Micellar electrokinetic chromatography for separation of a mixture of coptis alkaloids, scute flavonoids, and rhubarb anthraquinones and bianthrones.

Coptidis Rhizoma, Scutellariae Radix, and Rhei Rhizoma are three common Chinese herbs. There are many herbal formulas which contain either two or all three of the herbs mentioned above. Their bioactive components have already been identified, respectively. However, there is no report about separation of the 13 bioactive constituents of the three herbs at the same time. In order to assess these constituents of related Chinese herbal preparations, a micellar electrokinetic chromatography method was developed. While buffer pH and surfactant concentration affected the resolution of separation, acetonitrile percentage was found to significantly influence the resolution, peak shape, and elution window. Optimum separation of 13 compounds was achieved at pH 7.3 using a buffer mixture of 70% (v/v) 3 mM di-sodium tetraborate, 10 mM sodium dihydrogen phosphate, and 50 mM sodium deoxycholate with 30% (v/v) acetonitrile. When applying the developed method to analyze a model preparation, San-huang-xie-xin-tang, which contains all three herbs, 8 of the 13 bioactive constituents, could be determined. The present study proposed a method to assess San-huang-xie-xin-tang within short analysis time and also provided a possible starting point to evaluate related herbal preparations containing Coptidis Rhizoma, Scutellariae Radix, and Rhei Rhizoma.

Sensitivity improvement on detection of Coptidis alkaloids by sweeping in capillary electrophoresis.

A simple and rapid sweeping method for the online improvement of detection sensitivity of the main alkaloids of Coptidis Rhizoma has been developed in this work. Optimum separation conditions were found as follows: electrophoretic running solution comprising 100 mM phosphoric acid, 15 mM sodium dodecyl sulfate (SDS) and 10% (v/v) tetrahydrofurane with pH 1.82; running voltage of -25 kV; sample matrix composed of 50 mM phosphoric acid and sample injection at 1000 mbar for 60 s (sample injection volume ca. 2.75 microl). With this sweeping method, the concentration limits of detection of berberine, coptisine and palmatine were found to be 2.5 ppb (ng/ml), which was about 500 times lower than those from conventional sample injections. Baseline separation was achieved for the main alkaloids within 15 min. After validation, the developed method was applied to determine the quantity of berberine, coptisine and palmatine in a Coptidis Rhizoma sample. The method should be able to be used in identification and quantitative evaluation of the crude drugs requiring only a minor amount of sample.

Analysis of rhubarb anthraquinones and bianthrones by microemulsion electrokinetic chromatography.

In this work a method of microemulsion electrokinetic chromatography (MEEKC) has been developed for the analysis of nine anthraquinones and bianthrones in rhubarb. This study employed di-n-butyl tartrate as oil substance to make up the microemulsion. The composition of the microemulsion was 0.5% (w/w) di-n-butyl tartrate, 0.6% (w/w) SDS, 1.2% (w/w) 1-butanol and 97.7% (w/w) 10 mM sodium borate buffer, pH of the buffer being 9.2. Acetonitrile was added to the emulsion to improve the separation. The volume ratio between the emulsion solution and acetonitrile of an optimized separation was 70:30. With the optimized conditions all of the nine analytes were baseline-separated in peaks of good shapes within 20 min. After validation the method was used to analyze the components in a rhubarb sample. A solid-phase extraction procedure was employed. Five anthraquinones and two bianthrones had been detected in the sample and their amounts were determined. The method should be able to be used for the quantitative analysis of the main active components of rhubarb crude drugs.

Improved detection of Coptidis alkaloids by field-amplified sample stacking in capillary electrophoresis.

To improve the on-line detection of Coptidis alkaloids in capillary electrophoresis the field-amplified sample stacking was studied for them. In this work the peak height enhancements of stacking with hydrodynamic and electrokinetic injections were compared with respect to the conventional sample injection. It was found that the stacking efficiency of electrokinetic injection was more than ten times greater than that of hydrodynamic injection. No peak height enhancement was observed with the pre-injection of a short water plug before sample injection with electrokinetic injection. The concentration limits of detection of berberine, coptisine and palmatine obtained with electrokinetic injection were about 5 ng/ml (ppb), which was approximately 240 times lower than those from conventional sample injections. Baseline separation was also achieved for the main alkaloids. After validation the developed method was applied to determine the quantity of berberine, coptisine and palmatine in a Coptidis Rhizoma sample. The method is simple, rapid and should be able to be used in identification and quantitative evaluation of the crude drugs.

Pharmacokinetic and metabolic studies of retrojusticidin B, a potential anti-viral lignan, in rats.

The pharmacokinetics and metabolism of retrojusticidin B, an anti-HIV reverse transcriptase agent isolated from Phyllanthus myritifolius, were studied in rats. The phase II conjugated metabolites were characterized after solvolysis and enzymatic hydrolysis. The oral bioavailabilities of retrojusticidin B, suspended in Tween 80 and in corn oil, were found to be 22.1% and 33.1%, respectively. The elimination half-lives (T1/2) were 22.9 and 36.2 minutes, respectively. The T1/2, clearance, and the volume of distribution (Vz) of retrojusticidin B estimated from i.v. measurements were 24.5 min, 2.6 +/- 0.4 L/min, and 90.6 +/- 6.4 L, respectively. 9,9'-Secoretrojusticidin B was shown to be phase I metabolite.

Validated HPLC method for determination of sennosides A and B in senna tablets.

This study developed an efficient and reliable ion-pair liquid chromatographic method for quantitation of sennosides A and B in commercial senna tablets. Separation was conducted on a Hypersil C 18 column (250 x 4.6 mm, 5 microm) at a temperature of 40 degrees C, using a mixture of 0.1 M acetate buffer (pH 6.0) and acetonitrile (70:30, v/v) containing 5 mM tetrahexylammonium bromide as mobile phase. Sennosides A and B were completely separated from other constituents within 14 min. The developed method was validated. Both run-to-run repeatability (n=10) and day-to-day reproducibility (n=3) of peak area were below 0.4% RSD. Linearity of peak area was tested in the range 30-70 microg/ml (r>0.9997). Accuracy was assessed with recovery and the recoveries for sennosides A and B were 101.73+/-1.30% and 101.81+/-2.18% (n=3 x 6), respectively. Robustness of the analytical method was tested using a three-leveled Plackett-Burman design in which 11 factors were assessed with 23 experiments. Eight factors (column, concentration of ion pair reagent, % of organic modifier (acetonitrile), buffer pH, column temperature, flow rate, time constant and detection wavelength) were investigated in a specified range above and below the nominal method conditions. It was found that: (1) column and % acetonitrile affected significantly resolution and retention time, (2) column, % acetonitrile, column temperature, flow rate and time constant affected significantly the plate number of sennoside A, and (3) column and time constant affected significantly the tailing factor.