HPLC ENANTIOSEPARATION OF THE ALKALOID CANADINE AND DETERMINATION OF ENANTIOMERIC PURITY WITH CHIRAL/PHOTOMETRIC AND ACHIRAL/POLARIMETRIC DETECTION

Abstract

This work reports two methods developed for the determination of enantiomeric purity of canadine, the major alkaloid present in goldenseal extracts, by HPLC: 1) with physical separation of enantiomers using a chiral column with a stationary phase consisting of (S)-tert-leucine and (S)-1-(α-naphthyl)ethylamine, UV detection at 235 nm; and 2) without physical separation of enantiomers, using reverse-phase (C-18 column) and polarimetric detection at 830 nm (Diode-Laser).

Pyrkle-type chiral column and photometric detection was used to obtain base-line separation of canadine enantiomers (Rs = 1.78). A RP-18 achiral column and polarimetric detection provided a new nonchiral method to calculate enantiomeric purity.

Enantiomeric purity of 41.6% S-(−)-canadine and 58.4% R-(+)-canadine was obtained using Chirex-3019 column and photometric detection. Detection limit was 0.3 µg and linear range was 0.4 to 2 µg. RP-18 column and polarimetric detection provided a 1.5 µg detection limit and a dynamic range of 25.200 µg. Fluorimetric detection showed that R-(+)-canadine contained 7.4% S-(−)-canadine.

Assays using chiral column and photometric detection indicated that results obtained by both methods are concordant. The methods established are appropriate for the analysis of canadine enantiomers.

Keywords:

• alkaloids
• canadine
• chiral separation
• enantiomeric purity
• photometric/polarimetric detection
• reversed phase

ACKNOWLEDGMENT

We thank to the Organic Chemistry Department, in University of Málaga (Spain) for the supply of racemic and pure enantiomers of canadine.

Notes

A_ph, A_ph+, and A_ph− are peak areas of (+)- and (−)-canadine, respectively.

A_p is the area of polarimetric peaks, m₍₊₎ y m₍₋₎ are injected mass of pure enantiomers, and %m₍₋₎ is the percentage of (−)-Canadine in samples of 100 µg of total canadine.