Abstract
The couple of chiral sulfur compounds α-lipoic acid (ALA)/α-dihydrolipoic acid (DHALA) has attracted considerable attention in recent years owing to its remarkable anti-inflammatory and antioxidant properties. It is well known that the chirality of the C6 plays a key role in determining the biological activity of ALA. The natural occurring (R)-ALA enantiomer is an essential cofactor for key oxidative metabolism enzyme complexes and, after oral administration of the racemic mixture, it shows higher plasma concentration than (S)-ALA. Differently, the in vivo enantioselective action difference between the enantiomers of DHALA has not yet been studied. This lacking is perhaps due to the unavailability of analytical methods capable of determining the enantiomeric composition of biological samples during pharmacokinetic and pharmacodynamic events. In the present work, the direct and baseline enantioresolution of both chiral acids by HPLC on two amylose-derived chiral stationary phases is presented. The proposed chiral enantioselective protocol, therefore, does not require pre- or on-column derivatization. The performance of the coated Chiralpak AS-H CSP and the new immobilized Chiralpak IH-3 CSP, which have the same chiral selector amylose tris-[(S)-α-methylbenzylcarbamate], were compared using conventional normal-phase mobile phases containing ethanol or 2-propanol as alcoholic solvents and a fixed percentage of trifluoroacetic acid. Nonconventional eluents containing dichloromethane, ethyl acetate, and 2-methyltetrahydrofuran as organic cosolvents were applied in the separation of the enantiomers of two carboxylic acids on the immobilized Chiralpak IH-3 CSP. The effect of the column temperature was carefully evaluated in order to improve enantioselectivity. Adequate amounts of enantiomers were isolated by an analytical-size Chiralpak IH-3 column and submitted to chiroptical measurements. The absolute configuration assignment of the isolated enantiomers was determined by a multidisciplinary procedure based on the comparison of the experimental and calculated chiroptical properties.
Highlights
The polymeric selector is physically adsorbed onto 5 μm silica particles in the Chiralpak AS-H CSP, and it is chemically immobilized onto 3 μm silica particles in the Chiralpak IH-3 CSP
(iv) The resolution factor values for DHALA obtained with non-standard mobile phases on the immobilized Chiralpak IH-3 were higher than those observed using the coated Chiralpak AS-H CSP in standard elution mode
In this work it has been demonstrated that the amylose-based Chiralpak AS-H and Chiralpak IH-3 CSPs provide direct and complete resolution of ALA-enantiomers under normal-phase conditions
Summary
Chiralpak AS-H and Chiralpak IH-3 are commercially available CSPs based on amylose tris-[(S)-α-methylbenzylcarbamate]. The chromatographic enantioseparations of ALA and DHALA were initially performed using 25 cm length columns packed with the aforementioned CSPs in standard normal-phase mode To explore these conditions, the mobile phases n-hexane/2-propanol (IPA)/trifluoroacetic acid (TFA) 80:20:0.1 (v/v/v) and n-hexane/ethanol (EtOH)/TFA 85:15:0.1 (v/v/v) were selected. The enantioseparation of ALA and the complete solvent compatibility of the Chiralpak IH-3 CSP encouraged us to set a second series of enantioselection experiments based on the use of alternative mobile phases containing different percentages of IPA and non-standard cosolvents such as DCM, EA, tetrahydrofuran (THF), and 2-methyltetrahydrofuran (MTHF). Chemists are challenged in the search for cleaner, greener, and more sustainable solvents Along this line, we selected MTHF as an alternative solvent to THF and DCM in the preparation of non-standard normal-phase mobile phases.
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