Chemical-based species classification of rhubarb using simultaneous determination of five bioactive substances by HPLC and LDA analysis.

INTRODUCTION: Rhubarb is a traditional Chinese medicine derived from the rhizome of three species: Rheum tanguticum, Rheum palmatum and Rheum officinale. There are several species that are often misidentified as rhubarb. Taxonomical identification of these various species can be challenging. We have developed an HPLC-based species classification to identify rhubarb. OBJECTIVE: The objective of this study was to develop a simple HPLC method for the simultaneous determination of bioactive compounds and identification of medicinal rhubarb rhizome and non-medicinal species. METHODOLOGY: Quantitative analysis was performed on a C18-column using 0.05 M aqueous phosphoric acid and acetonitrile as the mobile phase under gradient conditions with ultraviolet detection at 280 nm. The method was validated with respect to linearity, accuracy, precision, and recovery. Statistical analysis was used to classify different groups of species. RESULTS: All calibration curves showed good linearity (r ≥ 0.9995). The method showed good repeatability with intra- and inter-day standard deviations of less than 1.13% and 1.32%, respectively. The accuracy and recovery of all marker compounds were in the ranges of 98.0 to 102.6% and 99.21 to 102.04%, respectively. Seventeen peaks were selected, and 39 known and 57 unknown samples were classified into five species based on linear discriminant analysis with an accuracy of 100%. CONCLUSION: A chemical-based species classification method of rhubarb using simultaneous determination of bioactive compounds by HPLC was developed with 39 known samples of five different species and successfully applied to identify 57 unknown samples collected from Korea and China.

Chiral HPLC studies on chemical behavior of 6-methoxydihydrosanguinarine in alcoholic solvent system.

All the enantiomers of methoxydihydrosanguinarine (MS), ethoxydihydrosanguinarine (ES) and iso-propoxydihydrosanguinarine (PS) were separated by chiral HPLC. They were further identified by comparing the retention times of authentic standards as well as LC-MS. Interestingly, the approximately same conversion rates for the formation MS from ES or PS and the slower conversion of MS in isopropanol compared to ethanol demonstrated two step mechanism in the reaction of alkoxysanguinarine in alcohols, which is composed of the initial formation of sanguinarine as a planar intermediate and the addition of alcohol to intermediate as possible rate limiting step. Thus, sanguinarine has a pivotal role in the chemical behavior of alkoxysanguinarine in alcoholic solvents. Such possible variation of the structure of sanguinarine may be the source of its diverse biological activities.

Quality control of a herbal medicinal preparation using high-performance liquid chromatographic and capillary electrophoretic methods.

Two methods based on high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were developed for the quality control of "samgiumgagambang" (SGMX), a new herbal medicinal preparation containing 14 herbs. Nine components from SGMX were selected as markers: 5-hydroxymethylfuraldehyde, geniposidic acid, chlorogenic acid, paeoniflorin, 20-hydroxyecdysone, coptisine, berberine, luteolin, and glycyrrhizic acid. The markers were identified and analyzed using HPLC coupled with a UV-diode-array detector and monitored at 250nm with a gradient elution of acetonitrile and water containing formic acid on a C(18) analytical column or using CE with a 70mM borate buffer (pH 9.5) containing 10% methanol on a 60-cm fused silica capillary monitored at 230nm. The marker components in SGMX were well separated using both methods and were readily determined within 60min using HPLC or 13min using CE with good precision and accuracy.

Reverse migration order of sibutramine enantiomers as a function of cyclodextrin concentration in capillary electrophoresis.

The current study demonstrates the reversal of enantiomer migration order (EMO) in capillary electrophoresis (CE) based separations of sibutramines (SIB) as a function of the concentration of two types of cyclodextrin (CD), native ß-CD and acetyl-ß-CD. At normal working concentrations (<10mM) of either CD, (S)-SIB migrated first. However, at CD concentrations greater than 10mM, (R)-SIB was the first to migrate. This study describes factors involved in determining EMO for sibutramine enantiomers at low and high concentrations of CDs. The reversal of EMO could be explained in terms of the opposing effects of the stability and the limiting complex mobility of the SIB-CD complexes. The enantioseparation of SIB with methyl- and 2-hydroxypropyl-ß-CD was possible based on differences in the binding constants of complexes. However, reverse EMO was not observed because of equal mobilities of SIB enantiomers complexed with methyl- and 2-hydroxypropyl-ß-CD.

Multiple component quantitative analysis for the pattern recognition and quality evaluation of Kalopanacis Cortex using HPLC.

A quantitative and pattern recognition analyses were conducted for quality evaluation of Kalopanacis Cortex (KC) using HPLC. For quantitative analysis, four bioactive compounds, liriodendrin, pinoresinol O-ß-D-glucopyranoside, acanthoside B and kalopanaxin B, were determined. The analysis method was optimized and validated using ODS column with mobile phase of methanol and aqueous phosphoric acid. The validation gave acceptable linearities (r > 0.9995), recoveries (98.4% to 101.9%) and precisions (RSD < 2.20). The limit of detection of compounds ranged from 0.4 to 0.9 µg/mL. Among the four compounds, liriodendrin was recommended as a marker compound for the quality control of KC. The pattern analysis was successfully carried out by analyzing thirty two samples from four species, and the authentic KC samples were completely discriminated from other inauthentic species by linear discriminant analysis. The results indicated that the method was suitable for the quantitative analysis of liriodendrin and the quality evaluation of KC.

HPLC-ELSD analysis of 18 platycosides from balloon flower roots (Platycodi Radix) sourced from various regions in Korea and geographical clustering of the cultivation areas.

An effective HPLC method to analyse platycosides from the balloon flower root was developed using ELSD. The optimum resolution of the platycosides was achieved on an ODS column with gradient elution of eluent A, 30mM ammonium acetate buffer (pH 4.81): methanol: acetonitrile=75:5:20 (v/v/v), and B, 69:5:26 (v/v/v). Amongst 18 platycosides, platycoside E showed the highest content, followed by polygalacin D2 and 3″-O-acetylplatyconic acid A. The sum of these three compounds was recommended for quality control of balloon flower root for medicinal purposes. The samples could be clustered into groups based on platycoside content. Group I, characterised by a high concentration of platycosides, was located near the west coast of Korea, whereas group II, characterised by a low concentration of platycosides, was located inland or in mountainous area. The method could be used to control the quality of balloon flower root.