32
September 2009
In Search of a Generic Chiral Strategy: 101 Separations WithOneMethod
Jim Thorn and John C. Hudson, Beckman Coulter, Inc., Fullerton, CA, USA
In any approach to drug discovery, the challenges presented to the analytical chemist are compounded when a product contains one or more chiral centres. Enantiomers are stereoisomers that display chirality, having one or more asymmetric carbon centres, allowing them to exist as non-superimposable mirror images of one another. These isomers are difficult to analyze as they are both physically and chemically identical and differ only in the way they bend plane-polarized light and in their behaviour in a chiral environment.
The key to separating enantiomers is to first create diastereomers from these enantiomers. Diastereomers may be created through chemical derivatization with a “chiral” reagent, or they may be formed transiently through interactions with chiral selectors. The latter, of course, is usually the most desirable as it is the easiest to employ. These chiral selectors have historically been introduced in the form of chromatographic media using HPLC, SFC or GC as the separation technique. Although chromatography has been a very effective methodology for chiral separations, the process of developing the methodology tends to be very expensive as development time is long, column lifetimes are short, and costs of chiral reagents are high.
Capillary electrophoresis has proven the most ideal analytical tool for this purpose, as it is simple to construct and modify a chiral environment within a capillary. The use of cyclodextrins for differential host- guest complexation of enantiomers is by far the most common “solution-based” chiral selector and is the basis of the chiral separation strategy that we propose. The primary strategy (1) focuses on the use of highly sulfated cyclodextrins (HSCDs) which are a family of three chiral reagents (2). This strategy first involves screening the compound for separation using all three (α,
β and γ) HSCDs and then optimizing the reagent which yields the best resolution.
The objective of the current study was to investigate the use of charged cyclodextrins in the on-going search for a generic strategy for the separation of
enantiomeric drug substances. A group of compounds selected from a set of drugs and metabolites of pharmaceutical and forensic interest was separated using HSCDs. This was a challenging group because it included many closely related metabolites of drug substances, in addition to the parent drugs. For simplicity, the screening strategy was designed to separate the enantiomers of individual compounds, although we present examples of separation of both drugs and their metabolites.
Materials and Methods Chemicals: Solutions of alpha-, beta and gamma-HSCD at a concentration of 20% w/v and all other reagents were obtained from Beckman Coulter, Inc., Fullerton, CA, USA. These reagents were prepared per the following table:
Boniface Hospital,Winnipeg, MB, Canada. Solutions of these drug and metabolite standards were purchased or prepared at a concentration of 1mg/mL and diluted to 25ppm (25ng/µL) in water.
Reference Marker: 1,3,6,8- Pyrenetetrasulfonate (PTS), 10mM in water: 2µL added to each sample.
Instrument: P/ACE™MDQ Capillary Electrophoresis System (Beckman Coulter, Inc.) equipped with a Photodiode Array Detector (PDAD) with detection at 200 nm (scanning190-350) and 32 Karat™ Version 7 Software.
Run Buffers: All chiral separations were performed in 5% HSCD in 25mM triethylammonium phosphate pH2.5, unless otherwise noted as either run in 2.5% or 7.5% of the HSCD.
The prepared solutions were dispensed to 2mL autosampler vials as shown in Figure 1.
Drug and Metabolite Standards: Standards were purchased from Cerilliant Corporation, Round Rock, TX, USA, or obtained as a gift from either the Royal Canadian Mounted Police, Forensic Laboratory,Winnipeg, MB, Canada or Dr. Robert Meatherall, St.
Capillaries and Conditioning: Fused-silica
capillaries, 50µm I.D. x30 cm (effective length 20 cm) were used in all separations. The columns are rinsed daily with 0.4% PEO (MW 300,000), 10% ethyleneglycol and adjusted to pH 4.75 to speed up column equilibration.
Applied Voltage: The voltage was set at 15kv (500v/cm), which resulted in running
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
