flow 3 High total throughput with less pressure Certified Quality 4 HPLC and GC Approved

Phenex is a trademark of Phenomenex, Inc.

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Separation of Generic PPIs in RP Mode [TN-1102]...... 51 Beta Blockers in NP, RP, and PO Modes [TN-1142]...... 54 Anti-Allergic Agents in NP, RP, and PO Modes [TN-1143]...... 58 Pain Relievers in NP, RP, and PO Modes [TN-1144]...... 62 Vasodilator Drugs in NP, RP, and PO Modes [TN-1145]...... 66 Anti-Anxiety and Antidepressive Drugs in NP, RP, and PO Modes [TN-1146]...... 70 Antifungal Drugs in NP, RP, and PO Modes [TN-1147]...... 74

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Enantiomeric Separation of Proton Pump Inhibitors Including Rabeprazole and Pantoprazole Using Lux® Polysaccharide-Based Chiral Stationary Phases in Reversed Phase Conditions Michael McCoy, Kari Kelly, Marc Jacob and Jeff Layne Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

Abstract We demonstrate in this technical note the successful separation The system flow rate was set to 1 mL/min and the column temperature of protein pump inhibitors (PPI) Omeprazole, Lansoprazole, was ambient. The mobile phases consisted of acetonitrile or Rabeprazole and Pantoprazole using Lux polysaccharide-based methanol with 0.1 % diethyl amine (DEA) (solvent A) and 20 mMoL columns. These chiral separations indicate the potential to ammonium bicarbonate with 0.1 % DEA (solvent B). prepare enantiomerically pure forms of racemic active ingredients Pantoprazole (Protonix) and Rabeprazole (AcipHex) using chiral chromatography. Results and Discussion Five different polysaccharide-based chiral stationary phases Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, and Introduction Lux Amylose-2 were explored in the reversed phase elution mode The competition for market share between PPI is fierce. Among for the enantioseparation of Omeprazole 2, Lansoprazole 4, Ra- therapeutic agents, PPI accounted for $13.6 billion of the total beprazole 5 and Pantoprazole 6 using mobile phases consistent $300.3 billion in sales for the year 20091. In terms of total United with LC/MS detection. States prescription revenues, the PPI Nexium2 is second only to Lipitor, a member of the drug class known as statins, used for lowering blood cholesterol. In order to reduce the volume of solvents used, the screening procedure was initially done on Lux 5 μm columns with dimension Esomeprazole 1, the active ingredient for the drug Nexium is the S of 150 x 4.6 mm. After the screening identified the best Lux enantiomer form of Omeprazole 2, the active ingredient for the drug polysaccharide phases, mobile phase conditions were further Prilosec. The patent for Prilosec, which posted sales of $5.6 billion optimized on 250 mm length columns of the same particle size and in 2001, expired in 2002. Generic Omeprazole may have eroded column internal diameter. The best results are shown in Figures the market share of Prilosec significantly had it not been for the 2-5; Rabeprazole 5 and Lansoprazole 4 show optimal resolution chiral separation, subsequent asymmetric synthesis, and timely on Lux Cellulose 4 whereas Omeprazole 2 and Pantoprazole 6 are marketing of Esomeprazole. By the end of 2002 combined sales of best resolved on Lux Cellulose 2 phase. Nexium and Prilosec were nearly $6.6 billion.

Figure 1. Structures of Analytes

This marketing patent-loss pattern has also been demonstrated 1. Esomeprazol (Nexium) 2. Omeprazole (Prilosec) with the introduction of Dexlansoprazole 3 (Kapidex) which is the R enantiomer of Lansoprazole 4 (Prevacid) and which was approved by the FDA in 2009 corresponding to the expiration of patent protection for the drug Prevacid.

A chiral screen was performed on Phenomenex Lux polysaccharide- based columns to identify chiral stationary phases (CSP) for 3. Dexlansoprazole (Kapidex) 4. Lansoprazole (Prevacid) possible preparative scale separation of the enantiomers of four benzimidazoles: Omeprazole 2, Lansoprazole 4, Rabeprazole 5 and Pantoprazole 6 under conditions suitable for mass spectroscopy (MS) detection. Optimization of the chromatographic conditions with respect to retention, enantioseparation, and resolution was achieved by variation of the mobile phase constituents at room temperature. The structures of the four analytes are depicted in Figure 1.

Materials and Methods ® All Analyses were performed using an HPLC Agilent 1100 series 5. Rabeprazole (Aciphex) 6. Pantoprazole (Protonix) (Agilent Technologies, Palo Alto, CA, USA).

Chiral chromatographic separations follwed by UV detection were performed using Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, and Lux Amylose-2 HPLC columns with dimensions 250 mm x 4.6 mm ID packed with 5 μm particles (Phenomenex, Torrance CA USA).

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Basic or acidic mobile phase additives are often required for im- to allow for chromatographic separation. This suggests that it proving resolution and peak shapes of ionisable analytes. Aqueous would be possible to isolate entiomerically pure compounds using mobile phase buffer at higher pH with ammonium salts such as ac- Lux polysaccharide-based chiral columns. etate or hydrogencarbonate (with ammonia) can be effective in the chiral separation of basic racemic compounds. Ammonium salts are thermolabile, hence fully compatible with MS detectors, and Conclusion even amenable to preparative purifications (as they can be easily The HPLC analysis of the four benzimidazoles Omeprazole, removed from the final product). Lansoprazole, Rabeprazole and Pantoprazole allows for fast and accurate identification of their enantiomers. The proven success of marketing enantiomeric pure pharmaceutical ingredients (such as The chromatogram for the racemic Lansoprazole 4 is shown in Esomeprazole and Dexlansoprazole) after expiration for the patent Figure 2a; while Figure 2b confirms the indentify of the single of their racemic formulations suggests that chiral purification of enantiomer of Lansoprazole as Dexlansoprazole. Pantoprazole and Rabeprazole will lead to similar success.

Likewise, the chromatogram in Figure 3b confirms the identity In this technical note, we described the successful separation of the single enantiomer of Omeprazole as Esomeprazole. The under reversed phase conditions of both Pantoprazole and chromatogram for the racemic Omeprazole is shown in Figure 3a. Rabeprazole. Based on previous work done at Phenomenex, the separation of Pantoprazole and Rabeprazole can be achieved without the use of base additives such as DEA3. Finally, these analytical reversed phase conditions can be Pantoprazole and Rabeprazole chromatograms shown in Figures 4 developed and scaled-up for the preparative chiral purification and 5 are racemic mixes. The Lux Cellulose-2 and Lux Cellulose-4 of enantiomerically pure forms of racemic active ingredients columns, respectively, provide more than enough enantioselectively Pantoprazole and Rabeprazole.

19719 Fig.19718 2a Lansoprazole (Prevacid) Fig. 2b Dexlansoprazole (Kapidex)

α = 2.1 App ID 19718 App ID 19719

0 2 4 6 8 10 min 0 2 4 6 8 10 min

Column: Lux® 5 µm Cellulose-4 Column: Lux® 5 µm Cellulose-4 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Part No.: 00G-4491-EO Part No.: 00G-4491-EO Injection Volume: 1 µL Injection Volume: 0.15 µL Concentration: 1 mg/mL Concentration: 3.75 mg/mL Mobile Phase: 90/10/0.1 Mobile Phase: 90/10/0.1

Methanol/20 mMoL of NH4 HCO3/DEA Methanol/20 mMoL of NH4 HCO3/DEA

52 Phenomenex l WEB: www.phenomenex.com TN-1102 APPLICATIONS 19721 19720 Fig. 3a Omeprazole (Prilosec) Fig. 3b Esomeprazole (Nexium)

α = 2.21 App ID 19721 App ID 19720

0 2 4 6 8 min 0 2 4 6 8 10 min

Column: Lux® 5 µm Cellulose-2 Column: Lux® 5 µm Cellulose-2 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Part No.: 00G-4457-EO Part No.: 00G-4457-EO Injection Volume: 1 µL Injection Volume: 0.5 µL Concentration: 1 mg/mL Concentration: 2.5 mg/mL Mobile Phase: 80/20/0.1 Mobile Phase: 80/20/0.1

Acetonitrile/20 mMoL NH4 HCO3/DEA Acetonitrile/20 mMoL NH4 HCO3/DEA 19717 19716

Fig. 4 Pantoprazole (Protonic) Fig. 5 Rabeprazole (Aciphex)

α = 1.53 α = 2.34 App ID 19717 App ID 19716

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 16 18 min

Column: Lux® 5 µm Cellulose-2 Column: Lux® 5 µm Cellulose-4 Dimensions: 250 x 4.6 mm Dimensions: 250 x 4.6 mm Part No.: 00G-4457-EO Part No.: 00G-4491-EO Injection Volume: 10 µL Injection Volume: 10 µL Concentration: 0.5 mg/mL Concentration: 0.5 mg/mL Mobile Phase: 60/40/10.1 Mobile Phase: 80/20/0.1

Acetonitrile/10 mMoL NH4 HCO3/DEA Methanol/10 mMoL NH4 HCO3/DEA

References

1. Source IMS Health, IMS National Sales Perspectives; Top Therapeutic Classes by U.S. Sales, 2009 2. Source IMS Health, IMS National Sales Perspectives; Top 15 U.S. Pharmaceutical Products by Sales 2009 3. Phenomenex Technical Note TN-1079: Method Development for Reversed Phase Chiral LC/MS/MS Analysis of Stereoisomeric Pharmaceutical Compounds with Polysaccharide-Based Stationary Phases.

Marc Jacob, Liming Peng, Michael McCoy, Michael Klein and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technical note, we report the chiral separation of various beta separation of enantiomers.1 A recent review pointed out that in blocker pharmaceutical drugs using Lux polysaccharide-based chi- 2007 more than 90 % of the HPLC methods used for the deter- ral stationary phases. The reported separations are the results of a mination of enantiomeric excess were performed on polysaccha- systematic screening of five different Lux phases in polar organic, ride-based chiral stationary phases.2 The polysaccharide-based normal phase, and reversed phase separation modes. CSPs are frequently used for preparative purifications because they are easily scaled-up from the analytical separations.3 Introduction Chiral separations can be performed by chromatographic sepa- Beta blocker drugs, also known as beta adrenergic receptor an- ration, enzymatic resolution, and crystallization. Chromatographic tagonists, are effective in the treatment of cardiovascular diseases enantioselective separation using chiral stationary phases (CSPs) such as hypertension. The various beta blockers analyzed in this for high performance liquid chromatography (HPLC) has signifi- study are depicted in Figure 1. The chiral separations presented cantly evolved during the past few decades and is recognized as are the results of a systematic screening of our five Lux polysac- the most popular and reliable tool for both analytical and prepara- charide-based CSPs (Cellulose-1, Cellulose-2, Cellulose-3, Cellu- tive separation of chiral compounds. Polysaccharide-based CSPs lose-4, and Amylose-2) under various separation modes. such as Lux are the most widely use CSPs for the chromatographic

Figure 1. Chemical Structure of 15 Beta Blocker Racemates H H N H N H N O HO HO H N O O O HO OH H N O O O O H N OH O

O N O H Betaxolol Bisoprolol Acebutolol Alprenolol Atenolol

H H H N N HN N HO HO O O O O O O N HO H HN O HO

O H N O N O O H O Bopindolol Carazolol Carvedilol Esmolol Metoprolol

O H S H N N H H O N N OH HO HO O

O O O OH OH O N N H H N H Oxprenolol Pindolol Propanolol Sotalol Toliprolol

54 Phenomenex l WEB: www.phenomenex.com TN-1142 APPLICATIONS

Material and Methods site for further research regarding each compound’s pharmaceu- All analyses were performed using an Agilent® 1100 series LC tical properties. The table summarizes the Lux phases used, the system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped selectivity, the retention time of the first enantiomer, as well as the with binary pump, in-line degasser, multi-wavelength UV detector isocratic conditions used for each compound. Lux columns are and autosampler. Lux® columns used for analysis were obtained quite successful at resolving chiral drugs of this type. All the beta from Phenomenex (Torrance, CA, USA). The HPLC column dimen- blockers tested are separated with selectivity greater than 1.1. sions were 250 x 4.6 mm ID and all columns were packed with In the last column, the corresponding Phenomenex application 5 µm particles. The flow rate was 1.0 mL/min and temperature was number is provided. Those applications are easily accessible on ambient. Standards were purchased from Sigma-Aldrich (St. Lou- our website (www.phenomenex.com/ChiralAppSearch) and can is, MO, USA). All solvents were purchased from EMD (San Diego, be searched by application number, structure, CID, or compound CA, USA). name.

Results and Discussion The chiral separations reported in Table 1 are baseline resolved Fifteen beta blocker racemates depicted in Figure 1 were ana- with a resolution greater than 1.5. The retention time for the first lyzed on five different Lux polysaccharide-based CSPs (Cellu- enantiomer is between 4 and 15 min and all the separations are lose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) in completed in less than 30 min. With amine derivatives such as normal phase (NP), polar organic (PO), and reversed phase (RP) beta blockers, we recommend to use 0.1 % of diethylamine (DEA) separation modes. After performing a systematic screening with as an additive. The presence of DEA favors dissociation of the various mobile phases, the best separation was selected, even amino group and improves peak shape. Interestingly, out of 15 though in most of the cases, alternative separation was obtained separations, 13 are most successful in NP separation mode. NP with other Lux phases and/or modes. mode is very similar in polarity and selectivity to Supercritical Fluid Chromatography (SFC) mode. In SFC mode, ammonium The racemic beta adrenergic receptor antagonists analyzed in hydroxide in MeOH, EtOH, or IPA can be used as basic addi- this study are listed in Table 1. For each beta blocker tested we tives to help peak shape.4 SFC mode is particularly attractive provide the chemical identification number (CID) of the racemate. for its high-throughput, low solvent consumption, low pressure This unique number can be linked to The PubChem Project web- drop, and high resolution. Another great advantage is the ease of scale–up to preparative scale, especially with our Axia™ packed preparative product line. Table 1. Chiral separations of Beta Blockers using Lux Polysaccharide-based CSPs

Beta Blocker CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Acebutolol 1978 Lux Amylose-2 4.21 4.91 PO ACN/IPA (95:5) DEA (0.1 %) 18130 Alprenolol 2119 Lux Cellulose-2 1.12 6.12 NP Hex/EtOH (95:5) DEA (0.1 %) 20443 Atenolol 2249 Lux Cellulose-1 1.39 10.55 NP Hex/EtOH (80:20) DEA (0.1 %) 20547 Betaxolol 2369 Lux Cellulose-2 1.28 6.33 NP Hex/EtOH (80:20) DEA (0.1 %) 20501 Bisoprolol 2405 Lux Cellulose-1 2.06 9.04 NP Hex/EtOH (80:20) DEA (0.1 %) 20261

Bopindolol 44112 Lux Cellulose-4 1.22 5.03 RP MeOH/20 mM NH4HCO3 (60:40) DEA (0.1 %) 20173 Carazolol 71739 Lux Cellulose-2 1.75 6.40 NP Hex/IPA (70:30) DEA (0.1 %) 20117 Carvedilol 2585 Lux Cellulose-4 1.74 6.79 NP Hex/IPA (40:60) DEA (0.1 %) 20422 Esmolol 59768 Lux Cellulose-1 2.04 6.10 NP Hex/IPA (80:20) DEA (0.1 %) 20403 Metoprolol 4171 Lux Cellulose-1 1.97 5.27 NP Hex/EtOH (80:20) DEA (0.1 %) 20470 Oxprenolol 4631 Lux Cellulose-1 3.09 5.25 NP Hex/EtOH (80:20) DEA (0.1 %) 20544 Pindolol 4828 Lux Cellulose-2 2.13 10.39 NP Hex/IPA (80:20) DEA (0.1 %) 20125

Propranolol 4946 Lux Cellulose-3 1.21 5.67 RP MeOH/20 mM NH4HCO3 (80:20) DEA (0.1 %) 20308 Sotalol 5253 Lux Cellulose-2 1.29 14.19 NP Hex/EtOH (90:10) DEA (0.1 %) 20550 Toliprolol 18047 Lux Amylose-2 1.17 5.97 NP Hex/EtOH (90:10) DEA (0.1 %) 20511

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol * To view the full application enter the App ID onto the search field on our website.

Phenomenex l WEB: www.phenomenex.com 55 TN-1142 APPLICATIONS

All of our Lux® products are pressure stable up to 300 bar and Conclusion compatible with SFC separation mode using an organic modi- In this study, we described the chiral separation of a variety fier such as MeOH, EtOH, IPA, or ACN. Two examples of chiral of beta blockers using Lux polysaccharide-based chiral sta- separation for beta blockers pindolol and oxprenolol are shown tionary phases. All enantiomeric separations reported showed in Figure 2. selectivity greater than 1.1 with the retention time for the first enantiomer below 15 min. Those separations can be used Figure 2. not only for analytical but for preparative purposes since our Representative chromatograms for the separation of Beta Blockers phases are available in various preparative formats such as ™ Pindolol on Lux 5 µm Cellulose-2 in NP Axia packed preparative columns or bulk media.

mAU 200 References 1 1. Chiral Separation Techniques: A Practical Approach, 3rd ed.; 100 2 Subramanian, G., Ed.; Wiley-VCH: Weinheim, Germany, 2007. a = 2.13 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. 0 4. Hamman, C.; Schmidt Jr., D. E.; Wong, M.; Hayes, M. J. Chromatogr. A App ID 20125 2011, 1218, 7886– 7894. 0 10 20 min

Oxprenolol on Lux 5 µm Cellulose-1 in NP

mAU

200 1

200 2 a = 3.09

0 App ID 20544

0 2 4 6 8 10 12 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 © 2012 Phenomenex, Inc. All rights reserved. 56 Phenomenex l WEB: www.phenomenex.com Get a Great Price on Your Vials

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Chromatographic Enantioseparation of Racemic Anti-Allergic Drugs using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA In this technical note, we report the chiral chromatographic CSPs such as Lux are the most widely used CSPs for the separation of various anti-allergic drugs using Lux polysaccha- chromatographic separation of enantiomers.1 A recent review ride-based chiral stationary phases. The reported enantiosepa- pointed out that in 2007 more than 90 % of the HPLC methods rations are the results of a systematic screening of five different used for the determination of enantiomeric excess were per- Lux phases in normal phase, polar organic, and reversed phase formed on polysaccharide-based chiral stationary phases.2 The separation modes. polysaccharide-based CSPs are frequently used for preparative purifications because they are easily scaled-up from the analytical Introduction separations.3 Chiral separations can be performed by chromatographic separation, enzymatic resolution, and crystallization. Chromatographic Anti-allergic drugs, also known as histamine antagonists, are ef- enantioselective separation using chiral stationary phases (CSPs) fective in the treatment of allergic reactions such as seasonal rhi- for high performance liquid chromatography (HPLC) has signifi- nitis and allergic dermatitis. The various anti-allergics analyzed cantly evolved during the past few decades and is recognized in this study are depicted in Figure 1. The chiral separations de- as the most popular and reliable tool for both analytical and pre- scribed in this application are the results of a systematic screen- parative separation of chiral compounds. Polysaccharide-based ing of our five Lux polysaccharide-based CSPs (Cellulose-1, Cel- lulose-2, Cellulose-3, Cellulose-4, and Amylose-2) under various Figure 1. Chemical structure of anti-allergic drugs racemic mixtures separation modes.

O OH N

N O N N N N

O N

N N N

CI CI Br CI Chlorpheniramine Dimetindene Brompheniramine Carbinoxamine Cetirizine

O N N

N O N N N N N N N N S CI CI

Doxylamine Ethopropazine Hydroxyzine Meclizine Mirtazapine

N N N HO

O N N N N O N H S N S

Mianserin Pheniramine Promethazine Propiomazine Terfenadine

58 Phenomenex l WEB: www.phenomenex.com TN-1143 APPLICATIONS

Material and Methods The table summarizes the Lux phases used, the selectivity, the All analyses were performed using an Agilent® 1100 series LC retention time of the first enantiomer, as well as the isocratic con- system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped ditions used for each compound. Lux columns are quite success- with quaternary pump, in-line degasser, multi-wavelength UV de- ful at resolving chiral drugs of this type. All the anti-allergic drugs tector, and autosampler. Lux® columns used for analysis were ob- tested are separated with selectivity greater than 1.1. In the last tained from Phenomenex (Torrance, CA, USA). The HPLC column column, the corresponding Phenomenex application number is dimensions were 250 x 4.6 mm ID and all columns were packed provided. Those applications are easily accessible on our website with 5 µm particles. The flow rate was 1.0 mL/min and tempera- (www.phenomenex.com/ChiralAppSearch) and can be searched ture was ambient. Standards were purchased from Sigma-Aldrich by application number, structure, CID, or compound name. (St. Louis, MO, USA). All solvents were purchased from EMD (San The chiral separations reported in Table 1 are baseline resolved Diego, CA, USA). with a resolution greater than 1.5. The retention time for the first enantiomer is between 5 and 10 min and all the separations are Results and Discussion completed in less than 30 min. With basic analytes such as an- Fifteen anti-allergic racemates depicted in Figure 1 were ana- ti-allergics, 0.1 % of diethylamine (DEA) is used as mobile phase lyzed on five different Lux polysaccharide-based CSPs (Cellu- additive. DEA is an ion-masking agent that reduces unwanted in- lose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) in teractions with residual silanols. DEA promotes improved peak normal phase (NP), polar organic (PO), and reversed phase (RP) shape by minimizing ion-exchange interactions between silanol separation modes. After performing a systematic screening with groups and basic analytes. Interestingly, out of 15 separations, various mobile phases, the best separation was selected, even 8 are most successful in NP separation mode. NP mode is very though in most of the cases, alternative separation was obtained similar in polarity and selectivity to supercritical fluid chromatog- with other Lux phases and/or modes. raphy (SFC) mode. In SFC mode, ammonium hydroxide in MeOH, The racemic anti-allergic drugs separated in this study are listed in EtOH, or IPA can be used as basic additives to help peak shape.4 Table 1. For each anti-allergic tested we provide the chemical SFC mode is particularly attractive for its high-throughput5, low identification number (CID) of the racemate. This unique num- solvent consumption, low pressure drop, and high resolution. ber can be linked to The PubChem Project website for further Another great advantage is the ease of scale–up to preparative research regarding each compound’s pharmaceutical properties. scale, especially with our Axia™ packed preparative product line.

Table 1. Chiral separations of anti-allergic drugs using Lux polysaccharide-based CSPs

Beta Blocker CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Brompheniramine 6834 Lux Amylose-2 1.42 6.30 NP Hex/IPA (90:10) DEA (0.1 %) 20082 Carbinoxamine 2564 Lux Amylose-2 1.28 6.69 NP Hex/EtOH (90:10) DEA (0.1 %) 20452

Cetirizine 55182 Lux Cellulose-3 1.29 8.06 RP ACN/20 mm NH4HCO3 (50:50) DEA (0.1 %) 19641 Chlorpheniramine 2725 Lux Amylose-2 1.98 6.94 NP Hex/EtOH (95:5) DEA (0.1 %) 20445 Dimetindene 21855 Lux Cellulose-1 1.25 7.07 NP Hex/EtOH (98:2) DEA (0.1 %) 20435 Doxylamine 3162 Lux Cellulose-4 1.91 6.04 NP Hex/IPA (90:10) DEA (0.1 %) 20346

Ethopropazine 3290 Lux Cellulose-3 1.30 7.14 RP MeOH/20 mm NH4HCO3 (95:5) DEA (0.1 %) 20303

Hydroxyzine 3658 Lux Cellulose-3 1.66 7.54 RP MeOH/20 mm NH4HCO3 (80:20) DEA (0.1 %) 20320 Meclizine 4034 Lux Cellulose-3 2.62 5.52 NP Hexane / EtOH (80:20) DEA (0.1 %) 20338 Mianserin 4184 Lux Cellulose-1 1.25 8.14 RP MeOH/20 mm NH4HCO3 (90:10) DEA (0.1 %) 20225 Mirtazapine 4205 Lux Cellulose-2 1.32 5.80 PO ACN/IPA (95:5) DEA (0.1 %) 20067 Pheniramine 4761 Lux Cellulose-3 1.17 5.47 NP Hexane/EtOH (95:5) DEA (0.1 %) 20429

Promethazine 4927 Lux Cellulose-3 1.34 9.01 RP MeOH/20 mm NH4HCO3 (95:5) DEA (0.1 %) 20306 Propiomazine 4940 Lux Cellulose-3 1.37 5.18 PO MeOH/IPA (90:10) DEA (0.1 %) 20556 Terfenadine 5405 Lux Cellulose-2 1.30 7.00 NP Hex/IPA (60:40) DEA (0.1 %) 20078

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol * To view the full application enter the App ID onto the search field on our website.

Phenomenex l WEB: www.phenomenex.com 59 TN-1143 APPLICATIONS

All of our Lux® products are pressure stable up to 300 bar and Conclusion compatible with SFC separation mode using an organic modi- In this study, we described the chiral separation of a variety fier such as MeOH, EtOH, IPA, or ACN. Two examples of chiral of anti-allergic drugs using Lux polysaccharide-based chi- separation for Brompheniramine and Ethopropazine are shown in ral stationary phases. All enantiomeric separations reported Figure 2. showed selectivity greater than 1.1 with the retention time for the first enantiomer below 10 min. Those separations can be Figure 2. Representative chromatograms for the chiral separation of used not only for analytical but for preparative purposes since anti-allergics 20082 our phases are available in various preparative formats such mAU Brompheniramine on Lux 5 µm Amylose-2 in NP as Axia™ packed preparative columns or bulk media. 600

a = 1.42 1 400 2 References 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). 200 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101.

App ID 20082 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. 0 4. Hamman, C.; Schmidt Jr., D. E.; Wong, M.; Hayes, M. J. Chromatogr. A 2011, 1218, 7886– 7894. -200 5. Miller L. J. Chromatogr. A 2012, 1250, 250. (Review). 20303 0 2 4 6 8 10 12 14 min

mAU Ethopropazine on Lux 5 µm Cellulose-3 in RP

600 1

a = 1.30 2

400

200 App ID 20303 0

0 2 4 6 8 10 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 © 2012 Phenomenex, Inc. All rights reserved. 60 Phenomenex l WEB: www.phenomenex.com Need help? Give us a call! • Application support • Method improvement and optimization • New method development • Pre-validation services • Scale-up and purification • On-site training and consulting … and more!

Phenomenex l WEB: www.phenomenex.com 61 TN-1144 APPLICATIONS Chromatographic Enantioseparation of Racemic Pain Relievers using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA In this technical note, we report the chiral chromatographic sepa- separation of enantiomers.1 A recent review pointed out that in 2007 ration of various anti-inflammatory agents and pain relievers using more than 90 % of the HPLC methods used for the determination Lux polysaccharide-based chiral stationary phases. The reported of enantiomeric excess were performed on polysaccharide-based enantioseparations are the results of a systematic screening of chiral stationary phases.2 The polysaccharide-based CSPs are fre- five different Lux phases in polar organic, normal phase, and re- quently used for preparative purifications because they are easily versed phase separation modes. scaled-up from the analytical separations.3

Introduction Anti-inflammatory and analgesic agents are effective in the treatment Chiral separations can be performed by chromatographic separa- of chronic arthritic conditions and certain soft tissue disorders asso- tion, enzymatic resolution, and crystallization. Chromatographic ciated with pain and inflammation. The pain relievers analyzed in this enantioselective separation using chiral stationary phases (CSPs) study are depicted in Figure 1. The chiral separations described in this for high performance liquid chromatography (HPLC) has signifi- application are the results of a systematic screening of our five Lux cantly evolved during the past few decades and is recognized as polysaccharide-based CSPs (Cellulose-1, Cellulose-2, Cellulose-3, the most popular and reliable tool for both analytical and prepara- Cellulose-4, and Amylose-2) under various separation modes. tive separation of chiral compounds. Polysaccharide-based CSPs such as Lux are the most widely use CSPs for the chromatographic

Figure 1. Chemical structures of pain relievers and anti-inflammatory agents

OH OH

O O

H O N H OH O N OH N O O OH CI O O Carprofen Etodolac Fenoprofen Ibuprofen Indoprofen

O O N N H O N O N CI HO O O O H OH Ketamine Ketoprofen Ketorolac Meptazinol Nefopam

O S

HO O

O N

HO Suprofen Tramadol

62 Phenomenex l WEB: www.phenomenex.com TN-1144 APPLICATIONS

Material and Methods All analyses were performed using an Agilent® 1100 series LC summarizes the Lux phases used, the selectivity, the retention time system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped of the first enantiomer, as well as the isocratic conditions used for with quaternary pump, in-line degasser, multi-wavelength UV de- each compound. Lux columns are quite successful at resolving tector and autosampler. Lux® columns used for analysis were ob- chiral drugs of this type. All the anti-inflammatory agents and pain tained from Phenomenex (Torrance, CA, USA). The HPLC column relievers tested are separated with selectivity greater than 1.1. dimensions were 250 x 4.6 mm ID and all columns were packed In the last column, the corresponding Phenomenex application with 5 μm particles. The flow rate was 1.0 mL/min and tempera- number is provided. Those applications are easily accessible on ture was ambient. Standards were purchased from Sigma-Aldrich our website (www.phenomenex.com/ChiralAppSearch) and can (St. Louis, MO, USA). All solvents were purchased from EMD (San be searched by application number, structure, CID, or compound Diego, CA, USA). name.

Results and Discussion The chiral separations reported in Table 1 are baseline resolved Pain reliever agents depicted in Figure 1 were analyzed on five with a resolution greater than 1.5. The retention time for the first different Lux polysaccharide-based CSPs (Cellulose-1, Cellu- enantiomer is between 5 and 14 min and all the separations are lose-2, Cellulose-3, Cellulose-4, and Amylose-2) in normal phase completed in less than 30 min. With basic pain relievers, 0.1 % of (NP), polar organic (PO), and reversed phase (RP) separation diethylamine (DEA) was used as an additive, whereas with acidic modes. After performing a systematic screening with various mo- derivatives, 0.1 % of formic acid (FA) was used as additive. Inter- bile phases, the best separation was selected, even though in estingly with pain reliever drugs, Lux Cellulose-3 phase was quite most of the cases alternative separation was obtained with other successful. Out of the 12 separations, 9 were most successful Lux phases and/or modes. with Lux Cellulose-3.

The racemic pain relievers analyzed in this study are listed in Ta- All of our Lux® products are pressure stable up to 300 bar and ble 1. For each compound tested we provide the chemical iden- compatible with SFC separation mode4 using an organic modifier tification number (CID) of the racemate. This unique number can such as MeOH, EtOH, IPA, or ACN. Two examples of chiral sepa- be linked to The PubChem Project website for further research ration for Ibuprofen and Ketoprofen are shown in Figure 2. regarding each compound’s pharmaceutical properties. The table

Table 1. Chiral separations of anti-inflammatory agents using Lux polysaccharide-based CSPs

Compound CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Carprofen 2581 Lux Cellulose-3 1.20 7.69 min NP Hex/EtOH (60:40) FA (0.1 %) 20385 Etodolac 3308 Lux Cellulose-3 1.21 7.38 min RP ACN/FA (0.1 %) (40:60) 20324 Fenoprofen 3342 Lux Amylose-2 1.57 8.06 min NP Hexane/EtOH (95:5) FA (0.1 %) 20453 Ibuprofen 3672 Lux Cellulose-3 1.22 9.1 min RP MeOH/FA (0.1 %) (80:20) 20310 Indoprofen 3718 Lux Cellulose-3 1.17 12.29 min RP MeOH/FA (0.1 %) (80:20) 20296

Ketamine 3821 Lux Cellulose-3 2.42 5.49 min RP MeOH/20 mM NH4HCO3 (90:10) DEA (0.1 %) 20287 Ketoprofen 3825 Lux Cellulose-3 1.13 8.09 min NP Hex/IPA (80:20) FA (0.1 %) 20099 Ketorolac 3826 Lux Cellulose-3 1.52 8.65 min PO MeOH/IPA (90:10) FA (0.1 %) 20367 Meptazinol 41049 Lux Cellulose-3 1.38 7.74 min NP Hex/IPA (90:10) DEA (0.1 %) 20392 Nefopam 4450 Lux Cellulose-4 1.64 13.31 min PO MeOH/IPA (90:10) DEA (0.1 %) 20376 Suprofen 5359 Lux Cellulose-3 1.31 7.06 min NP Hex/EtOH (60:40) FA (0.1 %) 20098

Tramadol 33741 Lux Cellulose-1 1.13 5.86 min RP ACN/20 mM NH4HCO3 (50:50) DEA (0.1 %) 20240 ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol, FA = Formic acid, DEA = Diethylamine * To view the full application enter the App ID onto the search field on our website.

Phenomenex l WEB: www.phenomenex.com 63 TN-1144 APPLICATIONS

Figure 2. Representative chromatograms for the chiral separation of Conclusion anti-inflammatory agents In this study, we described the chiral separation of a variety of anti-inflammatory agents using Lux polysaccharide-based Ibuprofen on Lux 5 µm Cellulose-3 in RP 20310 chiral stationary phases. All enantiomeric separations report- mAU ed showed selectivity greater than 1.1 with the retention time for the first enantiomer below 15 min. Those separations can 800 1 2 be used not only for analytical but for preparative purposes 600 since our phases are available in various preparative formats such as Axia™ packed preparative columns or bulk media. 400 a = 1.22

200 App ID 20310 References 0 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). 0 2 4 6 8 10 12 14 min 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. (Review) Ketoprofen20099 on Lux 5 µm Cellulose-3 in NP 4. Miller L. J. Chromatogr. A 2012, 1250, 250. (Review). mAU

600 1

2 400

= 1.13 200 a App ID 20099

0 2 4 6 8 10 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 © 2012 Phenomenex, Inc. All rights reserved. 64 Phenomenex l WEB: www.phenomenex.com ChiralFREE Screening

Polysaccharide Chiral Columns Services Dependable. Scalable. A ordable. Polysaccharide-Based Chiral Columns HPLC | SFC • High efficiency and loading capacity • Stability up to 300 bar • 3 µm, 5 µm, 10 μm and 20 µm

C-1 C-2 C-3 C-4 • StableA-2 in RP, NP, PO and SFC conditionsC-1 C-1 C-2 C-2 C-3 C-3 C-4 C-4 A-2 A-2

H H H H H H H C H Cl H HC CH H Cl Cl H C H H HC CH H H H H H H H H H H H H H H H H H C Cl C Cl H H C C Cl Cl C C Cl Cl C C C H H H H H H H H H

H H H C O O O C C H H H H H H

Cellulose-O-CONH Cellulose-O-CONH Cellulose-O Cellulose-O-CONH Amylose-O-CONH Cellulose-O-CONHCellulose-O-CONH Cellulose-O-CONHCellulose-O-CONH Cellulose-OCellulose-O Cellulose-O-CONHCellulose-O-CONH AmyloseAmylose-O-CONH-O-CONH Lux Amylose-2 Lux Cellulose-1 Lux Cellulose-2 Amylose tris(5-chloro-2-methylphenylcarbamate) Cellulose tris(3, 5-dimethylphenylcarbamate) Cellulose tris(3-chloro-4-methylphenylcarbamate)

C-1 C-1 C-2 C-2 C-3 C-3 C-4 C-4 A-2 A-2

H H H H H C H H C H Cl Cl H C H H C H H H H H H H H H H H H H C C Cl Cl C C Cl Cl C C H H H H H H

H H

O O C C H H H H

Cellulose-O-CONHCellulose-O-CONH Cellulose-O-CONHCellulose-O-CONH Cellulose-OCellulose-O Cellulose-O-CONHCellulose-O-CONH AmyloseAmylose-O-CONH-O-CONH Lux Cellulose-3 Lux Cellulose-4 Cellulose tris(4-methylbenzoate) Cellulose tris(4-chloro-3-methylphenylcarbamate)

Phenomenex l WEB: www.phenomenex.com 65 TN-1145 APPLICATIONS Chromatographic Enantioseparation of Racemic Vasodilator Drugs using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein, Tom Cleveland and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA In this technical note, we report the chiral chromatographic CSPs such as Lux are the most widely used CSPs for the chro- separation of various vasodilator drugs using Lux polysaccha- matographic separation of enantiomers.1 A recent review point- ride-based chiral stationary phases. The reported enantiosepa- ed out that in 2007 more than 90 % of the HPLC methods used rations are the results of a systematic screening of five different for the determination of enantiomeric excess were performed on Lux phases in normal phase, polar organic, and reversed phase polysaccharide-based chiral stationary phases.2 The polysaccha- separation modes. ride-based CSPs are frequently used for preparative purifications because they are easily scaled-up from the analytical separa- Introduction tions.3 Chiral separations can be performed by chromatographic separation, enzymatic resolution, and crystallization. Chromatographic Vasodilator drugs are effective in the treatment of cardiovascu- enantioselective separation using chiral stationary phases (CSPs) lar diseases such as hypertension, heart failure, and angina. The for high performance liquid chromatography (HPLC) has signifi- various vasodilator agents analyzed in this study are depicted in cantly evolved during the past few decades and is recognized Figure 1. The chiral separations described in this application are as the most popular and reliable tool for both analytical and pre- the results of a systematic screening of our five Lux polysaccha- parative separation of chiral compounds. Polysaccharide-based ride-based CSPs (Cellulose-1, Cellulose-2, Cellulose-3, Cellu- lose-4, and Amylose-2) under various separation modes. Figure 1. Chemical structure of vasodilator drugs

H N N

CI CI O O O O N O CI O O N O O O O S H N O H H O O O H N

H O N N O H O H H Amlodipine Carvedilol O Diltiazem Felodipine Ifenprodil

NO 2 H N N O H O O O NO2 O O O N N HN O O HO O O O O O N H O O O N H N H HO Isoxsuprine Isradipine Nicardipine Nisoldipine Oxprenolol

H O N H O N O O O N C N O CI O H N H O N O H Phenoxybenzamine Pindolol Propranolol Verapamil

66 Phenomenex l WEB: www.phenomenex.com TN-1145 APPLICATIONS

Material and Methods retention time of the first enantiomer, as well as the isocratic con- All analyses were performed using an Agilent® 1100 series LC ditions used for each compound. Lux columns are quite success- system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped ful at resolving chiral drugs of this type. All the vasodilator agents with quaternary pump, in-line degasser, multi-wavelength UV de- tested are separated with selectivity greater than 1.1. In the last tector and autosampler. Lux® columns used for analysis were ob- column, the corresponding Phenomenex application number is tained from Phenomenex (Torrance, CA, USA). The HPLC column provided. Those applications are easily accessible on our website dimensions were 250 x 4.6 mm ID and all columns were packed (www.phenomenex.com/ChiralAppSearch) and can be searched with 5 μm particles. The flow rate was 1.0 mL/min and tempera- by application number, structure, CID, or compound name. ture was ambient. Standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents were purchased from EMD (San The chiral separations reported in Table 1 are baseline resolved Diego, CA, USA). with a resolution greater than 1.5. The retention time for the first enantiomer is between 5 and 19 min and all the separations are Results and Discussion completed in less than 30 min. With basic analytes such as va- Fourteen vasodilator racemates depicted in Figure 1 were an- sodilators, 0.1% of diethylamine (DEA) is used as mobile phase alyzed on five different Lux polysaccharide-based CSPs (Cellu- additive. DEA is an ion-masking agent that reduces unwanted in- lose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) in teractions with residual silanols. DEA promotes improved peak normal phase (NP), polar organic (PO), and reversed phase (RP) shape by minimizing ion-exchange interactions between silanol separation modes. After performing a systematic screening with groups and basic analytes. Interestingly, out of 14 separations, various mobile phases, the best separation was selected, even 10 are most successful in NP separation mode. NP mode is very though in most of the cases, alternative separation was obtained similar in polarity and selectivity to supercritical fluid chromatog- with other Lux phases and/or modes. raphy (SFC) mode. In SFC mode, ammonium hydroxide in MeOH, EtOH, or IPA can be used as basic additives to help peak shape.4 The racemic vasodilator drugs separated in this study are listed SFC mode is particularly attractive for its high-throughput5, low in Table 1. For each vasodilator tested we provide the chemical solvent consumption, low pressure drop, and high resolution. identification number (CID) of the racemate. This unique num- Another great advantage is the ease of scale–up to preparative ber can be linked to The PubChem Project website for further scale, especially with our Axia™ packed preparative product line. research regarding each compound’s pharmaceutical properties. The table summarizes the Lux phases used, the selectivity, the

Table 1. Chiral separations of vasodilator drugs using Lux polysaccharide-based CSPs

Compound CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Amlodipine 2162 Lux Cellulose-4 1.78 5.83 min PO ACN/IPA (95:5) DEA (0.1 %) 20358 Carvedilol 2585 Lux Cellulose-4 1.74 6.79 min NP Hex/IPA (40:60) DEA (0.1 %) 20422 Diltiazem 39186 Lux Cellulose-4 2.24 7.17 min NP Hex/IPA (60:40) DEA (0.1 %) 20458

Felodipine 3333 Lux Cellulose-3 1.26 10.73 min RP MeOH/20 mM NH4HCO3 (80:20) DEA (0.1 %) 20307 Ifenprodil 3689 Lux Amylose-2 1.44 6.21 min NP Hex/EtOH (80:20) DEA (0.1 %) 20517 Isoxsuprine 3783 Lux Cellulose-4 1.16 5.84 min NP Hex/EtOH (80:20) DEA (0.1 %) 20541 Isradipine 3784 Lux Amylose-2 1.13 9.9 min NP Hex/IPA (90:10) DEA (0.1 %) 20089 Nicardipine 4474 Lux Cellulose-1 1.13 18.9 min NP Hex/IPA (90:10) DEA (0.1 %) 20075 Nisoldipine 4499 Lux Cellulose-1 1.11 9.69 min NP Hex/IPA (90:10) DEA (0.1 %) 20276 Oxprenolol 4631 Lux Cellulose-1 3.09 5.25 min NP Hex/EtOH (80:20) DEA (0.1 %) 20544

Phenoxybenzamine 4768 Lux Cellulose-2 1.14 10.28 min RP MeOH/20 mM NH4HCO3 (80:20) DEA (0.1 %) 20233

Pindolol 4828 Lux Cellulose-1 1.99 5.16 min RP MeOH/20 mM NH4Ac (80:20) DEA (0.1 %) 20198 Propranolol 4946 Lux Cellulose-1 1.35 6.9 min NP Hex/EtOH (80:20) DEA (0.1 %) 20477 Verapamil 2520 Lux Cellulose-3 1.38 6.25 min NP Hex/EtOH (60:40) DEA (0.1 %) 20114

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol, DEA = Diethylamine * To view the full application enter the App ID onto the search field on our website

Phenomenex l WEB: www.phenomenex.com 67 TN-1145 APPLICATIONS

All of our Lux® products are pressure stable up to 300 bar and com- Conclusion patible with SFC separation mode using an organic modifier such In this study, we described the chiral separation of a variety of as MeOH, EtOH, IPA, or ACN. Two examples of chiral separation vasodilator drugs using Lux polysaccharide-based chiral sta- for Diltiazem and Propanolol are shown in Figure 2. tionary phases. All enantiomeric separations reported showed selectivity greater than 1.1 with the retention time for the first enantiomer below 19 min. Those separations can be used not only for analytical but for preparative purposes since our Figure 2. phases are available in various preparative formats such as Representative chromatograms for the chiral separation of vasodilator agents. Axia™ packed preparative columns or bulk media.

20458

mAU 160 Diltiazem on Lux 5 µm Cellulose-4 in NP References 140 1 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). 120 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 100 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. 2 80 a = 2.24 4. Hamman, C.; Schmidt Jr., D. E.; Wong, M.; Hayes, M. J. Chromatogr. A 60 2011, 1218, 7886– 7894.

40 5. Miller L. J. Chromatogr. A 2012, 1250, 250. (Review).

20 App ID 20458 0

0 2 4 6 8 10 12 min

20477

mAU 600 Propranolol on Lux 5 µm Cellulose-1 in NP

400 2

a = 1.35

200

0 App ID 20477

0 2 4 6 8 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.

Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc.

Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383

Patented Hydraulic Piston Compression Technology

• High efficiency • Excellent reproducibility • Over 30 unique selectivities

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Chromatographic Enantioseparation of Racemic Antidepressive and Anti-Anxiety Drugs using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein, Tom Cleveland and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technical note, we report the chiral chromatographic sep- matographic separation of enantiomers.1 A recent review point- aration of various antidepressive and anti-anxiety drugs using Lux ed out that in 2007 more than 90 % of the HPLC methods used polysaccharide-based chiral stationary phases. The reported en- for the determination of enantiomeric excess were performed on antioseparations are the results of a systematic screening of five polysaccharide-based chiral stationary phases.2 The polysaccha- different Lux phases in normal phase, polar organic, and reversed ride-based CSPs are frequently used for preparative purifications phase separation modes. because they are easily scaled-up from the analytical separations.3 Introduction Chiral separations can be performed by chromatographic sepa- Antidepressive and anti-anxiety drugs are used to treat various ration, enzymatic resolution, and crystallization. Chromatographic disorders such as depression, obsessive compulsive disorders, enantioselective separation using chiral stationary phases (CSPs) eating disorder or chronic pain and in some case insomnia. The for high performance liquid chromatography (HPLC) has signifi- various antidepressive and anti-anxiety drugs analyzed in this cantly evolved during the past few decades and is recognized study are depicted in Figure 1. The chiral separations described as the most popular and reliable tool for both analytical and pre- in this application are the results of a systematic screening of our parative separation of chiral compounds. Polysaccharide-based five Lux polysaccharide-based CSPs (Cellulose-1, Cellulose-2, CSPs such as Lux are the most widely used CSPs for the chro- Cellulose-3, Cellulose-4, and Amylose-2) under various separation modes. Figure 1. Chemical structure of antidepressive and anti-anxiety drugs. F

F F

N O H N N O S

O O O O O CI

N Chlormezanone Citalopram Fluoxetine Kavain

H H N H N H C H 3 O N N

N N H N N N

Mianserin Milnacipran Mirtazapine Nomifensine

CI O O O O O N N O S N OH H N N 2 CI N H O O N H O NH O HO N O H

Oxazepam Reboxetine Sulpiride Temazepam Venlafaxine

70 Phenomenex l WEB: www.phenomenex.com TN-1146 APPLICATIONS

Material and Methods All analyses were performed using an Agilent® 1100 series LC provide the chemical identification number (CID) of the racemate. system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped This unique number can be linked to The PubChem Project web- with quaternary pump, in-line degasser, multi-wavelength UV de- site for further research regarding each compound’s pharmaceu- tector and autosampler. Lux® columns used for analysis were ob- tical properties. The table summarizes the Lux phases used, the tained from Phenomenex (Torrance, CA, USA). The HPLC column selectivity, the retention time of the first enantiomer, as well as dimensions were 250 x 4.6 mm ID and all columns were packed the isocratic conditions used for each compound. Lux columns with 5 μm particles. The flow rate was 1.0 mL/min and tempera- are quite successful at resolving chiral drugs of this type. All the ture was ambient. Standards were purchased from Sigma-Aldrich antidepressive and anti-anxiety agents tested are separated with (St. Louis, MO, USA). All solvents were purchased from EMD (San selectivity greater than 1.1. In the last column, the corresponding Diego, CA, USA). Phenomenex application number is provided. Those applications are easily accessible on our website (www.phenomenex.com/ Results and Discussion ChiralAppSearch) and can be searched by application number, Thirteen antidepressive and anti-anxiety racemates depict- structure, CID, or compound name. ed in Figure 1 were analyzed on five different Lux polysaccharide-based CSPs (Cellulose-1, Cellulose-2, Cellulose-3, Cellu- The chiral separations reported in Table 1 are baseline resolved lose-4, and Amylose-2) in normal phase (NP), polar organic (PO), with a resolution greater than 1.5. The retention time for the first and reversed phase (RP) separation modes. After performing a enantiomer is between 5 and 12 min and all the separations are systematic screening with various mobile phases, the best sepa- completed in less than 30 min. With basic analytes such as anti- ration was selected, even though in most of the cases, alternative depressive and anti-anxiety drugs, 0.1 % of diethylamine (DEA) is separation was obtained with other Lux phases and/or modes. used as mobile phase additive. DEA is an ion-masking agent that reduces unwanted interactions with residual silanols. DEA pro- The racemic antidepressive and anti-anxiety drugs separated in motes improved peak shape by minimizing ion-exchange interac- this study are listed in Table 1. For each compound tested we tions between silanol groups and basic analytes.

Table 1. Chiral separations of antidepressive and anti-anxiety drugs using Lux polysaccharide-based CSPs

Compound CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Chlormezanone 2717 Lux Cellulose-3 1.36 5.3 min PO MeOH/IPA (90:10) DEA (0.1 %) 20371 Citalopram 2771 Lux Cellulose-4 1.41 9.11 min NP Hex/IPA (80:20) DEA (0.1 %) 20424

Fluoxetine 3386 Lux Cellulose-1 1.3 8.94 min RP MeOH/20 mM NH4HCO3 (90:10) DEA (0.1 %) 20216 Kavain 5369129 Lux Cellulose-3 1.21 5.62 min PO MeOH/IPA (90:10) DEA (0.1 %) 20365

Mianserin 4184 Lux Cellulose-1 1.25 8.14 min RP MeOH/20 mM NH4HCO3 (90:10) DEA (0.1 %) 20225

Milnacipran 65833 Lux Cellulose-2 1.27 11.46 min RP MeOH/20 mM NH4HCO3 (60:40) DEA (0.1 %) 20227 Mirtazapine 4205 Lux Cellulose-4 1.64 5.61 min NP Hex/IPA (80:20) DEA (0.1 %) 20425

Nomifensine 4528 Lux Cellulose-3 1.76 5.84 min RP MeOH/20 mM NH4HCO3 (90:10) DEA (0.1 %) 20329

Oxazepam 4616 Lux Cellulose-1 2.32 5.94 min RP ACN/20 mM NH4HCO3 (60:40) DEA (0.1 %) 20232 Reboxetine 3022645 Lux Cellulose-1 1.57 11.4 min NP Hex/IPA (80:20) DEA (0.1 %) 20056 Sulpiride 5355 Lux Cellulose-3 1.1 10.87 min NP Hex/EtOH (80:20) DEA (0.1 %) 20463

Temazepam 5391 Lux Cellulose-1 1.32 6.08 min RP ACN/20 mM NH4HCO3 (60:40) DEA (0.1 %) 20236 Venlafaxine 5656 Lux Cellulose-2 1.11 6.47 min NP Hex/IPA (95:5) DEA (0.1 %) 20255

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol, DEA = Diethylamine * To view the full application enter the App ID onto the search field on our website

Phenomenex l WEB: www.phenomenex.com 71 TN-1146 APPLICATIONS

All of our Lux® products are pressure stable up to 300 bar and com- Conclusion patible with SFC separation mode using an organic modifier such In this study, we described the chiral separation of a variety of as MeOH, EtOH, IPA, or ACN. Two examples of chiral separation antidepressive and anti-anxity drugs using Lux polysaccha- for Fluoxetine and Milnacipran are shown in Figure 2. ride-based chiral stationary phases. All enantiomeric separations reported showed selectivity greater than 1.1 with the retention time for the first enantiomer below 12 min. Those Figure 2. separations can be used not only for analytical but for prepar- Representative chromatograms for the chiral separation of anti-depres- ative purposes since our phases are available in various pre- 20216 sants. parative formats such as Axia™ packed preparative columns or bulk media. 1 Fluoxetine on Lux 5 µm Cellulose-1 in RP References 2 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). a = 1.30 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. App ID 20216 202270 2 4 6 8 10 12 min

Milnacipran on Lux 5 µm Cellulose-2 in RP 1

a = 1.27 App ID 20227 0 2 4 6 8 10 12 14 16 18 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.

Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc.

Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383

• Multi 100-kilogram delivery worldwide Performance

• Over 15 durable HPLC bulk media offering unique and often orthogonal selectivities Service

• On site DAC packing assistance and technical seminars

Phenomenex l WEB: www.phenomenex.com 73 TN-1147 APPLICATIONS

Chromatographic Enantioseparation of Racemic Antifungal Drugs using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technical note, we report the chiral chromatographic matographic separation of enantiomers.1 A recent review point- separation of various antifungal agents using Lux polysaccha- ed out that in 2007 more than 90 % of the HPLC methods used ride-based chiral stationary phases. The reported enantiosepa- for the determination of enantiomeric excess were performed on rations are the results of a systematic screening of five different polysaccharide-based chiral stationary phases.2 The polysaccha- Lux phases in normal phase, polar organic, and reversed phase ride-based CSPs are frequently used for preparative purifications separation modes. because they are easily scaled-up from the analytical separations.3 Introduction Chiral separations can be performed by chromatographic sepa- Imidazole and triazole antifungal drugs inhibit the enzyme re- ration, enzymatic resolution, and crystallization. Chromatographic sponsible for converting lanosterol to ergosterol. Those drugs are enantioselective separation using chiral stationary phases (CSPs) effective in the treatment of fungal infections such as athlete’s for high performance liquid chromatography (HPLC) has signifi- foot and ring worm. The various antifungal agents analyzed in cantly evolved during the past few decades and is recognized this study are derived from imidazole or triazole and depicted in as the most popular and reliable tool for both analytical and pre- Figure 1. The chiral separations described in this application are parative separation of chiral compounds. Polysaccharide-based the results of a systematic screening of our five Lux polysaccha- CSPs such as Lux are the most widely used CSPs for the chro- ride-based CSPs (Cellulose-1, Cellulose-2, Cellulose-3, Cellu- lose-4, and Amylose-2) under various separation modes.

Figure 1. Chemical structure of antifungal agents.

CI N N N O N CI N CH2 N O O O CI N O N CI N O N CI

CI CI Bifonazole Econazole Enilconazole Ketoconazole

CI CI N N

N H CI O N O F N CI N N F CI N N OH N S N O2N N S N CI CI CI F Miconazole Ornidazole Sulconazole Tetramisole Voriconazole

74 Phenomenex l WEB: www.phenomenex.com TN-1147 APPLICATIONS

Material and Methods The table summarizes the Lux phases used, the selectivity, the All analyses were performed using an Agilent® 1100 series LC retention time of the first enantiomer, as well as the isocratic con- system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped ditions used for each compound. Lux columns are quite success- with quaternary pump, in-line degasser, multi-wavelength UV de- ful at resolving chiral drugs of this type. All the antifungal agents tector, and autosampler. Lux® columns used for analysis were ob- tested are separated with selectivity greater than 1.1. In the last tained from Phenomenex (Torrance, CA, USA). The HPLC column column, the corresponding Phenomenex application number is dimensions were 250 x 4.6 mm ID and all columns were packed provided. Those applications are easily accessible on our website with 5 μm particles. The flow rate was 1.0 mL/min and tempera- (www.phenomenex.com/ChiralAppSearch) and can be searched ture was ambient. Standards were purchased from Sigma-Aldrich by application number, structure, CID, or compound name. (St. Louis, MO, USA). All solvents were purchased from EMD (San Diego, CA, USA). The chiral separations reported in Table 1 are baseline resolved with a resolution greater than 1.5. The retention time for the first Results and Discussion enantiomer is between 5 and 14 min and all the separations are Nine antifungal agents racemates depicted in Figure 1 were an- completed in less than 30 min. With basic analytes such as antifun- alyzed on five different Lux polysaccharide-based CSPs (Cellu- gal agents, 0.1 % of diethylamine (DEA) is used as mobile phase lose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) in additive. DEA is an ion-masking agent that reduces unwanted in- normal phase (NP), polar organic (PO), and reversed phase (RP) teractions with residual silanols. DEA promotes improved peak separation modes. After performing a systematic screening with shape by minimizing ion-exchange interactions between silanol various mobile phases, the best separation was selected, even groups and basic analytes. Interestingly, out of 9 separations, 7 though in most of the cases, alternative separation was obtained are most successful in NP separation mode. NP mode is very with other Lux phases and/or modes. similar in polarity and selectivity to supercritical fluid chromatography (SFC) mode. In SFC mode, ammonium hydroxide in MeOH, The racemic antifungal agents separated in this study are list- EtOH, or IPA can be used as basic additives to help peak shape.4 ed in Table 1. For each antifungal agents tested we provide the SFC mode is particularly attractive for its high-throughput5, low chemical identification number (CID) of the racemate. This unique solvent consumption, low pressure drop, and high resolution. number can be linked to The PubChem Project website for further Another great advantage is the ease of scale–up to preparative research regarding each compound’s pharmaceutical properties. scale, especially with our Axia™ packed preparative product line.

Table 1. Chiral separations of antifungal agents using Lux polysaccharide-based CSPs

Compound CID CSPs (α) Rt (min) Mode Mobile Phase App ID* Bifonazole 2378 Lux Cellulose-2 1.57 8.9 min NP Hex/EtOH (80:20) DEA (0.1 %) 20506 Econazole 3198 Lux Cellulose-3 2.84 5.92 min NP Hex/EtOH (40/:60) DEA (0.1 %) 20110 Enilconazole 37175 Lux Cellulose-4 1.39 7.19 min NP Hex/IPA (60:40) DEA (0.1 %) 20427 Ketoconazole 3823 Lux Cellulose-1 1.25 13.71 min PO MeOH/IPA (90:10) DEA (0.1 %) 20353 Miconazole 4189 Lux Cellulose-3 2.18 5.21 min NP Hex/EtOH (96:4) DEA (0.1 %) 20129 Ornidazole 28061 Lux Amylose-2 5.36 5.46 min NP Hex/IPA (40:60) DEA (0.1 %) 20530 Sulconazole 5318 Lux Cellulose-2 1.67 12.25 min NP Hex/IPA (40:60) DEA (0.1 %) 20126 Tetramisole 3913 Lux Cellulose-2 1.48 6.66 min PO ACN/IPA (95:5) DEA (0.1 %) 20284 Voriconazole 5231054 Lux Cellulose-4 4.16 7.26 min NP Hex/EtOH (20:80) DEA (0.1 %) 20421

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, MeOH = Methanol, FA = Formic acid, DEA = Diethylamine * To view the full application enter the App ID onto the search field on our website.

Phenomenex l WEB: www.phenomenex.com 75 TN-1147 APPLICATIONS

All of our Lux® products are pressure stable up to 300 bar and Conclusion compatible with SFC separation mode using an organic modifier In this study, we described the chiral separation of a variety such as MeOH, EtOH, IPA, or ACN. Two examples of chiral sep- of antifungal agents using Lux® polysaccharide-based chi- aration for Bifonazole and Voriconazole are shown in Figure 2. ral stationary phases. All enantiomeric separations reported showed selectivity greater than 1.1 with the retention time for Figure 2. Representative chromatograms for the chiral separation of anti- the first enantiomer below 14 min. Those separations can be fungal agents. used not only for analytical but for preparative purposes since

Bifonazole20506 on Lux 5 µm Cellulose-2 in NP our phases are available in various preparative formats such mAU as Axia™ packed preparative columns or bulk media. 800 References a = 1.57 1 600 1. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). 2. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 2

400 3. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. 4. Hamman, C.; Schmidt Jr., D. E.; Wong, M.; Hayes, M. J. Chromatogr. A 2011, 1218, 7886– 7894. 200 5. Miller L. J. Chromatogr. A 2012, 1250, 250. (Review). App ID 20506

0 2 4 6 8 10 12 14 min

Voriconazole on Lux 5 µm Cellulose-4 in NP 20421 mAU

a = 4.16 1

100

0 App ID 20421

0 10 20 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Lux is a registered trademark and Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 © 2013 Phenomenex, Inc. All rights reserved. 76 Phenomenex l WEB: www.phenomenex.com Clinical Drugs Applications

Clinical Drugs Applications page

Synthetic Cannabinoids Metabolites [TN-1167]...... 78

Phenomenex l WEB: www.phenomenex.com 77 TN-1167 APPLICATIONS

Chiral LC/MS/MS Method for Analyzing Metabolites of the Synthetic Cannabinoids JWH-018 and AM2201 Contained in K2/Spice Herbal Mixtures using Strata™-X-Drug B SPE and Lux® Cellulose-3 Chiral Column

Marc Jacob, Matthew Trass, Art Miranda, Shahana Huq, Michael McCoy, JT Presley, and Erica Pike Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

In this technote, we describe a new targeted metabolomic approach This technote describes a novel LC/MS/MS method and SPE pro- for assessing human synthetic cannabinoid exposure and pharma- cedure capable of simultaneously resolving enantiomers as well as cology in blood and urine samples. The method utilizes a Solid Phase parent compounds and other related metabolites. Extraction (SPE) step followed by chiral LC/MS/MS analysis using a Lux polysaccharide-based chiral stationary column providing a Materials and Methods reliable and reproducible method that can be transferred to clinical Reagents and chemicals were obtained from Sigma-Aldrich research, forensic, and toxicology labs for analytical testing. (St. Louis, MO), Fisher Scientific (Pittsburgh, PA) and Hemostat Laboratories (Dixon, CA). All sample and analytical standards in- Introduction cluding chiral isomers of JWH-018-(ω-1)-OH and AM2201-(ω-1)- Herbal mixtures labeled as “K2” or “Spice” are often marketed OH were synthesized and provided by Cayman Chemical (Ann as legal marijuana substitutes to circumvent existing regulations Arbor, MI). Strata-X-Drug B polymeric strong cation-exchange and to avoid detection in standard drug screens. These products solid phase extraction cartridges, Lux Cellulose-3 analytical commonly contain the synthetic cannabinoid parent drugs JWH- column and SecurityGuard™ were obtained from Phenomenex 018 (Figure 1, Parent Drug 1) and AM2201 (Figure 1, Parent Drug (Torrance, CA). Samples were prepared using a Gilson Nebula 2), both aminoalkylindoles and potent cannabinoid receptor ago- 215 solid phase extraction system (Middleton, WI) and analyzed nists. With reports now indicating that 1 in 9 high school students using an Agilent® 1200 Series quaternary liquid chromatography experiment with synthetic cannabinoids and several medical re- system (Santa Clara, CA) interfaced with an API 4000™ QTRAP® ports specifically linking human injury and death to JWH-018 and tandem mass spectrometer (AB SCIEX, Framingham, MA). The AM2201, public health officials are increasingly concerned about operation of the HPLC system and mass spectrometer was con- abuse trends associated with these emerging cannabinoids. trolled by Analyst® software (version 1.5.1, AB SCIEX, Framing- ham, MA). Unfortunately, little is known about the metabolism and toxicology of these new drugs, but several clinical investigations identify Sample Pretreatment: the (ω -1)-hydroxyl metabolites enantiomers (Figure 1, Metabo- lites 1a/1b or 2a/2b), (ω)-hydroxyl (Metabolite 3) and (ω)-carboxyl Urine sample (Metabolite 4) as primary biomarkers. These metabolites are also See Reference 1 for internal standard preparation and complete known to retain significant in vitro and in vivo pharmacological ac- experimental details. tivity, which may offer a mechanistic explanation of the adverse effects associated with synthetic cannabinoid use. Since the Blood sample (ω-1)-hydroxyl metabolites of JWH-018 and AM2201 are chiral Pipette 50 µL of blood into 950 µL 0.1M sodium acetate buffer molecules, analytical procedures capable of low level quantifica- (pH 5.0) and spike with 10 µL of internal standard (IS) solution. tion of specific enantiomeric metabolites are required to further The sample was then subjected to the SPE method described understand the metabolic and toxicological consequences of syn- below. thetic cannabinoid use. SPE Procedure

Figure 1. Cartridge: Strata-X-Drug B, 30 mg/3 mL Parent drugs and metabolic oxidation compound structures. The circled Part No.: 8B-S128-TBJ compounds are chiral metabolites. Condition: NOT REQUIRED Equilibrate: NOT REQUIRED Load: 1 mL pretreated sample Wash: 1 mL Sodium acetate buffer Wash: 1 mL Sodium acetate buffer/Acetonitrile (70:30) Elute: 5 mL Ethyl acetate/Isopropanol (85:15) Dry: Dry down completely under a stream of nitrogen @ 60 °C Reconstitute: 100 µL Ethanol

78 Phenomenex l WEB: www.phenomenex.com TN-1167 APPLICATIONS

HPLC Conditions Figures 2 and 3 represent LC/MS/MS chromatograms produced Column: Lux® 3 μm Cellulose-3 from 10 ng/mL and 5 ng/mL (respectively) synthetic cannabinoid Dimensions: 150 x 2.0 mm quality control samples in human urine and blood (all synthetic Part No.: 00F-4492-B0 cannabinoids standard were provided by Cayman Chemical). The Mobile Phase: A: 20 mM Ammonium bicarbonate B: Acetonitrile chromatography was similar in all standards. The different color Gradient: Time (min) B (%) tracings are representative of the Specific Reaction Monitoring 0 40 (SRM) experiments for each specific metabolite (seeTable 1 for 10 95 SRM transitions). 12 95 15 40 Figure 2. 16 40 LC/MS/MS chromatograms produced from a representative 10 ng/mL Flow Rate: 0.5 mL/min quality control sample prepared in pooled human urine Temperature: 40 °C Detection: Tandem Mass Spectrometer (MS/MS) Detector: API 4000™ QTRAP® (AB SCIEX) ( ω)-hydroxyl 3 JWH-018 1 Table 1. AM2201 2 Mass Spectrometry Parameters for Selective Reaction Monitoring (SRM) AM2201 ( ω − 1 ) 2a/2b

Analyte Q1 (m/z) Q3 (m/z) ( ω)-carboxyl 4 155* JWH-018 ( ω − 1 ) 1a/1b AM2201 360 127† 155* (R)-(-)-AM2201-(ω-1)-OH 376 127† 2e+4 155* R R (S)-(+)-AM2201-(ω-1)-OH 376 † 7500 127 S S 155* JWH-018 342 5000 127† 2e+4 155* 2500 JWH-018-(ω)-OH 358 127† 155* JWH-018-(ω)-COOH 372 127† 1e+4 155* (R)-(-)-JWH-018-(ω-1)-OH 358 Signal (cps) 127† 155* (S)-(+)-JWH-018-(ω-1)-OH 358 127† 5e+3 *Quantification Ion † Confirmation Ion App ID 22141

2 4 6 8 min Results and Discussion Figure 3. JWH-018 is metabolized in humans to form the (ω)-monohydrox- LC/MS/MS chromatograms produced from a representative 5 ng/mL ylated, (ω)-carboxylated, and (ω-1)-monohydroxylated metabolites. quality control sample prepared in blood AM2201 exposure leads to the formation of common (ω)-JWH-018 metabolites but also the distinct (ω-1)-monohydroxylated AM2201 metabolites (Figure 1). A targeted metabolomic approach that ( ω)-hydroxyl 3 simultaneously measures each primary metabolite including the JWH-018 1 enantiomeric (ω-1)-metabolites is required to facilitate future clin- AM2201 2 ical studies designed to understand the relationship between drug AM2201 ( ω − 1 ) 2a/2b metabolism and clinical symptoms documented after JWH-018 ( ω)-carboxyl 4 and AM2201 use. This new chiral LC/MS/MS approach achieves JWH-018 ( ω − 1 ) 1a/1b this requirement by resolving all metabolites of interest, including the R and S enantiomers of the (ω-1)-monohydroxylated metabolites of JWH-018 and AM2201 in human urine and blood (Fig- ures 2 and 3). The chromatography of standards, QC samples, and unknown urine specimens is similar for all matrices evaluated. Retention times established for each analyte internal standard re- mained constant (±0.1 min). All calibration curves were linear over the tested analytical range, where r2 values were ≥0.99. The lower limits of quantification (LLOQ) for each analyte are comparable to previous LLOQ measurements reported with similar methods and mass spectra are consistent with reference libraries previously reported. 2,3 App ID 22142

Phenomenex l WEB: www.phenomenex.com 79 TN-1167 APPLICATIONS Figures 4a and 4b show representative LC/MS/MS chro- JWH-018 and AM2201 are both subjected to cytochrome-P450 matograms of human samples which tested positive for the mediated oxidation as well as uridine diphosphate glucuronyl- (ω-1)-monohydroxylated metabolite of JWH-018 (Figure 1, Me- transferase (UGT) conjugation during the metabolism process. tabolites 1a/1b) and for the (ω-1)-monohydroxylated metabolite Cytochrome-P450 metabolizes JWH-018 and AM2201 in the lung of AM2201 (Figure 1, Metabolites 2a/2b). As shown in Figure 1, and liver while UGT is thought to be responsible for conjugating the (ω-1)-monohydroxylated metabolites are unique biomarkers each metabolite with glucuronic acid. The pie chart inset com- for each respective synthetic cannabinoid. pares the total relative percentage of free cytochrome P450 metabolites versus the total relative percentage of glucuronic acid Figure 4. conjugates. The conjugation percentage was determined by mea- Representative LC/MS/MS chromatograms produced from (A) a human suring metabolite concentrations pre- and post-ω-glucuronidase sample positive for the (ω-1)-monohydroxylated metabolite of JWH-018, treatment (see Reference 1 for full details). These results show and (B) a human urine sample positive for the (ω-1)-monohydroxylated that when patients are exposed to only JWH-018 (Figure 5), the metabolite of AM2201 JWH-018 (ω-1)-monohydroxylated metabolite was excreted in a much higher concentration as compared to the other JWH-018 metabolites studied. In contrast, AM2201 (ω-1)-monohydroxylated enantiomers were not preferentially excreted. This indicates that UGTs may exhibit stereospecificity toward chiral synthetic cannabinoid metabolites.

Conclusion The LC/MS/MS method described in this technical note is capable of fully resolving and quantifying chiral metabolites of JWH-

App ID 22143 App ID 22144 018 and AM2201 as well as parent drugs. The precision and ac- curacy measurements are similar to previously developed clinical and forensic assays which make this method easily transferrable to clinical research, forensic, and toxicology labs for analytical testing. Moreover, this chiral method can help researchers in the In Figure 5, we demonstrate how this method was used to gener- understanding and evaluation of the clinical toxicity, pharmaco- ate the metabolic profile of a human urine specimen which tested dynamics and pharmacokinetics of achiral and chiral synthetic positive for JWH-018/AM2201 metabolites. The relative percent- cannabinoid metabolites produced from JWH-018 and AM2201. age of each metabolite is represented and the relative percentage of S or R enantiomers is provided above the bar of the corre- References sponding (ω-1)-monohydroxylated metabolite. 1. Moran, J. H. et al. Anal. Chem. 2013, 85, 9390− 9399.

2. Moran, J. H. et al. Anal. Chem. 2011, 83, 6381− 6388. Figure 5. Metabolic profile generated from a human urine sample which tested posi- 3. Moran, J. H. et al. Anal. Chem. 2011, 83, 4228− 4236. tive forJWH-018/AM2201 metabolites

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Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions. Parent Cannabinoids Trademarks Cytochrome P450 Metabolites Lux is a registered trademark and Strata-X, SecurityGuard, and Giga are trademarks of Phe- nomenex. Agilent is a registered trademark of Agilent Technologies. QTRAP and Analyst are UDP-UGT Metabolites registered trademarks and API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license. Strata-X is patented by Phenomenex, Inc. U.S. Patent No. 7,119,145 SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362 Caution: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges.

Pesticide Applications page

Enantioseparation of Racemic Herbicides [TN-1162]...... 82 Enantiomeric and Diastereoisomeric Resolutions of Chiral Triazole Fungicides [TN-1164]...... 86

Phenomenex l WEB: www.phenomenex.com 81 TN-1162 APPLICATIONS Chromatographic Enantioseparation of Racemic Herbicide Agents using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA In this technical note, we report the chiral chromatographic sepa- following a different pathway. The degradation difference of chiral ration of various herbicide agents using Lux polysaccharide-based herbicides, combined with possible enantiospecific toxicity can chiral stationary phases. The reported enantioseparations are the affect not only efficacy, but also exposure and risk to humans and results of a systematic screening of five different Lux phases in environment. In the pharmaceutical industry, mainly due to the normal phase and reversed phase separation modes. potential enantiospecific toxicity, chiral drugs are routinely tested for chiral purity, whereas pesticides generally are not. Introduction Herbicides have many positive uses such as increasing food Separations of chiral compounds can be performed by chiral production, decreasing damage to crops, reducing plant diseas- chromatography using chiral stationary phases (CSPs) in high es, and more, but they also pose risks to humans and the envi- performance liquid chromatography (HPLC). HPLC is recognized ronment. Of the 1,693 pesticides listed in a recent review,1 482 as the most popular and reliable tool for both analytical and pre- (28 %) are chiral (chemical compounds containing one or more parative separation of chiral compounds. As a matter of fact, centers of asymmetry) of which 150 are classified as herbicides. 76 % of the analytical chiral separations reported in the recent The mode of action for many herbicides is to interfere with chiral chiral pesticides review1 were performed by HPLC, and gas chro- plant hormones controlling growth and, therefore, the configura- matography (GC) was second with 18 % of the separations re- tion of the herbicides plays a role in efficacy. As a result, some ported. Polysaccharide-based CSPs such as Lux are the most of those herbicides, such as dichlorprop-methyl, diclofop-methyl, widely used phases for the chromatographic separation of en- fenoxaprop-ethyl, and haloxyfop-methyl are produced as single antiomers.3,4 Those CSPs show excellent success rate for chiral O or enriched stereoisomer formulation. Additionally, the degrada- separation of a broad range of chiral compounds, as well as high tion of those chiral herbicides by soil microbes is enantioselec- loading ability for preparative applications. The various herbicide O tive2 and each enantiomer will be eliminated from the environment agents analyzed in this study are depicted in Figure 1. O

Figure 1. Chemical structure of herbicides agents racemic mixtures separation modes O OHCI OH O CI O O O O O O O O O NH O O CI O O O CI O O CI O O CI CI CI CI O O O O Fenoxaprop-ethyl CI NH CI Diclofop-methyl Dichlorprop-methyl CI DiclofopF F CI F F CI CI F F F F F F F F F F F F F F O O N CI CI CI O N CI CI CI O F F F F F F O O F O NO N O NO N F O N O N O O O O O O O F OH O O F F F F F F O O O O O O O OO NO F F F O N + O O O O O O O O O O O O O N N HO N N N O Fluazifop-butyl HOHO O HOO O O O O OO O NH CI HaloxyfopO Haloxyfop-methyl O

O F F OH O O F O H H O N O N O Loctofen N + H H O N H H H N H O O N NO N O O N NO N N O O O O N N N N N N N O Imazaquin O O O OO O O O O OO O NH Imazamethabenz-methyl CI O

F F 82 Phenomenex WEB: www.phenomenex.com l F TN-1162 APPLICATIONS

Material and Methods provide the chemical identification number (CID) of the racemate. All analyses were performed using an Agilent® 1100 series LC This unique number can be linked to The PubChem Project web- system (Agilent Technologies Inc., Palo Alto, CA, USA) equipped site for further research regarding each compound’s pharmaceu- with quaternary pump, in-line degasser, multi-wavelength UV de- tical properties. The table summarizes the Lux phases used, the tector, and autosampler. Lux® columns used for analysis were ob- selectivity, the retention time of the first enantiomer, as well as the tained from Phenomenex (Torrance, CA, USA). The HPLC column isocratic conditions used for each compound. dimensions were 250 x 4.6 mm ID and all columns were packed with 5 μm particles. The flow rate was 1.0 mL/min and tempera- Lux columns are quite successful at resolving chiral compounds ture was ambient. Standards were purchased from Sigma-Aldrich of this type. All the herbicides agents tested are separated with (St. Louis, MO, USA). All solvents were purchased from EMD (San selectivity greater than 1.1. In the last column, the corresponding Diego, CA, USA). Phenomenex application number is provided. Those applications are easily accessible on our website (www.phenomenex.com/Chi- Results and Discussion ralAppSearch) and can be searched by application number, struc- Eleven racemic herbicide agents depicted in Figure 1 were an- ture, CID, or compound name. The chiral separations reported in alyzed on five different Lux polysaccharide-based CSPs (Cellu- Table 1 are baseline resolved with a resolution greater than 1.5. lose-1, Cellulose-2, Cellulose-3, Cellulose-4, and Amylose-2) in The retention time for the first enantiomer is between 5 and 19 min normal phase (NP) and reversed phase (RP) separation modes. and all the separations are completed in less than 21 min. With After performing a systematic screening with various mobile basic and neutral herbicides derivatives, 0.1 % of diethylamine phases, the best separation was selected, even though in most (DEA) was used as an additive whereas with acidic derivatives of the cases, alternative separation was obtained with other Lux 0.1 % of formic acid (FA) was used as the additive. The presence phases and/or modes. The racemic herbicide agents separated of DEA favors dissociation of the amino group and improves peak in this study are listed in Table 1. For each compound tested we shape. A similar effect is observed with formic acid as the additive with acidic pain reliever such as Dichlofop and Haloxyfop.

Table 1. Chiral separations of herbicides agents using Lux polysaccharide-based CSPs

Compound CID CSPs (α) Rt (min) Mode Mobile Phase App ID*

Dichlorprop-methyl 90988 Lux Amylose-2 1.06 19.22 RP ACN/20 mM NH4HCO3 (40:60) DEA (0.1 %) 21761 Diclofop 38687 Lux Amylose-2 1.25 8.94 NP Hex/IPA (80:20) FA (0.1 %) 21687 Diclofop-methyl 39985 Lux Cellulose-1 2.51 6.23 NP Hex/IPA (80:20) DEA (0.1 %) 21688 Fenoxaprop-ethyl 47938 Lux Cellulose-2 2.63 4.98 NP Hex/IPA (80:20) DEA (0.1 %) 21694

Fluazifop-butyl 50897 Lux Cellulose-3 1.31 7.89 RP ACN/20 mM NH4HCO3 (60:40) DEA (0.1 %) 21786

Haloxyfop 50895 Lux Cellulose-3 1.12 7.92 RP ACN / H2O (50:50) FA (0.1 %) 21793 Haloxyfop-methyl 50896 Lux Amylose-2 1.21 6.34 NP Hex/IPA (80:20) DEA (0.1 %) 21707 Imazamethabenz-methyl 54744 Lux Cellulose-4 1.24 7.75 NP Hex/IPA (80:20) DEA (0.1 %) 21711 Imazaquin 54739 Lux Cellulose-3 1.38 5.06 NP Hex/EtOH (60:40) DEA (0.1 %) 21714 Loctofen 62276 Lux Cellulose-2 1.37 7.11 NP Hex/IPA (80:20) DEA (0.1 %) 21716

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, Hex = Hexane, H2O = Water, FA = Formic acid, DEA = Diethylamine * To view the full application enter the App ID onto the search field on our website.

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All of our Lux® products are pressure stable up to 300 bar. Two ex- Conclusion amples of chiral separation for Fenoxaprop-ethyl and Haloxyfop In this study, we described the successful chiral separation of are shown in Figure 2. a variety of herbicide agents using Lux polysaccharide-based chiral stationary phases. All enantiomeric separations report- Figure 2. ed showed selectivity greater than 1.1 with the retention time Representative chromatograms for the chiral separation of herbicides. for the first enantiomer below 19 min. Those separations can be used not only for analytical but for preparative purposes

21694 Fenoxaprop-ethyl on Lux 5 µm Cellulose-2 in NP since our phases are available in various preparative formats mAU such as Axia™ packed preparative columns or bulk media. 800 4.981 References

600 1. Ulrich E.M.; Morrison C.N.; Goldsmith M.R.; Foreman W.T. Reviews of Envi- ronmental Contamination and Toxicology, Springer, New York, NY, 2012, 217, Chapter 1, 1-74. 400 8.703 = 2.63 2. Müller, M.D.; Buser, H.R. Environ. Sci. Technol. 1997, 31, 1953–1959

200 3. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review).

App ID 21694 4. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. 0

0 2 4 6 8 10 12 14 min

AppHaloxyfop ID 21793 on Lux 5 µm Cellulose-3 in RP mAU 400

7.916 8.499

= 1.12

200

0 App ID 21793

0 2 4 6 8 10 12 14 min

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.

Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc.

Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383

© 2013 Phenomenex, Inc. All rights reserved. 84 Phenomenex l WEB: www.phenomenex.com Need help? Give us a call! • Application support • Method improvement and optimization • New method development • Pre-validation services • Scale-up and purification • On-site training and consulting … and more!

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Enantiomeric and Diastereoisomeric Resolutions of Chiral Triazole Fungicides using Lux® Polysaccharide-Based Chiral Stationary Phases

Marc Jacob, Liming Peng, Michael Klein, Tom Cleveland, and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA In this technical note, we report the enantiomeric and diastereo- radation of those chiral fungicides by soil microbes is stereose- isomeric separations of five fungicides containing two stereogenic lective and each stereoisomer will be eliminated from the environ- centers using Lux polysaccharide-based chiral stationary phases. ment following a different pathway.2,3 The degradation difference The reported separations are the results of a systematic screening of chiral fungicides, combined with possible stereospecific tox- of five different Lux phases in normal phase and reversed phase icity can affect not only efficacy, but also exposure and risk to separation modes. For each compound screened, baseline res- humans and environment.3 In the pharmaceutical industry, mainly olution of the four different stereoisomers is provided with a run due to the potential stereospecific toxicity, chiral drugs are rou- time below 25 minutes. tinely tested for chiral purity, whereas pesticides generally are not.

Introduction In this application note, we present the enantiomeric and dias- Fungicides have many positive uses such as increasing food pro- tereoisomeric separations of five triazole fungicides: Bromuco- duction, decreasing damage to crops, reducing plant diseases, nazole, Cyproconazole, Difenoconazole, Propiconazole, and Tri- and more, but they also pose risks to humans and the environ- adimenol. The chemical structure for each fungicide is represent- ment. Of the 1693 pesticides listed in a recent review,1 482 (28 %) ed in Figure 1. are chiral (chemical compounds containing one or more centers of asymmetry) of which 104 are classified as fungicides. The deg-

Figure 1. Chemical structure of chiral fungicides

N O N * Br * N O * N N * O N N Cl * N OH N * Cl

Cl Cl Cl Bromuconazole Cyproconazole Difenoconazole

N N O * N * OH N * N * O O Cl

Cl Cl Propiconazole Triadimenol

86 Phenomenex l WEB: www.phenomenex.com TN-1164 APPLICATIONS

All triazole fungicides evaluated in this application contain two matter of fact, 76 % of the analytical chiral separations reported stereogenic centers and therefore can have four stereoisomers in the recent chiral pesticides review1 were performed by HPLC; as depicted in Figure 2 for the example of Difenoconazole. The gas chromatography (GC) was second with 18 % of the separa- stereoisomers that are mirror images are also called enantiomers tions reported. Polysaccharide-based CSPs such as Lux® are the (SS/RR and SR/RS). Enantiomers can be separated from each most widely used phases for the chromatographic separation of other by chiral chromatography using chiral stationary phases enantiomers.4,5 Those CSPs show excellent success rate for chiral (CSPs) in high performance liquid chromatography (HPLC). HPLC separation of a broad range of chiral compounds, as well as high is recognized as the most popular and reliable tool for both an- loading ability for preparative applications under both, HPLC6 and alytical and preparative separation of chiral compounds.4 As a supercritical fluid chromatography (SFC)7.

Figure 2. Structure of stereoisomeres for Difenoconazole

Diastereoisomers

H H

O O O O Cl Cl

N N N N O N Cl O N Cl

(2S, 4R) (2S, 4S) Enantiomers Enantiomers H H

O O O Cl O Cl

N N N N Cl O Cl O N N (2R, 4S) (2R, 4R)

Diastereoisomers

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Material and Methods All HPLC analyses were performed using an Agilent® 1100 se- each fungicide screened, we provide the chemical identification ries LC system (Agilent Technologies, Inc., Palo Alto, CA, USA) number (CID). This unique number can be linked to The PubChem equipped with quaternary pump, in-line degasser, multi-wave- Project website for further research regarding each compound’s length UV detector, and autosampler. Lux® columns used for anal- pharmaceutical properties. Additionally, the Lux phases used, the ysis were obtained from Phenomenex (Torrance, CA, USA). The retention time of the first and last stereoisomers, as well as the HPLC column dimensions were 250 x 4.6 mm ID and all columns isocratic conditions used for each compound are listed in Table were packed with 5 μm particles. The flow rate was 1.0 mL/min 1. As expected, polysaccharide-based Lux columns are quite and temperature was ambient. Standards were purchased from successful at resolving chiral compounds of this type. For each Sigma-Aldrich (St. Louis, MO, USA). All solvents were purchased fungicide tested, all the stereoisomers are separated with selec- from EMD (San Diego, CA, USA). tivity greater or equal to 1.1 between adjacent peaks. In the last column of the Table 1, the corresponding Phenomenex applica- Results and Discussion tion number is provided. Those applications are easily accessible The five triazole fungicides depicted inFigure 1 were analyzed on our website (www.phenomenex.com) and can be searched by on Lux polysaccharide-based CSPs (Cellulose-1, Cellulose-2, application number, structure, CID, or compound name. Cellulose-3, Cellulose-4, and Amylose-2) in normal phase (NP) and reversed phase (RP) separation modes. After performing a systematic screening, the separations that showed optimum resolution between all the peak were selected, even though in most of the cases, alternative separation was obtained with other Lux phases and/or modes. The separation results as well as the selectivity between each stereoisomer are summarized in Table 1. For

Table 1. Enantiomeric and diastereoisomeric separations of fungicides using Lux polysaccharide-based CSPs

Rt Rt α α α Analyte CID CSPs Mobile Phase 1 4 App ID (min) (min) (1,2) (2,3) (3,4)

Bromuconazole 3444 Lux Cellulose-2 ACN/20 mM NH4HCO3 (60:40) DEA (0.1 %) 14.63 23.18 1.36 1.14 1.11 21751

Cyproconazole 86132 Lux Cellulose-4 ACN/20 mM NH4HCO3 (60:40) DEA (0.1 %) 6.44 9.16 1.20 1.18 1.25 21755 Difenoconazole 86173 Lux Cellulose-3 Hexane/EtOH (85:15) DEA (0.1 %) 11.07 15.10 1.07 1.07 1.29 21681 Propiconazole 43234 Lux Cellulose-1 Hexane/IPA (80:20) DEA (0.1 %) 6.83 10.16 1.33 1.21 1.12 21726 Triadimenol 41368 Lux Cellulose-2 Hexane/IPA (80:20) DEA (0.1 %) 5.06 7.58 1.28 1.36 1.19 21739

ACN = Acetonitrile, IPA = Isopropanol, EtOH = Ethanol, DEA = Diethylamine, NH4HCO3 = Ammonium bicarbonate

88 Phenomenex l WEB: www.phenomenex.com TN-1164 APPLICATIONS

The enantiomeric and diastereoisomeric separations for the stereoisomers of Bromuconazole, Cyproconazole, Difenoconazole, Propiconazole and Triadimenol are respectively shown in Figure 3, 4, 5, 6 and 7.

Figure 3. Stereoselective HPLC analysis on the stereoisomers mix of Bromuconazole App ID 21751 mAU 140 N O N * Br * N 120 O * 14N .632 N * O N N Cl * N OH N * 100 Cl 18.931 21.166 80 23.175 O

60 Cl Cl Cl 40 Column: Lux® 5 µm Cellulose-2 Mobile Phase: 0.1 % Diethylamine Acetonitrile / 0.1 % Diethylamine in 20 mM Ammonium bicabonate (60:40) 20 N

N N App ID 21751 0 O * N * OH N * N * O O min 0 10 Cl 20

Figure 4. Stereoselective HPLC analysis on the stereoisomers mix of Cyproconazole App ID 21755 Cl Cl mAU N O * N N Br * N 400 O * N 6.437 * O N N 7.14Cl1 * N OH N * Cl 7.909 9.162 O

200 Cl Cl Cl

Column: Lux 5 µm Cellulose-4 Mobile Phase: 0.1 % Diethylamine Acetonitrile / N 0.1 % Diethylamine in 20 mM Ammonium bicabonate (60:40) N N O * N * OH App ID 21755 0 N * N * O O

0 Cl2 4 6 8 10 12 min

Cl Cl Phenomenex l WEB: www.phenomenex.com 89 TN-1164 APPLICATIONS

Figure 5. Stereoselective HPLC analysis on the stereoisomers mix of Difenoconazole 21681 mAU N O N 600 * Br * N 11.065 O * N N * O N N Cl 11.650 * N OH N * 12.297 Cl =1.07 400 15.097 O

Cl Cl N 200 Cl O N Column: Lux® 5 µm Cellulose-3 * Br * N O Mobile* Phase: 0.1 % DiethylamineN Hexane / N * O N 0.1 % Diethylamine in Ethanol (85:15) N * N Cl N OH App ID 21681 N * N 0 Cl N O * N * OH N N O * * 0 2 4 O 6 8 10 12 14O 16 18 min Cl Figure 6. Cl Cl Stereoselective HPLC analysis on the stereoisomers mix of Propiconazole Cl 21726 Cl Cl mAU N

600 N N O * N * OH N 6.829 * N * O O Cl 400 10.162

= 1.33 8.212 Cl Cl 9.391

200 Column: Lux 5 µm Cellulose-1 Mobile Phase: 0.1 % Diethylamine Hexane / 0.1 % Diethylamine in Isopropanol (80:20)

0 App ID 21726

0 2 4 6 8 10 12 min

90 Phenomenex l WEB: www.phenomenex.com N O N * Br * N O * N N * O N N Cl * N OH N * TN-1164 Cl

APPLICATIONS O

Figure 7. Cl Cl Stereoselective HPLC analysis on the stereoisomers mix of Triadimenol Cl 21739

N mAU 5.723 N N O * N * OH 400 N * N * O O Cl 7.576

= 1.28 200 Cl Cl 5.062

® Column: Lux 5 µm Cellulose-2 6.813 Mobile Phase: 0.1 % Diethylamine Hexane / 0.1 % Diethylamine in Isopropanol (80:20) App ID 21739 0

0 2 4 6 8 10 12 14 min

Conclusion References In this application note, we described the enantiomeric and 1. Ulrich E.M.; Morrison C.N.; Goldsmith M.R.; Foreman W.T. Reviews of Envi- diastereoisomeric resolution of five fungicide agents contain- ronmental Contamination and Toxicology, Springer, New York, NY, 2012, 217, ing 2 stereogenic centers using Lux polysaccharide-based Chapter 1, 1-74. chiral stationary phases. All stereoisomeric separations re- 2. Garrison, A.W.; Avants, J.K.; Jones, W.J. Environ. Sci. Technol. 2011, 45, ported showed baseline resolution between all stereoisomers 2186-2193. with run time below 25 min. Those separations can be used 3. Dong F. et al. Environ. Sci. Technol. 2013, 47, 3386-3394. not only for analytical but for preparative purposes since our 4. Chankvetadze, B. J. Chromatogr. A 2012, 1269, 26-51. (Review). phases are available in various preparative formats such as 5. Ikai, T.; Okamoto, Y. Chem. Rev. 2009, 109, 6077-6101. ™ Axia packed preparative columns or bulk media. These ana- 6. Francotte, E. J. Chromatogr. A 2001, 906, 379-397. (Review) lytical and preparative products can also be used under SFC mode for higher throughput.8 7. Miller L. J. Chromatogr. A 2012, 1250, 250. (Review). 8. Phenomenex TN-9002.

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.

Trademarks Lux is a registered trademark of Phenomenex. Axia is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc.

Disclaimer Comparative separations may not be representative of all applications. Phenomenex is not affiliated with Agilent.

Axia is patented by Phenomenex. U.S. Patent No. 7,674,383

Free Chiral Screening Services, provided by PhenoLogix www.phenomenex.com/PhenoLogix

Phenomenex l WEB: www.phenomenex.com 91 Axia Packed Preparative Columns

Axia packed preparative columns involve a single axial compression With Axia patented column packing technology, the ideal column bed step unlike conventional packed preparative columns like DAICEL density is custom calculated and automated for each LUX media and CHIRALCEL and CHIRALPAK prep columns. During the Axia pack- column dimension (21.2, 30 and 50 mm ID) producing both proper ing process, the packing piston is locked in place, eliminating any bed density and column uniformity every time. This solves common decompression and then re-compression of the media sorbent, thus lifetime and performance problems associated with conventional maintaining media and column bed integrity. packing processes for preparative columns.

Maximize Chiral Purification Performance

• Longer Column Lifetimes • Improved Column-to-Column Reproducibility • Recover Higher Compound Purity

Axia Packing Process Involves: Compression ➞ Final Column

Packing piston locked. No media Media maintains initial optimal or pressure escape packing density and shape

Silica media packed under piston pressure

View Axia Packing Process at www.AxiaPrep.com Column

Specialized computer If Axia packed columns do not provide control for ideal pressure at least an equivalent separation as compared to a competing preparative column of the same particle size, same phase, and dimensions, return the column with comparative data within 45 Conventional packing process involves: days for a FULL REFUND. Only applies to Compression ➞ Decompression ➞ Re-compression ➞ Final Column 21.2 mm ID columns.

Media density disruption Media escapes from and potential media crushing disconnected column Removal of escaped media Pump Column

“Axia packed column has a great efficiency for the separation of several classes of natural compounds. Due to its low back pressure Violent re-compression and therefore high flow work conditions, time for conditioning the Silica media packed of silica media under liquid pressure columns is sped up greatly! ” Sylvian Cretton - Europe.

Diagram from Waters Corporation U.S. Patent No. 7,399,410

92 Phenomenex l WEB: www.phenomenex.com Axia Outperforms All Other Prep Columns

Axia specialized preparative hardware shows higher performance columns. One column was packed using Axia technology and the than traditionally packed standard hardware preparative columns. other prep column was packed using the traditional slurry packing This revolutionary packing technology paired with Lux® polysaccha- process. ride-based chiral stationary phases provide purification results like no other chiral column can provide. The Axia packing technology had a substantial increase in column efficiency resulting in increased resolution over traditionally packed To better understand how much Axia technology improves column preparative columns. With increased resolution you are able to in- performance over traditionally slurry packed preparative columns we crease your sample load enabling you to purify more target com- scaled-up a 5 μm Lux® Cellulose-1 chiral media analytical column pound(s) per purification run. This equates to better throughput and and packed the same media into two different 150 x 21.2 mm I.D. economics.

Warfarin Chiral Purification in Normal Phase Mode Analytical

mAU Column: Lux 5 µm Cellulose-1 Dimensions: 150 x 4.6 mm 4000 Mobile Phase: Hexane/Ethanol (75:25) Flow Rate: 1 mL/ min 3000 Temperature: Ambient Inj. Volume: 100 µL

2000

1000

0 App ID 21922 -1000 0 1 2 3 4 5 6 7 min Warfarin

Standard Packing and Hardware Axia Technology and Hardware mV mV 2750 2750 2500 Rs = 3.72 2500 2250 2250 2000 2000 Rs = 2.85 1750 1750 30 % Increase in Resolution 1500 1500 1250 1250 1000 1000 750 750 500 500 250 250 App ID 21920 App ID 21921 0 0

0 1 2 3 4 5 6 7 min 0 1 2 3 4 5 6 7 8 min

Conditions for both PREP columns: Flow Rate: 20 mL/ min Media: Lux 5 µm Cellulose-1 Temperature: Ambient Dimensions: 150 x 21.2 mm Inj. Volume: 2 mL Mobile Phase: Hexane / Ethanol (75:25)

42 % Increase in Efficiency Analytical Standard Axia Column (mm) 150 x 4.6 150 x 21.2 150 x 21.2 Mass Loaded (mg) 2 40 40 Resolution* 1.5 2.85 3.72 Plates (N) 117 535 760

* Resolution calculated with peak width at baseline and center retention time due to the overloaded peaks being off-scale

“We have used Phenomenex Axia prep-HPLC columns for several Tip: If you are using CHIRALPAK® AD®, AD-H®, AD-3, years and they consistently provide excellent separation and AD-RH®, AD-3R then you must try Lux Amylose-2 reproducibility for a variety of different compounds.” for alternative selectivity. Jeremy R. Wolf. ABC Laboratories, USA.

Phenomenex l WEB: www.phenomenex.com 93 Column Protection

Less Frequent Chiral HPLC / PREP Column Replacement. HPLC Column Protection Save Time and Money. It’s a fact! Chemical contaminants and particulates are a natural part The easiest way to extend column performance is to remove these of any chromatographic analysis. contaminants and particulates with the SecurityGuard Cartridge Sys- tem before they reach your column and degrade your chromatography.

SecurityGuard:

• Protects and extends HPLC and PREP column lifetime

• Virtually no change in chromatography

• Simple to use

Fingertight connection to virtually any female inverted endfitting worldwide

Cut-away view showing cartridge – can be easily inspected for contaminants

Cartridge Replaced 110

100

Backpressure (Bar) Backpressure 30

20 Universal fingertight quick connection to 10 HPLC column – no wrenches required 0

-10 Column returns to initial backpressure Injections

Accelerated lifetime test using endogenous biomolecule matrix on a reversed phase C18 column, 5 µm, 50 x 4.6 mm with SecurityGuard C18 cartridges. Backpressure values represent additional backpressure contributed by SecurityGuard.

The SecurityGuard analytical cartridge holder (patented) directly Simply replace SecurityGuard cartridges instead of your ex- finger-tightens into virtually any manufacturer’s column with a fe- pensive HPLC columns. In this graph, once the expired Secu- male/inverted endfitting. Contaminants are retained by an inex- rityGuard cartridge was replaced, the pressure immediately pensive disposable cartridge instead of damaging your valuable dropped and the column performance was restored allowing for HPLC column investment. extended column use.

94 Phenomenex l WEB: www.phenomenex.com Helpful Chiral Resources Online Chiral Application Search Easily search over 2,000 chiral applications

Seach by: Application Structure Search by: Application Name www.phenomenex.com/ChiralStructureSearch www.phenomenex.com/ChiralNameSearch

Chiral and Prep Technotes • Over 20 detailed technotes to help give you greater in- sight into difficult chiral separations View them at: www.phenomenex.com/ Products/HPLCDetail/lux#technical-resources

Simplified Chiral Method Development Poster • Method Development walk-through for both HPLC and SFC conditions

• Convert your Lux column to different modes of Chiral Column Selection Guide chromatography • Discover the 3 Easy Ways to Choose the best Chiral Column for your application

Pick the RIGHT CHIRAL COLUMN The FIRST Time EVERY Time RISK-FREE*

See Inside for 3 Easy Ways

Polysaccharide Chiral Columns Dependable. Scalable. A ordable.

* See inside for complete ‘risk-free’ details.

Phenomenex l WEB: www.phenomenex.com 95 Ordering Information Lux Chiral Columns

Polysaccharide Chiral Columns Dependable. Scalable. A ordable.

3 µm Analytical Columns (mm) SecurityGuard™ Cartridges (mm) Phases 50 x 2.0 150 x 2.0 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 4 x 3.0* /10pk /10pk Cellulose-1 00B-4458-B0 00F-4458-B0 00B-4458-E0 00D-4458-E0 00F-4458-E0 00G-4458-E0 AJ0-8402 AJ0-8403 Cellulose-2 00B-4456-B0 00F-4456-B0 00B-4456-E0 00D-4456-E0 00F-4456-E0 00G-4456-E0 AJ0-8398 AJ0-8366 Cellulose-3 00B-4492-B0 00F-4492-B0 00B-4492-E0 00D-4492-E0 00F-4492-E0 00G-4492-E0 AJ0-8621 AJ0-8622 Cellulose-4 00B-4490-B0 00F-4490-B0 00B-4490-E0 00D-4490-E0 00F-4490-E0 00G-4490-E0 AJ0-8626 AJ0-8627 Amylose-2 00B-4471-B0 00F-4471-B0 00B-4471-E0 00D-4471-E0 00F-4471-E0 00G-4471-E0 AJ0-8471 AJ0-8470 for ID: 2.0 –3.0 mm 3.2–8.0 mm

5 µm Analytical Columns (mm) SecurityGuard™ Cartridges (mm) Phases 50 x 2.0 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 4 x 3.0* /10pk /10pk Cellulose-1 00B-4459-B0 00B-4459-E0 00D-4459-E0 00F-4459-E0 00G-4459-E0 AJ0-8402 AJ0-8403 Cellulose-2 00B-4457-B0 00B-4457-E0 00D-4457-E0 00F-4457-E0 00G-4457-E0 AJ0-8398 AJ0-8366 Cellulose-3 00B-4493-B0 00B-4493-E0 00D-4493-E0 00F-4493-E0 00G-4493-E0 AJ0-8621 AJ0-8622 Cellulose-4 00B-4491-B0 00B-4491-E0 00D-4491-E0 00F-4491-E0 00G-4491-E0 AJ0-8626 AJ0-8627 Amylose-2 00B-4472-B0 00B-4472-E0 00D-4472-E0 00F-4472-E0 00G-4472-E0 AJ0-8471 AJ0-8470 for ID: 2.0–3.0 mm 3.2–8.0 mm

SecurityGuard™ Bulk Media 5 µm Semi-Prep Columns (mm) Cartridges (mm) Phases 100 g 1 kg Phases 150 x 10.0 250 x 10.0 10 x 10.0‡ 10 µm m /3pk Cellulose-1 04G-4501 04K-4501 Cellulose-1† 00F-4459-N0 00G-4459-N0 AJ0-8404 Cellulose-2 04G-4502 04K-4502 Cellulose-2† 00F-4457-N0 00G-4457-N0 AJ0-8399 Cellulose-3 04G-4624 04K-4624 Cellulose-3 — 00G-4493-N0 AJ0-8623 Cellulose-4 04G-4625 04K-4625 Cellulose-4 — 00G-4491-N0 AJ0-8628 20 µm Amylose-2 00F-4472-N0 00G-4472-N0 AJ0-8472 Cellulose-1 04G-4473 04K-4473 for ID: 9–16 mm Cellulose-2 04G-4464 04K-4464 †Inquire for Lux 10 µm Cellulose-1 and Cellulose-2 columns. Cellulose-3 04G-4504 04K-4504 Cellulose-4 04G-4503 04K-4503 Please inquire for 20 µm Lux Amylose-2 media.

5 µm Axia™ Packed Preparative Columns (mm) SecurityGuard™ Cartridges (mm) Phases 150 x 21.2 250 x 21.2 250 x 30 250 x 50 15 x 21.2** 15 x 30.0♦ /ea /ea Cellulose-1† 00F-4459-P0-AX 00G-4459-P0-AX 00G-4459-U0-AX 00G-4459-V0-AX AJ0-8405 AJ0-8406 Cellulose-2† 00F-4457-P0-AX 00G-4457-P0-AX 00G-4457-U0-AX 00G-4457-V0-AX AJ0-8400 AJ0-8401 Cellulose-3 00F-4493-P0-AX 00G-4493-P0-AX 00G-4493-U0-AX 00G-4493-V0-AX AJ0-8624 AJ0-8625 Cellulose-4 00F-4491-P0-AX 00G-4491-P0-AX 00G-4491-U0-AX 00G-4491-V0-AX AJ0-8629 AJ0-8630 Amylose-2 00F-4472-P0-AX 00G-4472-P0-AX 00G-4472-U0-AX 00G-4472-V0-AX AJ0-8473 AJ0-8474 for ID: 18 –29 mm 30–49 mm *SecurityGuard Analytical Cartridges require holder, Part No. : KJ0-4282 **HPLC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8223 ‡SemiPrep SecurityGuard™ Cartridges require holder, Part No.: AJ0-7220 SFC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8617 ♦HPLC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8277 SFC PREP SecurityGuard Cartridges require holder, Part No. : AJ0-8618

Column Performance Check Standard Part No. Description Unit AL0-8412 Chiral Test Mix No. 5 (Lux) ea

If Lux analytical columns (≤ 4.6 mm ID) do not provide at least an equivalent or better separation as compared to a competing column of the same particle size, similar phase and dimensions, Lux Chiral Method Screening Kits are available. Please contact your Phenomenex representative for more information. return the column with comparative data within 45 days for a FULL REFUND.

96 Phenomenex l WEB: www.phenomenex.com Ordering Information Preparative Holder Replacement Parts and Accessories

Preparative LC Column Protection Preparative Holder (Two Sizes) SecurityGuard PREP For 21.2 mm ID cartridges, use with 18 to 29 mm ID columns PREP HPLC Column Prep Guard Cartridge Holder Part No. Description Unit AJ0-8223 HPLC Holder Kit for 21.2 mm ID cartridges, ea includes column coupler AJ0-8617 SFC Holder Kit for 21.2 mm ID cartridges, ea includes column coupler Holders Cartridges For 30.0 mm ID cartridges, use with 30 to 49 mm ID columns PREP SFC Prep Guard Cartridge Holder Part No. Description Unit AJ0-8277 HPLC Holder Kit for 30.0 mm ID cartridges, ea includes column coupler AJ0-8618 SFC Holder Kit for 30.0 mm ID cartridges, ea includes column coupler

21.2 mm ID 21.2 mm ID Cartridge Replacement Parts and Accessories HPLC Holder SFC Holder (15 x 21.2 mm ID) Part No. Description Unit AQ0-8374 PREP Coupler, SS w / PEEK Ferrule Inserts, 10-32 ea 1 Threads, /16 in. OD x 0.020 in. ID AQ0-8375 Replacement Ferrule Inserts, for PREP Coupler, 10/pk PEEK, 0.020 in. ID AQ0-8222 PREP Replacement O-Rings, Kalrez® 2/pk For 15 x 21.2 mm SG HPLC Holder, Size 2-021 AQ0-8318 PREP Replacement O-Rings, Kalrez® 2/pk For 15 x 30 mm SG HPLC Holder, Size 2-025 AQ0-8500 PREP Replacement O-Rings, Teflon® 2/pk For 15 x 21.2 mm SG SFC Holder, Size 2-021 30 mm ID 30 mm ID Cartridge AQ0-8501 PREP Replacement O-Rings, Teflon® 2/pk For 15 x 30 mm SG SFC Holder, Size 2-025 HPLC Holder SFC Holder (15 x 30.0 mm ID) AT0-0465 Capillary S.S. Tubing, 0.020 in. ID x 0.062 in. 5/pk 1 ( /16 in.) OD x 10 cm length O-Rings Coupler AT0-0466 Capillary S.S. Tubing, 0.020 in. ID x 0.062 in. 5/pk 1 ( /16 in.) OD x 20 cm length

Kalrez Teflon PREP Coupler O-Rings O-Rings

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions Trademarks Lux and Kinetex are registered trademarks and Phenex, Strata-X, Verex, Axia, and SecurityGuard are trademarks of Phenomenex. Agilent is a registered trademark of Agilent Technologies. Kalrez and Teflon are registered trademarks of E.I. du Pont de Nemours and Co. CHIRALCEL, CHIRALPAK, OD, OD-H, OZ-H, OJ, OJ-H, OX-HAY, AY-H, AD, AD-H, AD-RH, IB, and DAICEL are registered trademarks of DAICEL Corporation. Disclaimer Comparative separations may not be representative of all applications. Phenomenex is in no way affiliated with DAICEL Corporation. Axia is patented by Phenomenex. U.S. Patent No. 7,674,383 SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362 CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges. Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145 The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization. © 2014 Phenomenex, Inc. All rights reserved. Phenomenex l WEB: www.phenomenex.com 97 The Chiral Notebook Multiple Solutions for Chiral Applications

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