Y. Wang et al. / Chinese Chemical Letters 21 (2010) 860–863
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Fig. 1. Chemical structure of (À)clausenamide and (+)clausenamide.
1. Experimental
(À)Clausenamide, (+)clausenamide, and the racemate (Æ)clausenamide were supplied by Prof. Liang Huang from
the Department of Synthesis of Institute of Materia Medica, the Chinese Academy of Medical Sciences. Samples were
separated on a high performance liquid chromatography system with Waters 501 pump (Waters, USA), Rheodyne7125
injection valve (Rheodyne, USA), SPD-2A UV detector (Shimadzu, Japan), and Alltech Sage chromatography
workstation (Alltech, USA). Separation of clausenamide enantiomers was performed on the column of chiral-AGP
stationary phases with a guard column (150 mm  4.0 mm, and 10 mm  3.0 mm, 5 mm) from the Chromtech
company of Sweden.
2. Results and discussion
Separation of (À)clausenamide and (+)clausenamide has been studied on different chiral columns containing
Chromchiral OD-H, Kromasil CHI-TBB, Kromasil CHI-DMB, and Chromtech Chiral-AGP. Optimization results
displayed separation of (À)clausenamide and (+)calusenamide might be performed on the columns of Chromchiral
OD-H and Chromtech Chiral-AGP, whereas enantiomers had not been separated on the other two chiral columns. The
resolution factor of (À) and (+)calusenamide was 1.0 on the column of Chromchiral OD-H and more than 4 on the
chiral-AGP column. Based on the characteristics of accuracy to the determination and simplicity of action on the
column of chiral-AGP, separation of (À)clausenamide and (+)clausenamide has been chosen on a reversed-high
performance liquid chromatography system with a chiral-AGP column, but not with the column of Chromchiral OD-H
which is used in a normal high performance liquid chromatography system. Results have demonstrated that a1-acid
glycoprotein (AGP) as the stationary phase which is filled in the column has the capacity of reversible conjugation with
small molecular compounds. The conjugation capacity of APG possesses stereo-selectivity which has resulted in the
separation of clausenamide enantiomers. Optimization of the mobile phase was based on the resolution factor of
(À)clausenamide and (+)clausenamide on the column. By comparison with different mixtures as the mobile phases
such as methanol–water, methanol–isopropanol–water, propanol–acetate, isopropanol–phosphate, and isopropanol–
water, the resolution factor could be impacted seriously according to types and concentrations of the mobile phases.
Final separation of (À)clausenamide and (+)clausenamide was carried out in a mixture of isopropanol–water (5:95) as
the mobile phase with a resolution factor of 4.4. The numbers of theoretical plates of (À)clausenamide and
(+)clausenamide were 2957 and 2353, respectively.
Chromatographic behavior of the (Æ)clausenamide (0.4 mg/mL solution) separation on the chiral-AGP column
has been further investigated by the separation of a mixture solution with an equivalent amount of
(À)clausenamide (0.2 mg/mL) and (+)clausenamide (0.2 mg/mL), respectively. The results exhibited that the
chromatographic behaviors of (À)clausenamide and (+)clausenamide obtained from the racemate separation on
the chiral-AGP were in accord with those of (À)clausenamide and (+)clausenamide from the mixture solution
(Fig. 2). Hence, a method for the quantitative measurement of (À)clausenamide has been established and
validated which can be used to control the limitation of the (+)clausenamide in the (À)clausenamide. The limit of
detection (LOD) for (+)clausenamide is 15 ng (Fig. 3). The optical purity of (À)clausenamide for the clinical
trials should be more than 99%.