An Investigation of Enantiomer Resolution by Gas Chromatography: Application in Drug Metabolism Studies

Gilbert, Mary Telford (1975) An Investigation of Enantiomer Resolution by Gas Chromatography: Application in Drug Metabolism Studies. PhD thesis, University of Glasgow.

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The use of gas chromatography for the analytical resolution of optical enantiomers provides a means of assigning configurations, on a correlative basis, to small, and often impure, samples of biological origin. This problem has previously been approached in ;two ways, viz: direct resolution of optical enantiomers on chiral stationary phases, or the employment of chiral reagents to form diastereomeric derivatives which have been separated on conventional columns. In the present work, the latter approach was employed. It was considered that conferment of sufficiently distinctive chromatographic properties would depend on the enhancement of conformational differences between diastereomers. Accordingly, it seemed likely that reagents in which the chirality and the functional group were embodied in a rigid molecular skeleton would be useful as chromatographic resolving agents. On the basis of this hypothesis, a study of the suitability of terpenoid acids for the resolution of enantiomeric alcohols and amines was initiated. The two acids examined were the sesquiterpenoid drimanoic acid and the monoterpenoid chrysanthemic acid. Many good separations were in fact observed for a wide range of enantiomeric alcohols, amines and amino acid methyl esters. The results achieved with these derivatives on columns packed with a non-selective stationary phase (SE-30) are comparable with, or in many cases superior to, those reported with other chiral reagents on capillary columns. An observation that the alpha-phenylbutyryl derivatives of (R)-alpha-phenylethylamine were well separated on conventional packed columns prompted an investigation into the use of alpha-substituted phenylacetic acids as gas chromatographic resolving agents for amines and amino acid methyl esters. Within the series studied, a resolution was achieved for every enantiomeric pair as their amides derived from at least one, and in many cases all of the chiral phenylacetic acids. The mechanisms involved in the resolution are still open to speculation, but examination of the separations obtained within a series of related diastereomeric amides indicated some trends. In general, the degree of resolution achieved increased with the size of the substituent on the chiral acid centre (i. e. R = Me < Et < iPr). Introduction of a more polar substituent (as in alpha-methoxy-phenylacetic acid) produced resolutions comparable to those obtained with the isopropylphenylacetic acid but some effects which could be attributed to the increased polarity of the substituent were noted. The derivatives of alpha-chlorophenylacetic acid showed satisfactory resolutions, but this reagent was judged unsuitable because of racemisation during acylation. All the derivatives were easily prepared, in good yield, without racemisation (except for the alpha-chlorophenyl-acetamides). The diastereomers were thermally and chromatographically stable, as shown by analysis using combined gas chromatography-mass spectrometry (GC-MS). The considerable degree of regularity observed in the elution order of these derivatives rendered the correlative assignment of configuration a feasible proposition. The techniques developed during the examination of enantiomer resolution were subsequently applied to a study of the in vivo metabolism of the oral anti-rheumatic drug (RS)-2-(4-isobutylphenyl) propionic acid ("ibuprofen") in humans. Pour metabolites, resulting from oxidation of the isobutyl side chain, were identified by GLC and GC-MS. Gas chromatographic resolution of diastereomeric amides formed with (R)-(+)-alpha-phenylethylamine showed that the excreted drug was enriched in the (S)-(+)-enantiomer. Similarly, two of the hydroxylated metabolites were shown to be enriched in the (S)-form. Synthesis of one of the minor hydroxylated metabolites, 2,4'-(1-hydroxy-2-methylpropyl) phenylpropionic acid, allowed complete characterisation of the metabolite. Separation of the enantiomeric alcohols as esters of (R)-(-)-alpha-phenylbutyric acid indicated some stereoselectivity in the metabolic hydroxylation. A brief examination of the metabolism of the aldosterone antagonist, spironolactone was also undertaken.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Analytical chemistry
Date of Award: 1975
Depositing User: Enlighten Team
Unique ID: glathesis:1975-78693
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:01
Last Modified: 30 Jan 2020 15:01

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