Analytical Applications - Pharmaceutical Technology

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Analytical Applications
A Technical Forum Moderated by Patricia Van Arnum, featuring contributions from PerkinElmer, BioTools, Chiral Technologies, Shimadzu Scientific Instruments, GE Analytical Instruments, and Waters. This article is part of a special issue on analytical technology.

Pharmaceutical Technology
pp. s38-s46

Determination of the absolute configuration of resolved enantiomers

Bo Wang is a research scientist, BioTools, and Laurence A. Nafie is chief technology officer, BioTools and distinguished professor emeritus, Syracuse University. Elena Eksteen is senior manager, business development and planning, Chiral Technologies

During the small-molecule drug development process, chromatographic resolution is an effective way for separating racemic compounds into single enantiomers for use in bioassays and toxicology studies. Chromatography also is a tool that can be used to establish pharmacokinetic and toxicological properties of candidate drug compounds (1). Vibrational circular dichroism (VCD) technology is an important tool for determining the absolute configuration (AC) of enantiomers and is used by pharmaceutical companies for molecular structure characterization of chiral molecules. Knowledge of the AC is vital in drug discovery, development, and the regulatory requirements for investigational new drug submissions (2).

Figure 1 (Chiral): Separation of a racemic mixture. (FIGURES 1–3 (CHIRAL) ARE COURTESY OF THE AUTHORS)
VCD is the small difference in the IR absorbance of a chiral molecule for left versus right circularly polarized light. The determination of AC using VCD is a method for assigning the absolute stereochemistry of chiral molecules. It supplements, or in some cases, replaces the previous gold-standard method of anomalous X-ray diffraction that requires a pure single crystal of one enantiomer of the chiral molecule. VCD requires no crystallization and no chemical modification or derivatization of the chiral molecule. Assignments of AC are made by comparing the solution-state VCD and IR absorbance spectra to the corresponding quantum chemical calculations of the same spectra. When the VCD spectra agree, the AC chosen for the calculations is the same as that of the sample. If the signs of the calculated VCD are opposite to that of the measured VCD, the sample has the opposite AC. In addition, valuable information about the conformation or conformations of the chiral molecule in solution also may be obtained. Instrumentation for the measurement of VCD (Chiral IR-2X, BioTools) and software (Gaussian 09) for the calculation of VCD are commercially available as are services for performing AC determinations using VCD.

Figure 2 (Chiral): IR absorbance (bottom), vibrational circular dichroism VCD (middle) and VCD noise spectra (top) of the two enantiomers before baseline subtraction. The two enantiomers show identical IR spectra, as expected, but opposite-signed VCD spectra for every IR band.
Case study. A client requested enantiomer separation of a racemic mixture to obtain pure enantiomers needed for toxicology studies. In preparation for clinical trials, the client also requested AC measurements for an IND submission. The racemic compound was screened against several chiral stationary phases to identify a method appropriate for the separation of 50 to 100 g of the mixture. Figure 1 (Chiral) shows the separation of the racemic mixture using a Chiralpak IA column (Chiral Technologies).

Figure 3 (Chiral): Comparison between observed vibrational circular dichroism (VCD) for one enantiomer and the corresponding calculated VCD for the S,S-configuration of this enantiomer. The agreement in signs between the two spectra yields an absolute configuration assignment of S,S for the enantiomer.
Using the conditions of the separation method for scale-up, 80 g were separated, and pure enantiomers were isolated for toxicology studies and for VCD-based AC measurements of the pure enantiomers. VCD spectra of the resolved enantiomers were measured using a Chiral IR-2x instrument (BioTool) The IR and VCD spectra are shown in Figure 2 (Chiral). Figure 3 (Chiral) demonstrates the comparison of experimental to theoretical spectra.

Chiral references

1. K. Valko, Eur. Pharm. Rev. Issue 5, 40 (2010).

2. H. Yanan et al., Appl. Spectrosc. 65 (7), 699–723 (2011).


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