Simultaneous determination of iridoid glycosides and flavanoids in Lamionphlomis rotate and its herbal preparation by a simple and rapid capillary zone electrophoresis method.

Iridoid glycosides and flavanoids are two main effective components of Lamiophlomis rotata (Benth.) kudo. However, there is no method for simultaneous analysis of iridoid glycosides and flavanoids in L. rotata and its pharmaceutical preparations. A simple and rapid capillary zone electrophoresis (CZE) method was developed and validated for simultaneous determination of two iridoid glycosides (8-O-acetylshanzhiside methylester and 8-deoxyshanzhiside) and three flavanoids (apigenin, quercetin and luteolin) in L. rotata. Operational variables, such as the voltage, buffer concentration and pH were optimized, the final optimum separation condition was 10 mM sodium tetraborate-20 mM NaH(2) PO(4) (pH 8.5)-15% (v/v) methanol, 238 nm UV detection, 18 kV applied voltage. The linearity and the recovery of the proposed method were very satisfactory (correlation coefficients were 0.9994-0.9998 and the recoveries were 94.5-108.8% for the analytes) and the method allowed analytes in real samples to be determined within 9 min. The proposed CZE method can be used for quality control of iridoid glycosides and flavanoids in L. rotata and its herbal preparation.

An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis.

In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-µL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 µg/mL and 6.9 µg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.

On-line combination of single-drop liquid-liquid-liquid microextraction with capillary electrophoresis for sample cleanup and preconcentration: a simple and efficient approach to determining trace analyte in real matrices.

In order to improve the sensitivity of capillary electrophoresis (CE) and overcome the deficiency of commercial CE instruments in handling complex matrices directly, we proposed a novel technique which combined single-drop liquid-liquid-liquid microextraction (SD-LLLME) with CE on-line. In this technique, SD-LLLME was realized using a commercial CE instrument and, to further concentrate the target analyte, large-volume sample stacking combined sweeping without polarity switching was utilized. Even though without agitating the donor phase in the extraction process, the model compound, adenine was enriched 550-fold in only 10 min. The enrichment factors were 760 and 1030 when the extraction time was extended to 30 and 60 min, respectively. The relative standard deviations (RSDs) of adenine were 5.24% and 2.29% for peak area and migration time, respectively, which indicated that this method was much more reproducible compared to the existing methods that combined sample-preparation strategies with CE. In addition, this approach was selective while cleaning up target analyte. These mentioned advantages allowed the developed method to be an attractive approach to determining trace target compounds in complex real samples.

Micelle to trapping solution stacking in micellar electrokinetic chromatography.

An analytical strategy micelle to trapping solution stacking (MSS) was developed in acidic buffer in micellar electrokinetic chromatography (MEKC). The stacking mechanism is based on the transport, release, capturing of molecules bound to micelle carriers that are made to collapse into trapping solution (TS) to serve as the medium to contain and stacking the analytes. Tetrandrine and fangchinoline were selected as model mixture using sodium dodecyl sulfate (SDS) micelles as carrier to demonstrate this stacking method. The experiments by MSS-MEKC were carried out and further compared with those by normal MEKC. The results reveal that 113-123-fold improvements in the detection sensitivity was obtained for the analytes, and separation and determination of tetrandrine and fangchinoline in Stephaniae tetrandrae S. Moore and Fengtongan capsules were finished under optimum conditions using the sample matrix containing 8.0mM SDS and TS containing 50mM H(3)PO(4)-55% (v/v) ethanol.

Preparation and characterization of a novel fluorescent-magnetic nanomaterial.

For the first time, a new multifunctional nanomaterial which combined the superparamagnetic property of iron oxide nanoparticles with the fluorescent property of morin-Al3+ complex was prepared. Iron oxide nanoparticles were first coated with SiO2 which could isolate them from the complex and the surrounding. Subsequently, Al2O3 . nH2O which provided active aluminum atom were deposited on the silica shell. Finally, morin which would react with the reactive aluminum atom was added into the reaction mixture to prepare the final multifunctional nanomaterial. The obtained product was characterized by X-ray powder diffraction, transmission electron microscopy, fourier transform infrared spectra, photoluminescence spectra, fluorescence microscopy, and vibration sample magnetometer. The characterization results showed that the final samples had an average size of 60 nm with spherical shape. The saturation magnetization of this new synthesized material was about 8.5 emu g(-1). Its excitation and emission wavelengths were 420 and 513 nm, respectively.

Low-temperature bath/high-conductivity zone/stacking micellar electrokinetic chromatography for the analysis of phenolic acids in coffee drink.

Two stacking methods in micellar electrokinetic chromatography (MEKC) were investigated in this article in an attempt to increase the amount of sample injected, as well as to focus analytes onto a small zone. One employed a "high-conductivity zone", which was inserted between the sample zone and background solution to build an unequal conductivity gradient. The other employed a "low-temperature bath". A portion of the capillary was immersed in a low-temperature bath, which served as a "pseudo-low-conductivity zone". As a result, a large volume of sample injection can be achieved. Using three phenolic acids-chlorogenic acid (CGA), caffeic acid (CA) and ferulic acid (FA) in coffee as model compounds, the limit of detection (LoD) was determined to be 0.31microg/ml (S/N=3) for CGA by means of normal MEKC under suppressed electroosmotic flow (EOF). The LoD could be improved to 2.8 x10(-2), 5.3 x10(-3) and 6.0 x10(-3) microg/ml, respectively, when normal MEKC-stacking, high-conductivity zone MEKC-stacking and the low-temperature zone MEKC-stacking methods were applied. Furthermore, the high conductivity zone and the low-temperature bath were operated simultaneously on one capillary to investigate the synergism effect. The results showed that there did exist synergism effect for CGA and CA when the two were hyphenated. The stacking efficiency was higher than that of the single one used. However, there was not synergism effect for FA.

Characterization of interaction between bergenin and human serum albumin in membrane mimetic environments.

The interaction between bergenin and human serum albumin (HSA) in AOT/isooctane/water microemulsions was studied by fluorescence quenching technique in combination with UV absorption spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique. Fluorescence data in omega (o) 20 microemulsions revealed the presence of a binding site of bergenin on HSA and its binding constants (K) were 1.64 x 10(4), 1.44 x 10(4), 1.26 x 10(4) and 1.09 x 10(4) M(-1) at 289, 296, 303, and 310 K, respectively. The binding of bergenin with HSA in microemulsions was stronger than that in buffer solution. The alterations of protein secondary structure in the microemulsions in the absence and presence of bergenin compared with the free form of HSA in buffer were qualitatively and quantitatively analyzed by the evidence from CD spectra. Enthalpy and entropy changes for the reaction were calculated to be -14.45 kJ mol(-1) and 30.76 J mol(-1) K(-1). These results indicated that bergenin bound to HSA mainly by a hydrophobic interaction in microemulsions which was in agreement with the result of the molecular modeling study. The DLS data suggested that HSA may locate at the interface of the microemulsion and bergenin could interact with them.

[Determination of tetrandrine and fangchinoline in Radix Stephaniae tetrandrae and its preparation by nonaqueous capillary chromatography].

OBJECTIVE: To establish a new method for the determination of fangchinoline and tetrandrine in Stephania tetrandra and Fengtongan capsule by noanqueous capillary electrophoresis. METHOD: Separation was carried out in an uncoated fused capillary (50 cm x 75 microm i.d.) with a running buffer containing 50 mmol x L(-1) ammonium acetate, 1.0% acetic acid and 20% acetonitrile in methanol. A separation voltage of 20 kV and a UV detector wavelength at 214 nm were adopted. Sample was introduced from the anode. RESULT: The calibration ranges were 1.00, 500 mg x L(-1) for both analytes. Under the optimum conditions, the relative standard deviation (RSD, n = 6) for the migration time of each analyte were 0.09%, 1.9% (intra-day) and 0.63%, 1.9% (inter-day); The RSD for the peak area of each analyte were 0.45%, 5.9% (intra-day) and 2.3%, 5.6% (inter-day), respectively. The contents of the analytes were determined easily with average recoveries 102% for fangchinoline and 105% for tetrandrine in S. tetrandra and 94.6% for fangchinoline and 98.7% for tetrandrine in Fengtongan capsules, respectively. CONCLUSION: The proposed method is simple, rapid, accurate and higher repeatable, and can be used to control of the quality of S. tetrandra and Fengtongan capsules.

Comparison of two sample preconcentration strategies for the sensitivity enhancement of flavonoids found in Chinese herbal medicine in micellar electrokinetic chromatography with UV detection.

Two on-column preconcentration techniques named stacking with reverse migrating micelles (SRMM) and anion selective electrokinetic injection and a water plug-sweeping with reverse migrating micelles (ASIW-sweep-RMM) were used and compared for concentration and separation of flavonoids in Chinese herbs using reverse migration micellar electrokinetic chromatography (RM-MEKC). The optimal background electrolyte (BGE) used for separation and preconcentration was a solution composed of 20mM phosphoric acid (H(3)PO(4))-100mM sodium dodecyl sulfate (SDS)-20% (v/v) acetonitrile (ACN) buffer (pH 2.0), the applied voltage was -15kV. To achieve reasonable results of the two techniques, the conditions which affected preconcentration were examined. A comparison of used techniques with normal hydrodynamic injection (5s), concerning enhancement factors and limits of detection (LODs) was presented. Under the optimum stacking conditions, about 27-37- and 45-194-fold improvement in the detection sensitivity was obtained for SRMM and ASIW-sweep-RMM, respectively, compared to usual hydrodynamic sample injection (5s). The LODs (S/N=3) for SRMM and ASIW-sweep-RMM in terms of peak height, can reach down to 1.15 x 10(-2) microg/ml for hesperetin and 2.4 x 10(-3) microg/ml for nobiletin, respectively. Finally, the amounts of the six flavonoids in extract of Fructus aurantii Immaturus were successfully determined using ASIW-sweep-RMM. The six analytes were baseline separated with sample matrix under the optimum ASIW-sweep-RMM conditions and the experimental results showed that preconcentration was well achieved after the dilution of sample solutions.

Separation and determination of psoralen and isopsoralen by microemulsion electrokinetic chromatography.

The use of microemulsion electrokinetic chromatography was proposed to separate psoralen and isopsoralen in Psoralea corylitolia L. and its preparations. After conducting a series of optimizations, baseline separation was obtained for the analytes under the optimum conditions [sodium dodecyl sulfate 1.05% (m/v), ethyl acetate 0.96% (v/v), butan-1-ol 0.24% (v/v), 25 mm borate, pH 8.5, applied voltage 17.5 kV and detection at 254 nm]. Regression equations revealed linear relationships (correlation coefficients 0.9997 for psoralen and 0.9999 for isopsoralen) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal-to-noise ratio of about 3) were 0.42 microg/mL for psoralen and 0.32 microg/mL for isopsoralen, respectively. The analytes were successfully determined with recoveries ranging from 95.50 to 102.03%. The method has been successfully applied for the analysis of psoralen and isopsoralen in medical samples. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 20 min three times, was used for sample preparation.

Separation and determination of three water-soluble compounds in Salvia miltiorrhiza Bunge and two related traditional medicinal preparations by flow injection-capillary electrophoresis.

A sensitive and reproducible method was developed for the separation and determination of three water-soluble components-protocatechuic aldehyde (PAH), beta-(3,4-dihydroxyphenyl) lactic acid (DSS) and protocatechuic acid (PA) in medicine plant Salvia miltiorrhiza Bunge and two related traditional medicinal preparations using flow injection-capillary electrophoresis system. This analysis was carried out by using an unmodified fused-silica capillary (28.4 cm x 75 microm i.d.x 375 microm o.d., 25 cm effective separation length) and direct ultraviolet detection at 214 nm, 7.0 kV applied voltage. With boric acid (200 mM) adjusted to pH 7.8 as a background electrolyte. The separation was achieved in 9 min. The sample throughput rate could reach up to 15 h(-1). Calibration curves showed good linearity with correlation coefficients (r) more than 0.9986. The repeatability (defined as R.S.D.) were 0.20%, 0.46%, 0.47% with migration time evaluation and 0.62%, 3.66%, 1.50% with peak area evaluation for PAH, DSS and PA, respectively. The limits of detection (S/N=3) were 0.36 microg/mL, 0.84 microg/mL, and 0.73 microg/mL for PAH, DSS, and PA, respectively. The mean recoveries of PAH, DSS and PA were 103.2%, 98.1% and 100.5%, respectively. This method has been applied successfully to monitor these three components in Salvia miltiorrhiza Bunge and its two traditional medicinal preparations.

Separation and determination of epinephrine and dopamine in traditional Chinese medicines by micellar electrokinetic capillary chromatography with laser induced fluorescence detection.

A micellar electrokinetic capillary chromatography method with laser-induced fluorescence detection was developed for the analysis of epinephrine and dopamine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The optimum derivatization conditions were: 30 mM sodium borate (pH adjusted to 8.0 with 1.0 M HCl), reaction time 30 min at 60 degrees C. Baseline separation was achieved within 14 min with a running buffer composed of 10 mM sodium borate + 25 mM sodium dodecyl sulfate (pH adjusted to 9.5 with 0.1 M NaOH) and an applied voltage of 15 kV. Good linearity relationships (correlation coefficients: 0.9991 for epinephrine and 0.9985 for dopamine) between peak areas and concentrations of the analytes were obtained. The detection limits and quantification limits for epinephrine and dopamine were 0.0038 mg/L and 0.013 mg/L, and 0.065 mg/L and 0.020 mg/L, respectively. The method was applied to the analysis of the two compounds in two Chinese medicines with recoveries in the range of 92.6-108.7%.

Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive.

Microemulsion electrokinetic chromatography (MEEKC) using 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) ionic liquid (IL) as additive was developed for the analysis of baicalin, wogonin and baicalein in Scutellariae radix and its preparation. After conducting a series of optimizations, baseline separation was obtained for the analytes within 5min under the optimum conditions (sodium dodecyl sulfate (SDS) 0.88% (m/v) ethyl acetate 0.8% (v/v) butan-1-ol 0.2% (v/v) and the buffer composition were 25% acetonitrile (v/v), 7.5 mM BMIM-BF4 and 10 mM NaH2PO4, pH 8.2, applied voltage 17.5 kV and detection at 254 nm), the method has been successfully applied to the determination and quantification of the analytes in the extracts of S. radix (cooked), S. radix (raw) and Qingfeiyihuowan which was the preparation including S. radix.

Determination of five anthraquinones in medicinal plants by capillary zone electrophoresis with beta-cyclodextrin addition.

A simple and rapid method for the simultaneous determination of five anthraquinone derivatives including aloe-emodin, emodin, chrysophanol, physcoin and rhein in Rheum species and Polygonum cuspidatum was established by capillary zone electrophoresis (CZE) using beta-cyclodextrin (CD) as modifier and urea to enhance its solubility. The apparent binding constants of these derivatives with beta-CD were evaluated. After an optimization study, the best conditions were selected using 35 mM phosphate buffer (pH 11.0) containing 20 mM beta-CD and 2 M urea, applied voltage 20 kV and detection at 254 nm. Under such conditions, all of the five anthraquinones were baseline-separated within a short analysis time of 12 min with symmetrical peaks and high theoretical plate numbers (189,000-314,000). The RSD values of the migration times and peak areas were 0.6-1.1, 1.3-1.9% (intra-day) and 0.6-1.5, 1.3-2.8% (inter-day, for a 5-day period), respectively. The limits of detection for the analytes (S/N = 3) were 0.33-0.62 microg/ml. The recoveries were ranged from 93.37 to 107.69%. The proposed method was successfully applied to the determination of anthraquinones in ethanol extracts of two kinds of Rheum plants (R. palmatum and R. hotaoense) and P. cuspidatum.

Separation and determination of honokiol and magnolol in herbal medicines by flow injection-capillary electrophoresis.

A simple, rapid, and accurate method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of flow injection (FI) and capillary zone electrophoresis (CZE). The analysis was carried out using an unmodified fused-silica capillary (50-microm I.D.; total length 7.5 cm; effective length 4.5 cm). A series of optimization steps afforded the following conditions: the sample solvent consisted of 150 mM NaOH and a running buffer composed of 10 mM sodium tetraborate/10 mM sodium dihydrogenphosphate (NaH2PO4) at pH 12 was applied for the separation of the analytes. The separation could be achieved within 5 min with a sample throughput rate of up to 28 h(-1). The repeatability (defined as the relative standard deviation, RSD) for honokiol and magnolol was 2.0% and 1.6% with peak area evaluation, 3.6% and 2.0% with peak height evaluation, and 2.0% and 1.4% with migration time evaluation, respectively. Regression equations revealed linear relationships (r = 0.9991-0.9998) between the peak area of each analyte and the concentration.

Simultaneous determination of bioactive flavone derivatives in Chinese herb extraction by capillary electrophoresis used different electrolyte systems--borate and ionic liquids.

In this paper, novel capillary electrophoresis (CE) methods, which used ionic liquids (1-ethyl-3-methylimidazoium tetrafluoroborate, 1-butyl-3-methylimidazoium tetrafluoroborate), were established to separate and determine some bioactive flavone derivatives in Chinese herb Seriphidium santolinum (Schrenk) Poljak. In order to investigate the traits of ionic liquids in CE, borate was also used as electrolyte to compare with. And the excellence of CE, which used ionic liquids as main running electrolyte and beta-cyclodextrin (beta-CD) as modifier, was illuminated as well. As a result of the study, the difference of ionic liquids and borate in CE was discussed and the advantage of CE, which used ionic liquids as electrolytes for separation, was shown. The analysis was obtained within short time (5-6 min). From the result, it was found that the system, which used ionic liquids, was robust because the joule heating was small. The method of CE, which used ionic liquids, has lower detection limits (0.137-0.642 microg/mL) than that of borate (0.762-1.036 microg/mL). And the CE, which used ionic liquids method, has lower limit of linear range (1.100-2.656 microg/mL), while that of CE, which used borate method, was 2.188-5.313 microg/mL.

Separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation by nonaqueous capillary electrophoresis.

An easy, rapid method for simultaneous determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation was developed by nonaqueous capillary electrophoresis (NACE) without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cmx75 microm i.d.) and a running buffer containing 30 mM ammonium acetate, 1.0% acetic acid and 15% acetonitrile (ACN) in methanol medium. The applied voltage was 30.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. The established method with sophoridine as internal standard was linear in the range of 5-1000 mg/mL for both strychnine and brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for determination with recoveries ranging from 94.5 to 104%.

Rapid separation and determination of aconitine alkaloids in traditional Chinese herbs by capillary electrophoresis using 1-butyl-3-methylimidazoium-based ionic liquid as running electrolyte.

A novel and very simple capillary electrophoretic method for analyzing aconitine components in Aconitum plants was developed using 1-butyl-3-methylimidazoium tetrafluoroborate (1B-3MI-TFB)-based ionic liquid as running electrolyte solution for the first time. The optimum conditions were 35 mM 1B-3MI-TFB solution (pH 8.50) and 15 kV applied voltage. The detection was performed at 254 nm. Aconitine, meaconitine and hypaconitine in Aconitum plants were separated and identified within 5 min. The recoveries were 91.0-103.0% for hypaconitine, 92.8-96.2% for aconitine and 96.0-106.6% for mesaconitine, respectively. Compared with other methods, the analytical time was decreased 4-8-fold and the effect of Joule heating was weaker because the current was smaller.

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.

Novel and simple nonaqueous capillary electrophoresis separation and determination bioactive triterpenes in Chinese herbs.

Three bioactive triterpenes ursolic acid, oleanolic acid and 2alpha,3beta,24-trihydroxy-urs-12-en-28-oic acid were simultaneously separated by nonaqueous capillary electrophoresis (NACE) with methanol:acetonitrile (65:35 v/v) mixture containing 90 mm trishydroxymethylaminomethane (Tris) at an applied voltage of +25 kV and a hydrodynamic injection of 5s. The effect of solvent composition, electrolyte nature and concentration on the electrophoretic behavior of the analytes were systematically studied. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation and correlation coefficients were obtained. Meanwhile, the method was applied to separation and determination the three components in six Chinese herbs extraction. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive triterpenes in Chinese herbs.