Capillary Electrophoresis Aids QC Labs
Nearly 75 percent of all new drugs in development are chiral, i.e. they exist as mirror-image optical isomers. Many of these drugs are being developed as single-isomers, which presents interesting manufacturing and quality control problems. For example the "wrong" isomer is often now considered a contaminant, whereas 10 years ago it coexisted in equal quantities with the more active or less toxic isomer.
Discovery, development, and manufacturing technologies are moving quickly to adjust to the chiral phenomenon, but analytical instruments lag. Chiral gas chromatography (GC) is one costly option, but of limited versatility: Once you purchase several $1200 chiral columns and they don't work, then what?
Luckily chiral capillary electrophoresis (CE), a versatile, inexpensive technique borrowed from experimental biology laboratories, can help. CCE separates molecules based on their mobility in an electric field. The chiral "twist" comes from addition of chiral mediators, such as cyclodextrins, to the capillary. With a wide range of cheap cyclodextrins and buffers available, separations can be tried out for pennies without risk of damaging or not using thousand-dollar chromatography columns. And chiral analyses generally take less than 10 minutes, so real-time QC is possible.
Hewlett-Packard Corp. (HP), which manufactures CE equipment and chiral reagents, recently published an application note detailing separation of four stereoisomers of troglitazone, an oral insulin enhancer with promise for treating diabetes. Troglitazone exists in two diastereomeric (non-superimposable) forms, each with its own pair of enantiomers. Since diastereomers are energetically different, their chromatography is not nearly as problematic as that of enantiomers, which are isoenergetic. This is obvious from Figure 1, a run using "first pass" separation conditions.
Figure 1: First-pass separations at various cyclodextrin (CD) concentrations
Experimental
HP Engineers used an HP 3D Capillary Electrophoresis system equipped with a diode array detector and an HP ChemStation for system control, data acquisition, and data analysis. Fused silica capillaries, 50 microns in diameter with effective lengths of either 56 or 72 cm, were cooled with a Peltier cooling unit. The applied voltage was 30 kV. Separation conditions for the successful separation (Figure 2) were as follows:
- Inlet buffer: 2.5 mM sodium borate (pH 9.0), 20 mM sodium dodecyl sulfate (SDS), 56 mM heptakis (2,3,6-tri-O-methyl)-beta cyclodextrin
- Outlet buffer: 2.5 mM sodium borate, pH 9.4, 20 mM SDS
- Injection pressure: 50 mbar for 3 seconds
- Operating temperature: 10 degrees C
- Detection at 200 nm
Results
Optimized separation of the four isomers, using conditions described above, is shown in Figure 2. Achieving this result required significant development work. Other cyclodextrins were not nearly as effective as the less polar 2,3,6-tri-O-methyl cyclodextrin; temperature, capillary length, and cyclodextrin concentration also affected results.
Figure 2: Optimized separation of the four steroisomers of troglitazone
But the lesson here is that chiral methods development takes far less time and far less money when done in CE than in chiral GC. Run times are comparable, but a new CE "column" takes just a few minutes to prepare, vs. half an hour for changing a GC column. Plus CE affords nearly infinite possibilities (using capillary length, buffer, temperature, chiral agent, and chiral agent concentrations as the variables) for creating novel, tailored separation systems for the cost of a few microliters of reagent. Chiral GC columns, because of their cost, are best suited to separations where they are known to work.
For more information, contact: Doug Forsyth, Hewlett Packard Co., 3000 Hanover St., Palo Alto, CA 94304. Tel: 650-857-5603.
By Angelo DePalma