Rapid analysis of melamine in infant formula by sweeping-micellar electrokinetic chromatography.

In the present study, an analytical method using capillary electrophoresis with on-line preconcentration technique was developed for rapid determination of melamine in infant formula. Both stacking and sweeping preconcentration techniques had been investigated for the comparison of their effectiveness in melamine analysis. The limit of detection of melamine standard was 0.5 ng/mL for the field amplified sample stacking (FASS) technique and 9.2 ng/mL for the sweeping technique. Although the FASS technique provided better concentration efficacy than the sweeping technique, the matrix effect was more profound with the former. Matrix effect was evaluated by comparing the enhancement factor (EF) of melamine standard and post-extraction spiked infant formula solution. The EF was changed from 429.86 +/- 9.81 to the level less than 133.31 with significant peak distortion in the FASS system, and it was remained unchanged in the sweeping system. Sweeping-micellar electrokinetic chromatography (sweeping-MEKC) was demonstrated to be most suitable for real sample analysis. Under optimum sweeping-MEKC conditions, melamine content in infant formulas could be determined within 6 min. The developed solid phase extraction (SPE) procedures coupled with the sweeping-MEKC method was subjected to method validation. Run-to-run repeatability (n = 3) and day-to-day reproducibility (n = 3) of peak area were within 3.6% and 4.8% RSD, respectively. The accuracy was tested by spiking 0.5 and 2 microg/mL of melamine standard in the melamine contaminated milk powder provided by the European Commission, and the recoveries were 93.4 +/- 0.5% and 98.7 +/- 0.4%, respectively. Results of this study show a great potential for the sweeping-MEKC method as a tool for the fast screening of melamine in infant formulas.

Cyclodextrin-modified microemulsion electrokinetic chromatography for separation of alpha-, gamma-, delta-tocopherol and alpha-tocopherol acetate.

Different forms of tocopherols, together with tocotrienols, are collectively named as vitamin E, and each possesses different degree of medical, biological and physiochemical significance. The main difficulty of separating different forms of tocopherols lay in their highly structural similarities and hydrophobicities. Microemulsion electrokinetic chromatography (MEEKC), claimed to attain high peak efficiency with great solubilization power, has not previously been applied to the separation of tocopherols. The effects that various parameters, such as buffer system, type and concentration of cyclodextrins, temperature, and sample matrix, have on the separation of tocopherols by MEEKC have been investigated. By using a buffer mixture of 4% (w/w) sodium dodecyl sulfate (SDS), 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane, 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25mM, pH 2.5), and 25mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), the separation of alpha-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, as well as the antioxidant butylated hydroxytoluene (BHT) at -26kV, 25 degrees C was completed within 35min. The practical potential of the present approach has been further validated by the determination of tocopherols in a vitamin E preparation, with the result of 132.63 (RSD 1.25%), 176.51 (RSD 0.29%), and 64.32mg (RSD 3.34%) per 500mg capsule for alpha-, gamma- and delta-tocopherol, respectively.

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.

Effects of calcineurin inhibitors on sirolimus pharmacokinetics during staggered administration in renal transplant recipients.

STUDY OBJECTIVE: To compare the effects of different calcineurin inhibitors on sirolimus pharmacokinetics during long-term, staggered administration in kidney transplant recipients. Design. Randomized, open-label, parallel-group trial. SETTING: A medical center and one of its teaching hospitals in Taiwan. PATIENTS: Twenty-two de novo kidney transplant recipients. INTERVENTION: Patients received cyclosporine microemulsion or tacrolimus capsules twice/day in combination with once-daily sirolimus solution and corticosteroids. Sirolimus was administered 6 hours after the morning dose of cyclosporine or tacrolimus. After receiving a 6-mg loading dose of sirolimus, participants received sirolimus 2 mg/day for at least 7 days. Neither the cyclosporine nor the tacrolimus dosage was adjusted for at least 3 days before and during blood sampling for pharmacokinetic profiling. MEASUREMENTS AND MAIN RESULTS: One patient dropped out because of trimethoprim-sulfamethoxazole-related hepatotoxicity. We observed no differences between the two patient groups in terms of their demographic data, renal and liver function, or dosage of sirolimus during the study. During multiple-dose administration, the area under the whole-blood concentration-time curve and the peak and trough concentrations of sirolimus in the cyclosporine group were, respectively, 1.46 (95% confidence interval [CI] 1.21-1.71), 1.42 (95% CI 1.08-1.76), and 1.42 (95% CI 1.09-1.76) times higher than those of the tacrolimus group, even though sirolimus was administered 6 hours after the other agents. CONCLUSION: Sirolimus pharmacokinetics may change significantly when calcineurin inhibitors are switched, even with staggered administration, which may not completely prevent a drug interaction between cyclosporine and sirolimus solution.

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.

Effects of conversion from sirolimus oral solution to tablets in stable Taiwanese renal transplant recipients.

BACKGROUND AND PURPOSE: Sirolimus (SRL) has a considerable inter- and intra- individual variability in clearance. Steady-state trough concentration (C(0)) is a reliable index of SRL exposure. This study assessed the effect of conversion of SRL oral solution to tablet form on C(0) in stable renal transplant recipients. METHODS: Twenty two stable renal transplant recipients who had received calcineurin inhibitor (CNI)/SRL solution/ steroid for more than 3 months before conversion from SRL solution to tablets were included. C(0) values of SRL were compared for the periods of use of each dosage form. The relation between liver function and SRL levels was also assessed. RESULTS: With a dose of 0.03 mg/kg/day, SRL solution and tablets achieved a similar dose-adjusted C(0) (mean +/- SEM, 2.9 +/- 0.3 ng/mL/mg) upon conversion. Similar results were found when multiple SRL C(0) values from different dosage form periods were compared. Four patients with persistent liver enzyme elevation had significantly higher dose-adjusted SRL C(0) values with both the solution (mean +/- SEM, 4.5 +/- 0.7 vs 2.3 +/- 0.1 ng/mL/mg; p < 0.01) and the tablet formulation (4.0 +/- 0.5 vs 2.6 +/- 0.2 ng/mL/mg; p < 0.05). CONCLUSIONS: Conversion from SRL solution to SRL tablets did not significantly affect the dose-adjusted SRL C(0). The dose-adjusted C(0) of SRL in patients with persistent liver enzyme elevation was significantly higher than in those with normal liver function.

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.

Electrophoretic behavior and pKa determination of quinolones with a piperazinyl substituent by capillary zone electrophoresis.

Electrophoretic behavior and pKa determination of six quinolones with a piperazinyl substituent, together with two quinolones without a piperazinyl substituent and 1-phenylpiperazine, were investigated by capillary zone electrophoresis. The results indicate that quinolones with a piperazinyl substituent involve three protonation/deprotonation equilibria. The results also suggest that the contribution of the zwitterionic species of these quinolones to the effective mobility may not be neglected. This is probably due to a slightly incomplete protonation of the piperazinyl moiety in the pH range of 6.0-8.0, compared with the complete dissociation of the carboxylic group. Consequently, the zwitterionic species of ciprofloxacin, in particular, is slightly negatively charged. With the aid of computer simulation, three pKa values were determined for quinolones with a piperazinyl substituent, thus allowing us to rationalize precisely the influence of pH on the electrophoretic behavior of these compounds.

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.

Determination of ursodeoxycholic acid in pharmaceutical preparations by capillary electrophoresis with indirect UV detection.

A simple and rapid capillary electrophoretic method, with indirect UV detection, for the quantification of ursodeoxycholic acid (UDCA) in pharmaceutical preparations was developed in this study. Sodium p-hydroxy benzoate was used as background electrolyte (BGE) (5 mM, pH 8.0) and visualization agent. Separation was carried out on a fused-silica capillary (50 microm x 72 cm) at a potential of 25 kV under ambient temperature and detected at 250 nm. Glycocholic acid was used as internal standard for quantification. Both run-to-run repeatability and day-to-day reproducibility of migration time were below 0.1% relative standard deviation (R.S.D.). Repeatability and reproducibility of relative peak height were 3.3 and 3.8% R.S.D., respectively. Accuracy was tested by spiking two pharmaceutical tablets with standards and the recoveries were 101.9+/-9.87 and 99.6+/-9.60% (n=3), respectively. Linearity of relative peak height was tested in the range 20-100 microg/ml. Limit of detection (LOD) was 3 microg/ml. The method could be used to assay UDCA raw materials and formulation products.

Analysis of lignans using micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) was used to separate twelve lignan compounds originating from Phyllanthus plants. To increase the reliability of peak identification, two micellar systems, the sodium dodecyl sulfate (SDS) and sodium deoxycholate (SDC) systems, were investigated. Because of the high lipophilicity of the lignan analytes, tetrahydrofuran was added to the SDS micellar system to increase its separating ability. In contrast to SDS system, no organic solvent was needed with SDC micelles. Both micellar systems gave a satisfactory separation within a reasonable analysis time. On considering accuracy for quantitation, the SDS method was validated and then used to determine the content of the lignans in two Phyllanthus plants. The selectivity (elution order of the lignans) was significantly different between the SDS and SDC micellar systems. Retention in SDC-MEKC seemed to be dominated by the hydrophobicity of the lignan solutes, while in SDS-MEKC, retention was greatly influenced by hydrogen bonding interactions.

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.

Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography.

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.