[Near-infrared spectroscopy technology for online monitoring of the column separation and purification process of active components of Centella asiatica L. Urban].

The present paper is to study and develop a method for online monitoring of the column separation and purification process of active components that are madecassoside and asiaticoside of Centella asiatica L. Urban using near-infrared (NIR) spectroscopy technology. After collecting 50%-ethanol eluant, we detected their NIR spectra and developed the high performance liquid chromatography (HPLC) assay method of active components. Then, partial least square (PLS) was used to develop linear correlation between their NIR spectra and contents. During modeling, correlation coefficient (R2) and root mean square errors of cross-validation (RMSECV) were regarded as the indexes to select optimal wavenumbers and preprocessing methods. The optimal wavenumbers of madecassoside and asiaticoside were in the range of 12 000.8-7 499.8 cm(-1) and 12 000.8-9 750.3 cm(-1), respectively; R2 were 96.44 and 96.07, respectively, and RMSECV were 0.084 80 and 0.000 99, respectively. The above developed model was used for online monitoring of the contents of madecassoside and asiaticoside during the column separation and purification process of Centella asiatica L. Urban. The predicted results were satisfactory. This method was proved to be fast, convenient and precise. It can be used in online monitoring and quality control of the manufacturing of madecassoside and asiaticoside.

Optimization and validation of an ion-pair RP-HPLC-UV method for the determination of total free iodine in rabbit plasma: application to a pharmacokinetic study.

An ion-pair reverse-phase high performance liquid chromatographic method with UV-vis detection has been developed for the determination of total free iodine in rabbit plasma after vaginal administration of povidone-iodine (PVP-I). Sample preparation was done by protein precipitation with acetonitrile in 96-well format and aspirin was used as the internal standard. The 100 microL sodium thiosulfate solution (5 g L(-1)) was added to 100 microL plasma sample before protein precipitation, to convert the total free iodine in plasma to iodide (I(-)). Separation was performed on a C(18) column (200 x 4.6 mm i.d., 5 microm). The mobile phase consisting of a mixture of water phase (containing 10 mmol L(-1) 18-crown-6 ether, 5 mmol L(-1) octylamine and 5 mmol L(-1) sodium dihydrogen phosphate, pH adjusted to 6.0 with phosphoric acid) and acetonitrile in the ratio 70:30 (v/v) was delivered isocraticly at a flow rate of 1.0 mL min(-1). The method was sensitive with a lower limit of quantification of 0.005 microg mL(-1), with good linearity (r(2) > 0.9990) over the linear range of 0.005-2 microg mL(-1). All the validation data, such as linearity, accuracy and precision, were within the required limits. The method was successfully applied to study the pharmacokinetic of PVP-I in rabbits after vaginal administration.

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development, validation and application to a pharmacokinetic study of curcumin liposome.

This paper describes a sensitive, specific and rapid high-performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96-well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse-phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C(18) analytical column (4.6 x 100 mm, 5 microm) using acetonitrile-5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r(2) >or= 0.999) over the linear range 1-500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high-throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats.

[Study on purification of total flavones from Trollius ledebouri by macroporous resin].

OBJECTIVE: To study the optimal technologies for the purification of total flavones from Trollius ledebouri with macroporous resin. METHODS: Static and dynamic adsorption-desorption methods were adopted to choose the optimal type of resin. The adsorption function of D101 macroporous resin and the effects of sample concentration, pH, flow rate and eluant etc. were studied. Then the orthogonal design L9 (3(4)) was used to select the optimum purification process conditions. RESULTS: The appropriate technological conditions were as follows: the sample concentration was 0.5 g/mL, pH 6.0, the ratio of sample to D101 macroporous resin was 1:1 (W/W), 4 BV of water was used as purificant and 6 BV of 30% alcohol was used as eluant. Under these conditions, the average content of total flavonoids extract was over 60%.

Determination of porcine fibrinogen in rat and dog plasma after intraperitoneal injection of a porcine-derived fibrin glue by fluorescein-labeled assay method: Comparison with isotope-labeled assay method.

A sensitive and specific fluorescein isothiocyanate (FITC) label coupled with size-exclusion high-performance liquid chromatography-fluorescence detection (SE-HPLC-FLD) method was developed and validated for the estimation of the pharmacokinetic profiles of porcine fibrinogen after intraperitoneal injection of a porcine-derived fibrin glue (FG) to SD rats and beagle dogs with three single doses. Porcine fibrinogen, the major composition of the FG, was labeled with FITC. The FG containing FITC-labeled porcine fibrinogen was intraperitoneally administered to SD rats at three single dosages (100, 200, 400mg/kg of porcine fibrinogen), and the collected plasma was then detected by SE-HPLC-FLD method. The present technique was compared to the previously introduced isotope-labeled assay method for the pharmacokinetic studies in SD rats. The pharmacokinetic studies in SD rats showed that the correlation coefficient between the FITC-labeled assay and (125)I-labeled assay methods was r(2)=0.989. Thus, this FITC-labeled assay method performed well and demonstrated high concordance with the previous (125)I-labeled assay method, suggesting that FITC-labeled assay could substitute the (125)I-labeled assay as a method of choice for quantification in beagle dogs. Then the plasma levels of porcine fibrinogen in beagle dogs were studied by the FITC-labeled assay method with three single doses (15, 30, 60mg/kg of porcine fibrinogen). The method validation showed that the FITC label coupled with SE-HPLC-FLD method was suitable for the quantification of porcine fibrinogen in plasma samples with satisfactory linear (r(2)>0.999), precision (<12%), accuracy (95.5-104.9%) and recovery (>88%). The results showed linear disposition of porcine fibrinogen at the examined dosage range in SD rats or beagle dogs.

Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis.

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.