Using Blends of Solvents and Additives to Enhance Sfc Chiral Method Development Screening

USING BLENDS OF SOLVENTS AND ADDITIVES TO ENHANCE SFC CHIRAL METHOD DEVELOPMENT SCREENING

Thomas Swann*; Kenneth Berthelette; Jacob Fairchild; Jason Hill Waters Corporation, CBU Technology Advancement, 34 Maple Street, Milford, MA 01757

were further diluted with isopropanol and heptane to achieve When employing only Trefoil™ CEL1, the OP screen with INTRODUCTION working solutions in the range of 400 ug/mL to 2400 ug/mL. solvent mixtures gave the same success as when only single The working solutions were injected in volumes of 0.7—2 uL. solvents were used, Fig 4. Using only Trefoil™ CEL2, Fig 5, SFC chiral method development usually involves This concentration range allowed the chromatogram UV also provided OP screens that were equivalent between solvent screening numerous chiral stationary phases responses to be kept in the range of ca. 0.5 to 1.5 AU. mixtures and single solvents. From these data, cellulose based (CSPs) in search of one providing adequate Each of the fifty five probe compounds was analyzed using all CEL1 and CEL2 do not seem to respond in the same way to a selectivity between stereoisomers. To keep CSP forty four blends across each of the ACQUITY UPC2 Trefoil™ chiral recognition modulation effect of solvent blends, screening simple, many analysts employ single columns for a total of 55 x 44 x 3 = 7,260 runs. compared to AMY1.

solvents instead of mixtures and avoid All % % Total Single % % Total Step Step additives. However, this approach forgoes the RESULTS & DISCUSSION Combos Salvage Success Combos Salvage Success benefits of selectivity/resolution modulation 1 MI-TFA ---- 45.5 % 1 A-TFA ---- 43.2 % Optimal Combinations and Optimal Path Screen 2 A-TFA 11.4 % 56.8 % 2 I-TFA 13.6 % 56.8 % from judicious blending of solvents and 3 I-TFA 4.5 % 61.4 % 3 M-AmOH 4.5 % 61.3 % additives. Blending two or more solvents, with Each racemate probe compound represents a problem needing a solution: the resolution of the enantiomers. This study 4 M-AmOH 2.3 % 63.6 % 4 E-TFA 2.3 % 63.6 % or without additives, has been occasionally investigated the impact of different mobile phase blends on % Success by Step described in SFC but little investigated for chiral resolution. We evaluated the 7,260 chromatograms 100.0% For CEL1 alone, solvent mixtures improving selectivity/resolution between 90.0% obtained from the 3 Trefoil™ CSPs, with the above 55 80.0% were essentially equivalent to racemate probes and the 44 blends, using this “Pass” criterion: 70.0% single solvents enantiomers on chiral stationary phases. 60.0% All Combos 50.0% Single Combos

In this presentation, we report a systematic If Enantiomer Resolution (Rs) > 1.2 Success Total 40.0% 30.0% study with forty four different blends of solvents then the compound is “solved” 1 2 3 4 and additives to chromatograph fifty five We found that 44 racemate probe compounds were “solved” by Step diverse racemate probe compounds on 2.5 um at least one Combination ("Combo") of a Trefoil™ CSP & a Fig. 4. CEL1 only OP Screens particle ACQUITY UPC² Trefoil™ columns. The blend of solvent(s) plus additive. This is the “Solved Racemate All % % Total Single % % Total Set”. Step Step objective was to determine if blends could Combos Salvage Success Combos Salvage Success facilitate SFC chiral resolution and thereby For the “Solved Racemate Set”, we asked: 1 E-TFA ---- 34.1 % 1 E-TFA ---- 34.1 % enhance SFC chiral method development  Which are the Optimal Combinations (OCs) for Trefoil™ 2 A-TFA 9.1 % 43.2 % 2 A-TFA 9.1 % 43.2 % screening. columns that resolve the greatest number of racemates? 3 IA-AmOH 6.8 % 50.0 % 3 M-AmAc 4.5 % 47.7 %  What is the shortest and highest success screening path, 4 M-AmAc 2.3 % 52.3 % 4 I-TFA 2.3 % 50.0 % the “Optimal Path (OP) Screen”, using Trefoil™ columns? 5 A-AmOH 2.3 % 54.5 % 5 A-AmOH 2.3 % 52.3 % METHODS To search for answers, we started with the resolution data for % Success by Step all the “unsolved” probe compounds and looked for the Optimal Chiral (Racemate) Probe Compounds (55) 100.0% For CEL2 alone, solvent mixtures Combination (OC) of column and blend that gave the highest 90.0% were essentially equivalent to  cyclopentylphenyl acetic acid, 2-phenylbutyric acid, 2- 80.0% % success, Step 1. This “solves” the highest single percentage 70.0% single solvents 60.0% phenylpropionic acid, etodolac, fenoprofen, ibuprofen, All Combos of the racemates. We then excluded the “solved” compounds 50.0% Single Combos

indoprofen, ketoprofen, sulindac, 1-acenaphthenol, 1- Success Total 40.0% and applied the OC with the next highest % success, Step 2. 30.0% indanol, 1-phenylethanol, trans-stilbene oxide, 1,1'-bi-2- This “solved” additional compounds that failed in Step 1. The 1 2 3 4 5 naphthol, guaifenesin, warfarin, naringenin, flavanone, Step exclusion of solved compounds and application of Optimal benzoin, methyl mandelate, bucetin, praziquantel, Combinations was iterated until all of the compounds in the Fig. 5. CEL2 only OP Screens phenylthiohydantoin norleucine, chlorthalidone, indapamide, “Solved Racemate Set” are assigned to an Optimal sulpiride, acebutolol, atenolol, metoprolol, oxprenolol, All Trefoil™ Columns Optimal Path Screens Combination step. This process is illustrated by the cartoon in pindolol, practolol, propranolol, salbutamol, epinephrine, Fig. 2. A useful chiral method development screen requires more than arterenol, tetramisole, promethazine, thioridazine, a single CSP. Fig. 6 demonstrates that an OP Screen using all mianserin, troger's base, chlorpheniramine, oxybutynin, three Trefoil™ columns favors use of solvent mixtures over atropine, bupivacaine, 4-chlorophenylalanine methyl ester, Step 1 “solves” single solvent alone (more successful by ca. 14% after 4 econazole, fluoxetine, hydroxyzine, ketamine, lansoprazole, X% of cmpds (fails on all others) steps). prilocaine, terfenadine, trimebutine, verapamil. All % % Total Single % % Total Solvent Mixtures (15) Step Step Combos Salvage Success Combos Salvage Success  15 Mixtures of 4 Solvents: Methanol (M), Ethanol (E), 1 AMY1 ---- 45.5 % 1 CEL1 ---- 43.2 % Isopropanol (I), Acetonitrile (A) in EVEN v/v proportions EIA-AmAc E-TFA (i.e. 1, 1:1, 1:1:1, 1:1:1:1). All solvents were HPLC grade. “Unsolved” compounds Step 2 “solves” 2 CEL1 27.3 % 72.7 % 2 AMY1 22.7 % 65.9 % additional Y% MI-TFA E-AmOH pH Additives (3) (“salvages” Step 1 failures) 3 CEL2 13.6 % 86.4 % 3 CEL2 11.4 % 77.3 %  3 pH Additives (Low, Mid & High pH): 0.2% Trifluroacetic EA-TFA E-TFA Acid (TFA), 20 mM Ammonium Acetate (AmAc), 20 mM 4 AMY1 9.1 % 95.5 % 4 AMY1 4.5 % 81.8 % EI-TFA I-AmAc etc. Ammonium Hydroxide (AmOH). 5 CEL1 4.5 % 100.0% 5 AMY1 4.5 % 86.4 % Solvent/Additive Blends (44) I-TFA E-TFA Step 3 “solves” % Success by Step AMY1+CEL1+CEL2:  15 Solvent Mixtures X 3 pH Additives - 1 insoluble additional Z% Recommend solvent 100.0% mixtures combination (A-AmAc) = 44 Solvent/Additive Blends (“salvages” Step 2 failures) 90.0% 80.0% Columns (3) 70.0% 60.0% 2 Fig. 2. Search Approach for OCs and OP Screens For 3 CSPs, solvent mixtures were better All Combos 50.0%  ACQUITY UPC Trefoil™ AMY1 2.1x50, 2.5 um than single solvents (ca. 14%) Single Combos Total Success Total 40.0% [Amylose tris-(3,5-dimethylphenylcarbamate)] The resulting sequence of Optimal Combinations represented 30.0% 2 1 2 3 4 5 6  ACQUITY UPC Trefoil™ CEL1 2.1x50, 2.5 um an Optimal Path Screen. We applied this approach, first to Step [Cellulose tris-(3,5-dimethylphenylcarbamate)] “Solved Racemate Set” data for each Trefoil™ column alone  ACQUITY UPC2 Trefoil™ CEL2 2.1x50, 2.5 um then to the “Solved Racemate Set” data set with all three Fig. 6. AMY1 + CEL1 + CEL2 OP Screens [Cellulose tris-(3-chloro-4-methylphenylcarbamate) ] Trefoil™ columns. Here, when all three Trefoil™ columns and all blends are Single Trefoil™ Column Optimal Path Screens considered, the Step 1 Optimal Combination is now resolving a group of racemates that includes those previously solved by With only the Trefoil™ AMY1 column and considering either all CEL1 and CEL2 alone with single solvents (Fig. 4 and 5, resp.). blends or only blends using a single solvent, Fig. 3 shows that The “failures” of this Step 1 therefore are enriched with using solvent mixtures ("All Combos") gives an OP screen that compounds that benefit from CEL1 or CEL2 with solvent is more successful (by ca. 16% after 4 steps) than single mixtures, causing the Step 2 and 3 All Combos of Fig. 6 to solvents alone ("Single Combos"). Amylose based AMY1, is have higher % success. In contrast with Fig. 4 and 5, where responding to the ability of solvent blends to favorably alter the cellulose columns were examined in isolation, the Step 1 enantiomer selectivity. with AMY1 in Fig. 6 is revealing a smaller but still present

All % % Total Single % % Total favorable modulation of enantiomer selection by blends with Step Step Combos Salvage Success Combos Salvage Success CEL1 and CEL2. 1 EA-AmAc ---- 45.5 % 1 E-AmOH ---- 36.4 % Fig. 1. CSP Structures 2 EI-TFA 13.6 % 59.1 % 2 I-TFA 11.4 % 47.7 % CONCLUSION 3 IA-AmOH 9.1 % 68.2 % 3 E-TFA 4.5 % 52.3 % Instrument and Conditions 4 MI-AmOH 4.5 % 72.7 % 4 A-TFA 4.5 % 56.8 % Optimal selection of solvent/additive blends can enhance chiral 2  ACQUITY UPC with PDA and Empower™ 3 Data Collection 5 I-AmOH 2.3 % 75.0 % 5 M-AmOH 2.3 % 59.1 % resolution on Trefoil™ AMY1 and, to a lesser degree, CEL1 and CEL2 columns. Use of the three Trefoil™ column Optimal Path  6 A-TFA 2.3 % 77.3 % 6 I-AmAc 2.3 % 61.4 % Detection @ UV 220 nm 2  Mobile Phase: A = CO , B = Solvent / pH Additive blends Screen on ACQUITY UPC should give a rapid and more 2 % Success by Step  1.2 mL/min with ABPR = 3200 psi, Col. Temp. = 40° C successful SFC chiral method development. 100.0% For AMY1 alone, solvent mixtures were 90.0%  Equilibrate at 3% B for 0.5 min. Gradient from 3 to 60% B better than single solvents (ca. 16%) 2 Minute Screening Method 80.0% Optimal Path Screen Trefoil 2.1 x 50 mm columns @ 40° C over 1.5 min. Hold at 60% B for 0.5 min 70.0% Flow rate: 1.2 mL/min 60.0% Solv ABPR: 3,200 psi All Combos Step Columns & Blends 50.0% Line Single Combos Use indicated blend with:

Total Success Total 40.0% 1 AMY1-EtOH/IPA/ACN-AmAc B1 • 20 mM AmAc OR Experimental 30.0% • 0.2% TFA 1 2 3 4 5 6 2 CEL1-MeOH/IPA-TFA B2 Step Equilibrate: 3% B for 0.5 min The forty four solvent/additive blends were prepared. Each 3 CEL2-EtOH/ACN-TFA B3 Gradient: 3 to 60% B over 1.5 min Hold: 60% B for 0.5 min probe compound was individually dissolved in methanol to 4 AMY1-EtOH/IPA-TFA B4 Fig. 3. AMY1 only OP Screens 2.5 min Cycle Time per Step create a stock solution of 2 or 4 mg/mL. These stock solutions