[Improvement of reproducibility in capillary electrophoretic characterization of rhubarb by normalization of migration time]

Abstract

The principle of the normalization of migration time and its application on the traditional Chinese medicine (TCM) analysis by capillary electrophoresis (CE) are presented. It is the core of the normalization of migration time that the fluctuation of apparent migration velocity for each component at different runs is attributed to the difference of electroosmotic flow velocity. To transform migration time (t) to normalized migration time, one peak or two peaks in the original electropherogram are selected as internal peak. The normalization of migration time is therefore classified into two types based on the number of selected internal peaks, one-peak and two-peak approaches. The migration times processed by one-peak normalization and by two-peak normalization are conducted by the following equations, respectively: (t'(i))(j) = 1/ [1/(t(i))(j) - [1/(t(istd))(j) - 1/(t(istd))1]] and (t"(i)(j) = 1/[1/(t(i))(j) - [[1/(t(istd, l))(j) - 1/(t(istd, l))(l)] + [1/(t(istd, end))(j) - 1/(t(istd, end))(l)]]/2], where (t'(i))(j) and (t"(i))(j) are the normalized migration times obtained by one-peak and two-peak approaches in the jth run for component i, respectively; (t(i))(j) and (t(istd))(j) are the migration times in the jth run for component i and the selected internal standard in the sample, respectively; (t(istd, l))(j) and (t(istd, l))(l) are the migration times for the first peak selected as internal peak in the jth run and the first run, respectively; (t(istd, end))(j) and (t(istd, end))(l) are the migration times for the last peak selected as internal peak in the jth run and the first run, respectively. One of the commonly used traditional Chinese medicines (TCM), rhubarb, was chosen as a model to verify the advantage of migration time normalizations in improving reproducibility of CE. Both of the experiments, the five parallel micellar electrokinetic chromatography for rhubarb extract and the capillary zone electrophoresis for the samples prepared by mixing rhubarb extract with various solutions, showed RSD(t") < RSD(t') < RSD(t/t(istd)) and RSD(t). The RSDs(t") of the five parallel micellar electrokinetic chromatography were in the range of 0.07% -0.58%, and the RSDs(t") of the capillary zone electrophoresis were in the range of 0.31% - 0.57%. The results showed that the qualitative precision improvements for rhubarb characterized with CE can be carried out by the normalization of migration time. The normalization of migration time will be gained much more attention to TCM characterized by CE due to its simplicity and reliability.