Chiral substances, enantiomeric purity

The development of a single enantiomer as a new active substance should be described in the same manner as for any other new chemical entity. Studies should be carried out with the single enantiomer, but if development began with the race-mate then these studies may also be taken into account. Chiral conversion should be considered early on so that enantiospecific bioanalytical methods may be developed. These methods should be described in chemistry and pharmacy part of the dossier. If the opposite enantiomer is formed in vivo, then it should be evaluated in the same way as other metabolites. For endogenous human chiral compounds, enantiospecific analysis may not be necessary. The enantiomeric purity of the active ingredient used in preclinical and clinical studies should be stated. 

As already mentioned, chiral cations are involved in many areas of chemistry and, unfortunately, only few simple methods are available to determine their optical purity with precision. In the last decades, NMR has evolved as one of the methods of choice for the measurement of the enantiomeric purity of chiral species [ 110,111 ]. Anionic substances have an advantage over neutral reagents to behave as NMR chiral shift agents for chiral cations. They can form dia-stereomeric contact pairs directly and the short-range interactions that result can lead to clear differences in the NMR spectra of the diastereomeric salts. 

Organosulfur chemistry is presently a particularly dynamic subject area. The stereochemical aspects of this field are surveyed by M. Mikojajczyk and J. Drabowicz. in the fifth chapter, entitled Qural Organosulfur Compounds. The synthesis, resolution, and application of a wide range of chiral sulfur compounds are described as are the determination of absolute configuration and of enantiomeric purity of these substances. A discussion of the dynamic stereochemistry of chiral sulfur compounds including racemization processes follows. Finally, nucleophilic substitution on and reaction of such compounds with electrophiles, their use in asymmetric synthesis, and asymmetric induction in the transfer of chirality from sulfur to other centers is discussed in a chapter that should be of interest to chemists in several disciplines, in particular synthetic and natural product chemistry. 

This method amounts to a complete resolution of the type described in Section 19-3D, but on an analytical scale. For example, assume that you have a partially resolved compound, A, consisting of unequal amounts of the enantiomers Aand A. By reaction with a second chiral enantiomerically pure substance, B+, A is converted to a mixture of diastereomers A+B+ and A B+. Because these diastereomers are chemically and physically different, the mixture usually can be analyzed by gas-liquid chromatography (Section 9-2A). If the reaction of B+ with A+ and A was quantitative, the relative areas of the two peaks eluting from the column correspond to the ratio of the diastereomers A,.B+/AJB+, and thus to the ratio of enantiomers A+/A, from which the enantiomeric purity of the partially resolved mixture can be calculated. 

The measurement of the optical rotatory power of chiral substances has been of major importance in the characterization of the enantiomeric purity. A number of computational techniques have been developed in the last year to evaluate this property. A recent review [142] shows in detail the advances in this field. Application of the new implementation of the evaluation of the optical rotatory power has allowed to the study of the conformational [143-146] and solvent effects [147,148] on the magnitude and sign of the optical rotation power. 

For all analytical methods the quality of the results ultimately relates back to the chemical purity of the very best available SRM and to the linearity of the correlation curve for the experimentally measured property vs. the SRM concentration. For substances that are naturally chiral there is the additional very serious concern about enantiomeric purity. The determination of an enantiomer whether for an enantiomeric purity test, or for an enantiomeric ratio or excess test in the study of a partial racemic mixture, is one of the more difficult analytical problems. To actually report the enantiomeric purity of an enantiomer as better than 99% is truly beyond the capability of current analytical methodology [31], for after all few substances ever have a chemical purity that is guaranteed to be greater than 99%. So, as mentioned earlier, one has to accept the fact that the results are measured relative to an enantiopurity of an SRM that is defined to be 100%. This limitation of course impacts on the true meaning of a calculated enantioexcess, and to a much lesser degree perhaps, in assays of chiral substances extracted from plant materials using calibration data that were obtained for synthetic SRM s. 

The article is a brief review of the applications of CD as a detector in both preparative and analytical liquid chromatography. The objectives are to identify elution orders for enantiomers, to measure enantiomeric purities and enantiomeric excesses, to analytically determine diastereoisomers, and to selectively determine chiral analytes when present as components in mixtures with achiral substances. 

In its broadest terms the discussion of HPLC detection for chiral species must include the analysis of mixtures with achiral substances as well as the quality testing of, for example, the enantiomeric purity of a chemically pure drug form. The distinction between the definitions of chemical purity versus optical purity can not be overemphasized. In an efficient chiral HPLC system the latter problem is trivial, and if retention times are significantly different then any conventional detector such as RI, electrochemical, absorption, etc., could be used. Co-elutions are a major experimental concern in separations of mixtures and at this juncture it is not only prudent but absolutely necessary to involve a chiroptical detector to preferentially identify the chiral analyte. 

Several lactic acid derivatives were used by Gessner et al. for the determination of the enantiomeric purity of flavor substances such as chiral alcohols from natural sources. Diastereomeric 0-acetyl-, propionyl-, and hexanoyllactic acid esters of the chiral alcohols were separated by GLC (155). A report from the same laboratories described characterization of several chiral aroma substances that are S-lactones. The lactones were hydrolyzed to the corresponding hydroxy acids, and the acid moiety was esterified to the isopropyl ester. The remaining hydroxyl group was esterified with (R)-2-phenylpropionic add chloride or [30], and the diastereomeric derivatives were separated using preparative silica gel LC. The derivatives were also separated on an analytical scale by GLC (156). 

For all types of chemical analysis, the quality of the results ultimately relates to the chemical purity of the best available SRM. For naturally chiral substances, there is the additional more serious concern over what constitutes absolute enantiomeric purity. Not even mass spectroscopy, which provides assurance that a substance is chemically pure, can be used to report absolute enantiomeric purities. To actually report an enantiomeric purity higher than 99% is truly beyond the capability of current analytical methodology. ° As noted previously, the fact is that results are measured relative to an enantiopurity defined to be 100%. Chemical purities aside, the measurement of enantiomeric purity and enantiomeric excess is technically the same, the difference being the extent of race-mization. There are only two experimental options, either enantiomeric separations or multivariate spectroscopic analyses, that involve either two distinct detectors or multiple-wavelength detection for a single detector, as noted above. The newly described derivati-zation reactions fulfill the second option. 

HPLC provides reliable quantitative precision and accuracy, along with a linear dynamic range (LDR) sufficient to allow for the determination of the API and related substances in the same run using a variety of detectors, and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide array of compound types by judicious choice of HPLC column chemistry. Major modes of HPLC include reversed phase and normal phase for the analysis of small (<2000 Da) organic molecules, ion chromatography for the analysis of ions, size exclusion chromatography for the separation of polymers, and chiral HPLC for the determination of enantiomeric purity. Numerous chemically different columns are available within each broad classification, to further aid method development. 

Many chiral saturated hydrocarbons are known in nonracemic form. Some are natural products or derivatives thereof others are products of synthesis. Absolute configurations have been established by chemical correlations with substances of known configuration by changes not affecting the stereogenic atomsIn many cases the enantiomeric composition can be estimated by comparisons with samples that have been fully resolved or by extended correlations with substances that have been analyzed by modern methods for the assessment of enantiomeric purity. This permits calculation of maximum rotations and these are, where possible, reported here. Observed rotations, ol, have been corrected for tube length (/ = 1 dcm) when necessary and are reported as such when corrections for density or concentration (p in gml" ) have not been made by authors. Specific rotations ... 

Gyllenhaal, O. Packed column supercritical fluid chromatography of a peroxysome proliferator-activating receptor agonist drug Achiral and chiral purity of substance, formulation assay and its enantiomeric purity. J. Chromatogr. 2004 , 1042,173-180. 

---