Enantiomer-Specific Analyses

It is nowadays a truism (but therefore also true) to say that life is a chiral process. The implication for the subject of this book is that those pharmaceutical compounds that contain one or more chiral centers could weU show differences between enantiomers with respect to their efficacy. Since most pharmaceutical drugs are administered as race-mates, it is sometimes important in drug development to be able to track the enantiomers separately, and this presents a significant challenge to the analyst in addition to the usual demands of validation and GLP etc. Since mass spectrometry can not in general distinguish between enantiomers, the chiral selectivity must be provided by chromatographic separation (Section 4.4.Id). 

Time (min) Flow rate (ml/min) A(%) B(%) 10-Port valve Event 

Analyte recoveries were evaluated (Xia 2003a) by comparing the response for a spiked plasma extracted and analyzed on-line as described above, with that obtained by spiking a blank plasma extract at the same nominal 

Matrix matched calibrators were prepared in blank rat plasma for each enantiomer separately and also for racemic propranolol. Prior to online extraction-analysis each 25p,L plasma sample was spiked with an equal volume of a solution (250ng.mL ) of the internal standard (d7-propranolol) in 20% methanol 80% 0.5 M formic acid in water, so the internal standard concenh ation in all anal54ical samples was 125ng.mL. To monitor the accuracy and precision of the assay, fom different sets 

Already it is evident that the requirements placed upon validation of a chiral LC-MS method are appreciably more demanding than those for an achiral assay, for 

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Koppen R, Becker R, Esslinger S, Nehls I. Enantiomer-specific analysis of hexa-bromocyclododecane in fish from Etnefjorden (Norway). Chemosphere 2010 80(10) 1241-5. 

Finally, there is upwelling in cyclonic eddies. For example, enantiomer-specific analysis of a-hexachlorocyclohexane (a-HCH) showed that samples from the East Greenland Sea (75°N around the meridian) had much lower enantiomer ratios, such as typically encountered in deeper, older Arctic waters (Jantunen and Bidleman, 1996). This could be interpreted as surface waters from 75 N represented older/deeper water masses that had surfaced. 

Care should be exercised when attempting to interpret in vivo pharmacological data in terms of specific chemical—biological interactions for a series of asymmetric compounds, particularly when this interaction is the only parameter considered in the analysis (10). It is important to recognize that the observed difference in activity between optical antipodes is not simply a result of the association of the compound with an enzyme or receptor target. Enantiomers differ in absorption rates across membranes, especially where active transport mechanisms are involved (11). They bind with different affinities to plasma proteins (12) and undergo alternative metaboHc and detoxification processes (13). This ultimately leads to one enantiomer being more available to produce a therapeutic effect. 

An interesting and practical example of the use of thermodynamic analysis is to explain and predict certain features that arise in the application of chromatography to chiral separations. The separation of enantiomers is achieved by making one or both phases chirally active so that different enantiomers will interact slightly differently with the one or both phases. In practice, it is usual to make the stationary phase comprise one specific isomer so that it offers specific selectivity to one enantiomer of the chiral solute pair. The basis of the selectivity is thought to be spatial, in that one enantiomer can approach the stationary phase closer than the other. If there is no chiral selectivity in the stationary phase, both enantiomers (being chemically identical) will coelute and will provide identical log(Vr ) against 1/T curve. If, however, one... 

There will be a continued need for enantiospecific methods of preparation and analysis, not only to ensure the quality of the final drug substance and reference materials, but also to control starting materials used for their manufacture, and key intermediates during synthesis. Likewise, specific and sensitive bioanalytical methods will be required to follow the fate of individual enantiomers after their administration. 

Examples of specific methods important to neurochemists include separation and quantification of R- and S-fluoxetine and R- and S-norfluoxetine in brain tissue and body fluids using derivatization with (—)-(S)-N-(trifluoracetyl)prolyl chloride, a chiral derivatizing agent (Torok-Both et al., 1992 Aspeslet et al., 1994). A similar method has been used to separate the enantiomers of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) (Hegadoren et al., 1993). Eluoxetine and norfluoxetine enantiomers have also been separated on a chiral column in series with a nonchiral column with NPD detections (Ulrich, 2003). Reviews of the analysis of enantiomers of several drugs of abuse are available (Jirovsky et al., 1998 Tao and Zeng, 2002 Liu and Liu, 2002). 

The nitroaldol-lipase DCR process could not only amplify specific (3-nitroalcohol derivatives, but also lead to their asymmetric discrimination. HPLC analysis proved that the enantioselectivity of the process is very high, resulting in products of very high optical purity. The R-enantiomer of the ester 45 was resolved to 99% ee, and the R-enantiomer of the ester 46 to 98% ee. 

The analysis of IR spectra of different synthetic mixtures of C-labeled and non-labeled enantiomeric actetates poses no problems. After applying an automated baseline correction to the spectra and correcting the absorbance of one enantiomer in the synthetic mixtures by the absorbance of the other enantiomer at this position, the accuracy of the /i.seMJo-enantiomeric system is excellent, specifically, within +3% in comparison to the ee values determined by chiral GC 101). Using commercially available HTS-FTIR systems, high-throughput measurements are easily possible. The analysis can be performed on a Tensor 27 FTIR spectrometer coupled to a HTS-XT system, which is able to analyze the samples on 96- or... 

More recently, enantiomer ratios have been used as evidence of adulteration in natural foods and essential oils. If the enantiomer distribution of achiral component of a natural food does not agree with that of a questionable sample, then adulteration can be suspected. Chiral GC analysis alone may not provide adequate evidence of adulteration, so it is often used in conjunction with other instrumental methods to completely authenticate the source of a natural food. These methods include isotope ratio mass spectrometry (IRMS), which determines an overall 13C/12C ratio (Mosandl, 1995), and site-specific natural isotope fractionation measured by nuclear magnetic resonance spectroscopy (SNIF-NMR), which determines a 2H/ H ratio at different sites in a molecule (Martin et al 1993), which have largely replaced more traditional analytical methods using GC, GC-MS, and HPLC. 

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