Establishment and application of characteristic degradation fingerprint for the quality control of Compound Banlangen Granules polysaccharides.

Compound Chinese medicine preparation is a complex multi-component system. The traditional methods such as physicochemical identification and quantification of several main index components cannot provide adequate quality evaluation for Compound Banlangen Granules. The objective of this work was to establish a characteristic degradation fingerprint of Compound Banlangen Granules polysaccharides, and the reference fingerprint was obtained from the model samples prepared using prescription medicinal herbs from different origins. The partial degradation products of Compound Banlangen Granules polysaccharides were profiled by capillary zone electrophoresis, and the quality difference of polysaccharides of these preparations was compared by cluster analysis and principal component analysis. It was found that the contents and the characteristic degradation fingerprints of the polysaccharides from 25 batches of Compound Banlangen Granules of 17 manufacturers were significantly different. The quality of Compound Banlangen Granules polysaccharides was evaluated by the characteristic degradation fingerprint tool with satisfactory results. The present method provides a reference for the quality control strategy development of polysaccharides in other compound Chinese medicine preparations.

Separation and purification of glycosides from medicinal plants based on strong polar separation medium with online closed-loop mode.

Natural glycosides widely distributed in medicinal plants are valuable sources of therapeutic agents, showing various pharmacological effects. The separation and purification of natural glycosides are meaningful for their pharmacological research, which face with great challenges due to the complex of medicinal plants samples. In this work, two kinds of functional monolithic separation mediums A and S were fabricated and fully applied in the online extraction, separation and purification of active glycoside components from medicinal plants with a simple-procedure closed-loop mode. Chrysophanol glucoside and physcion glucoside were detected and separated from Rhei Radix et Rhizoma using separation medium A as a solid-phase extraction adsorbent. Rhapontin was isolated and purified from Rheum hotaoense C. Y. Cheng et Kao using separation medium S as the stationary phase of high-performance liquid chromatography. Compared to the reported literatures, high yield of 5.68, 1.20 and 4.76 mg g-1 of these three products were obtained with high purity. These two online closed-loop mode methods were carried out using high-performance liquid chromatography system, in which the sample injection, isolation and purification procedures are all online mode, and reduced loss compared to offline extraction and purification procedures, thus achieving high recovery and high purity.

Extraction and determination of terpenoids from Zexie Decoction based on a porous organic cage-doped monolithic cartridge.

A monolithic solid-phase extraction (SPE) cartridge packed with a composite adsorbent was fabricated via polymerization using dodecene as the monomer with the porous organic cage (POC) material doped, combing with an analytical column through a high-performance liquid chromatography (HPLC) instrument, which was used for the online extraction and separation of 23-acetyl alismol C, atractylodes lactone II and atractylodes lactone III from Zexie Decoction. The POC-doped adsorbent shows porous structure with a relatively high specific surface area of 85.50 m2/g, which was obtained from the characterizations of a scanning electron microscope and an automatic surface area and porosity analyser. Efficient extraction and separation of three target terpenoids was achieved by an online SPE-HPLC method based on the POC-doped cartridge, which exhibits strong matrix-removal ability and good terpenoids-retention ability with a high adsorption capacity, due to the interactions of hydrogen bond and hydrophobicity between the terpenoids and the POC-doped adsorbent. Method validation shows good linearity (r ≥ 0.9998) of the regression equation, and high accuracy with the spiked recovery in the range of 99.2 %-100.8 % of the proposed method. Compared to the generally disposable adsorbent, this work fabricated a reusable monolithic cartridge, which can be used for at least 100 times, with the RSD based on the peak area of the three terpenoids less than 6.6 %.

Fabrication of a monolithic, macroporous diallyl maleate-based material and its application for fast separation of intact proteins from human plasma with reversed-phase chromatography.

A monolithic diallyl maleate-based column was developed and used to separate intact proteins from complex bio-samples with high-performance liquid chromatography. The resulting monolith exhibited a relatively uniform porous structure with macro-pores. A sample of standard proteins mixture was used to explore the reversed-phase mechanism for fast separation on the homemade monolithic column. The observed retention time increased with the values of the diameters, relative hydrophobicities and molecular weights of the standard proteins, which mainly depends on the relative hydrophobicity of the proteins. It is worth mentioning that the mask of the three most abundant proteins in human plasma can be avoided, thus providing an opportunity for the middle- and low-abundance proteins to be detected. According to this exploration, the fast separation of human plasma proteins on the diallyl maleate-based monolithic column was achieved in 15 min. The chromatographic fractions were identified by liquid chromatography-tandem mass spectrometry, and the results indicate that the present method is an outstanding method for the fast and efficient fractionation of human plasma that will be significant sense for plasma proteomics research, especially for exploring new disease marker and drug target.

In situ synthesis of a monolithic material with multi-sized pores and its chromatographic properties for the separation of intact proteins from human plasma.

The fabrication of a monolithic allyl phenoxyacetate-based material was proposed via the in situ radical polymerization using ethylene dimethacrylate as the crosslinker and 2,2'-azobisisobutyronitrile as the initiator within a stainless steel column (50 mm × 4.6 mm i.d.). The effects of the porogen composition, the crosslinker amount and the monomer type on the resulting monoliths were investigated. The morphology of the monoliths was characterized using scanning electron microscopy and a nitrogen adsorption-desorption instrument, and the pore structure was characterized using mercury intrusion porosimetry. The results indicate that the optimized monolith has a micro-, meso- and macro- multi-sized pore structure with a high specific surface area of 260.66 m2 g-1. The resulting monoliths were used as stationary phases for the separation of proteins from bio-samples, including a mixture of six standard proteins, chicken egg whites, snailase and human plasma, using high-performance liquid chromatography. Compared to optimized glycidyl methacrylate-based and styrene-based monolithic columns, the allyl phenoxyacetate-based monolithic column exhibited improved selectivity in the separation of proteins. Furthermore, the present method avoids the masking of highly abundant proteins, such as human serum albumin, immunoglobulin G and human fibrinogen, in the detection of middle- or low-abundance proteins in human plasma. The protein identification results obtained from liquid chromatography/mass spectrometry indicate that the present method is an outstanding method for efficient fractionation of human plasma, which will be especially useful in future plasma proteomics research.

Preparation of a hydroxyethyl-based monolithic column and its application in the isolation of intact proteins from complex bio-samples.

In this work, a monolithic hydroxyethyl-based column was fabricated in a stainless-steel column (50 mm × 4.6 mm i.d.) via radical polymerization technique using hydroxyethyl methylacrylate as the monomer. The morphology and pore size distribution indicate that the optimized monolith has a relatively uniform structure with macro-pores. The homemade monolith was used as the stationary phase of high performance liquid chromatography for the separation of intact proteins from complex bio-samples, including human plasma, egg white and snailase. The resulting monolith shows excellent selectivity for intact proteins mainly depending on the different relative hydrophobicity of the objective proteins with reversed-phase liquid chromatographic mechanism. Besides, the hydrogen-bond interaction and electrostatic interaction were the additional interactions in the chromatographic separation owing to hydroxyl groups present in the surface of monolithic material.

A novel poly (NMA-co-DEA-co-EDMA) monolithic column as a sorbent for online solid-phase extraction and its application in the determination of ß-sitosterol in plant oil samples.

A novel monolithic column was prepared by in-situ free radical polymerization using N-methylolacrylamide (NMA) and N,N-diethylacrylamide (DEA) as co-monomers. The monolith was characterized by scanning electron microscopy (SEM) and its nitrogen adsorption-desorption isotherm, and it was used as a solid phase extraction (SPE) absorbent for the online enrichment of ß-sitosterol by high performance liquid chromatography. The optimized method had good linearity, and the linear regression coefficient was 0.998. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.006 mg/mL and 0.02 mg/mL, respectively. The interday and intraday accuracies were less than 7.28%. The spiked recoveries of ß-sitosterol in the six plant oil were 90.96-103.56%. The maximum amount of ß-sitosterol adsorbed on the monolithic column was 12.69 mg/g, and the enrichment factor of ß-sitosterol was 78. The results showed that the monolith could be used as an online SPE absorbent for the determination of ß-sitosterol in plant oil samples.

Identification of Confusable Herbal Medicines by Mapping of Partial Degradation Products from Herbal Medicine Polysaccharides.

Partial degradation products (PDPs) of herbal medicine (HM) polysaccharides with precolumn derivatization using 1-phenyl-3-methy-5-pyrazolone (PMP) were mapped by high performance liquid chromatography (HPLC), and three groups of confusable HMs were differentiated using the PDP fingerprints assisted with cluster analysis (CA). Three variables of HPLC mobile phase, i.e. acetonitrile proportion, buffer concentration and pH value were optimized with PDP of ß-cyclodextrin. Radix Glehniae and Radix Adenophorae; Radix Sophorae Tokinensis and Rhizoma Menispermi; Radix Achyranthis Bidentatae and Radix Cyathulae were successfully distinguished by the method, respectively. The results involving mass spectrometry analysis showed that these PDPs primarily included oligosaccharides and a few monosaccharides. The method can be used as an effective approach for the identification and quality control of HMs, and can also facilitate the in-depth study of biological activity and further development of HM polysaccharides to some extent.

Amino acid and ionic liquid modified polyhedral oligomeric silsesquioxane-based hybrid monolithic column for high-efficiency capillary liquid chromatography.

In this study, a novel amino acid and ionic liquid dual organically functionalized reagents modified polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) based hybrid monolithic column (POSS-VBI-Cys) was designed and reported. With amino acid L-cysteine and ionic liquid 1-vinyl-3-butylimidazolium bromide as dual monomers, POSS-MA as the crosslinker, the new POSS-VBI-Cys hybrid monolithic column could be facilely fabricated via the "one-pot" free radical copolymerization and thiol-ene click reaction. Because of the introduction of polar amino acid L-cysteine, the new POSS-VBI-Cys column exhibited attenuated hydrophobicity in reversed-phase liquid chromatography separation. Polar amides, nucleosides and nucleic acid bases displayed strong retention on the POSS-VBI-Cys column and could be successfully separated. Furthermore, the new POSS-VBI-Cys column displayed good separation selectivity for model glycoproteins and non-glycoproteins mixture and it was also successfully used for the purification and separation of TARG1 protein from its originally expressed sample. In the future research, we will further exploit its performances for separation of intact proteins and in-depth proteome applications.

[Chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis].

We developed a new method for chiral separation of fourteen amino alcohols by nonaqueous capillary electrophoresis (NACE) with the D-(+)-gluconic acid δ-lactone-boric acid complex as chiral selector. In order to achieve good enantioseparation, the effects of D-(+)-gluconic acid δ-lactone and boric acid concentrations, triethylamine concentration, as well as capillary temperature were systematically investigated. The optimized conditions were identified as follows: an uncoated fused silica capillary of 50 µm ID with a total length (L(tot)) of 55 cm and an effective length (L(eff)) of 45 cm; 200 mmol·L(-1) D-(+)-gluconic acid δ-lactone, 80 mmol·L(-1) boric acid, and 57.4 mmol·L(-1) triethylamine in methanol; positive pressure injection at 2.9 psi for 2 s; capillary temperature, 25 ± 0.2 ℃; applied voltage, +15 k V; detection wavelength, 214 nm. Under the optimized conditions, a good chiral resolution was achieved in most of the tested drugs. This method provides a foundation for the development and application of new chiral selectors of polyhydroxy compound-boric acid complexes in chiral drugs analysis by NACE.

Optimized hydrolysis and analysis of Radix Asparagi polysaccharide monosaccharide composition by capillary zone electrophoresis.

Using orthogonal design, optimized conditions for the hydrolysis of the polysaccharide from Radix Asparagi were determined, as well as its monosaccharide composition. Optimized hydrolysis conditions were a temperature of 100°C in 1.5 M sulfuric acid solution for 5 h. The resulting monosaccharides were derivatized with 1-phenyl-3-methyl-5-pyrazolone, then separated by capillary zone electrophoresis in 40 mM sodium tetraborate buffer (pH 10.1), and detected by ultraviolet absorption at 245 nm. Results indicate that the polysaccharide from Radix Asparagi is composed of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid, and galacturonic acid, which differs from published findings. Moreover, xylose, glucuronic acid, and galacturonic acid have not been previously reported in Radix Asparagi polysaccharide. This method is simple, fast, and yields a highly efficient separation. As well, these findings can be applied to quality control of Radix Asparagi and for in-depth study of the biological activity of Radix Asparagi polysaccharide.

Extraction optimization of the polysaccharide from Adenophorae Radix by central composite design.

The central composite design (CCD) was applied to optimize the water extraction of the polysaccharide from Adenophorae Radix in the paper. The three variables of extraction temperature (X1), extraction time (X2) and ratio of water to raw material (X3) were investigated by single factor analysis first. Since the presence of active polysaccharides and the imperfect of its extraction, the purpose of the paper was to evaluate the effects of selected variables on the yield of polysaccharide, which was expected to obtain the maximum yield. By variance and regression analysis, the quadratic regression equation was established as a predicted model. The R(2) of 0.9825 indicated that the equation was well-fitted. The optimal conditions were 72.5°C, 133min, 1:35 (g/mL) and the predicted maximum yield of the polysaccharide was 5.78%. The predicted value was verified in triplicates under the optimum conditions, which was 5.68% and well matched with the predictive yield.

[Dissolution determination of Shuanghuanglian capsules by HPLC analysis assisted with principal component analysis].

OBJECTIVE: To develop a quality analysis method based on self-reference principal for dissolution determination of Shuanghuanglian capsules. METHOD: Dissolution of Shuanghuanglian capsules was determined by principal component analysis consociated HPLC method. RESULT: The liner of regression equation was good. The average recovery rates of quality assurance samples (QA) and quality control samples (QC) were all no less than 96. 0%. Dissolution curves of Shuanghuanlian capsules of different manufacturers and different batches of the same manufacturer had obvious disparity. CONCLUSION: The method can better evaluate the dissolution conditions of Shuanghuanglian capsules. The prospect of the method is expected for assessing the dissolution of other oral solid dosage of traditional Chinese medicines.

[Identification of traditional Chinese medicines by oligosaccharide electrophoresis analysis assisted with cluster analysis].

The structure of polysaccharide in traditional Chinese medicine (TCM) is complex and characteristic. A method of oligosaccharide electrophoresis analysis assisted with cluster analysis (CA) was established to simultaneously identify TCMs. Six TCMs from three families were selected as experimental subjects and their polysaccharides were extracted. The obtained oligosaccharides via incompletely degraded TCM polysaccharides were derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP). The PMP-oligosaccharide derivatives were separated and analyzed by capillary zone electrophoresis (CZE). The six TCMs were identified by the featured information of oligosaccharide electropherograms with CA. The electrophoresis conditions were as follows: uncoated fused silica capillary column (49 cm/40 cm (total length/effective length) x 50 microm); running buffer solution, 50 mmol/L phosphate buffer solution (pH 2.5); detection wavelength, 245 nm; operating voltage, 15 kV; hydrodynamic pressure injection, 10 cm x 4 s. The results showed that the six TCMs from three families were effectively identified by the method of TCM oligosaccharide electropherograms combined with CA. This method is a promising tool to identify TCM with good reliability and reproducibility.

[Separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of traditional Chinese medicine oligosaccharides by capillary zone electrophoresis].

A simple and practical capillary zone electrophoretic (CZE) method has been developed for the separation of traditional Chinese medicine oligosaccharides according to their relative molecular masses (M(r)). To optimize the conditions of the method, the concentration and pH value of the running buffer, and the applied voltage were evaluated. The optimized conditions were as follows: 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of the oligosaccharides were separated with 50 mmol/L phosphate buffer (pH 2.5) as running buffer and the voltage was 15 kV. The detection was performed with an ultraviolet detector at 245 nm. An uncoated fused-silica capillary of 50 microm i. d. and 38/48 cm length (effective length/total length) was employed, and a hydrodynamic pressure injection (10 cm x 2 s) was applied. In order to examine the practicability of the method for the analysis of actual traditional Chinese medicine oligosaccharide samples, a complex sample consisting of some usual monosaccharides and oligosaccharides degraded from beta-cyclodextrin was separated under the electrophoretic conditions. And then, the method was applied to the analysis of the controlled degradation products of Indigowoad Root polysaccharide. The results indicated that the oligosaccharide sample could fully be separated from low to high M(r). This method is efficient and practical. In addition, the separation hypothesis of PMP derivatives of monosaccharides and oligosaccharides in pH 2.5 running buffer is also discussed, which would be helpful for us to understand the unusual migration of the PMP derivatives of rhamnose and mannose.

[Influence of different compatibility on contents of calcium and magnesium dissolution in maxing shigan decoction].

OBJECTIVE: To establish a method for the determination of gypsum and magnesium in decoction by capillary zone electrophoresis and study the influence of different compatibility on the contents of calcium and magnesium dissolution. METHOD: Nine decoctions with gypsum were prepared and analysed by L9 (3(4)) orthogonal design and the contents of calcium and magnesium ions were determined by the set method. The conditions of the experiment were a running buffer of 6.4 mmol x L(-1) imidazole solution (glacial acetic acid ajusted pH to 3.50) and an applied voltage of 10 kV (room temperature). Samples were introduced by hydrodynamic injection (8 cm x 7 s) and determined with on-column UV monitoring at 214 nm. Copper sulphate was chosen as the internal standard. RESULTS: The linear responses covered the range from 0.009 68 mg x L(-1) to 9.68 mg x L(-1) (r = 0.998 1) for calcium, the detection limits (S/N = 3) and the quantitation limits (S/N = 10) of calcium were shown to be 0.002 1 mg x L(-1) and 0.007 1 mg x L(-1), respectively. The average recovery for calcium was 100.4%. The linear response covered the range from 0.01 mg x L(-1) to 10 mg x L(-1) (r = 0.995 9) for magnesium. The detection limits (S/N = 3) and the quantitation limits (S/N = 10) of magnesium showed to be 0.002 8 mg x L(-1) and 0.008 9 mg x L(-1), respectively. The average recovery for magnesium was 96.4%. CONCLUSION: The method is simple, rapid, cost-effective and precise with satisfactory results. The influence of Ephedra and Semen Armeniacae Amarum on the contents of gypsum dissolution are significant, while that of Radix Glycyrrhizae on the contents of gypsum dissolution is insignificant. The influence of Ephedra on the contents of magnesium dissolution are significant, while that of Semen Armeniacae Amarum and Radix Glycyrrhizae on the contents of magnesium dissolution are insignificant.

[Determination of ephedrine and pseudoephedrine in Herba Ephedrae and Maxing Shigan Tang by capillary zone electrophoresis].

OBJECTIVE: To establish a method for the determination of ephedrine and pseudoephedrine in Herba Ephedrae and Maxing Shigan Tang by capillary zone electrophoresis. METHOD: The conditions of the experiment were optimized with a fused-silica capillary of 60 cm x 50 microm (50 cm effective length) in a running buffer of 50 mmol x L(-1) borax-20 mmol x L(-1) threonine (pH 9.27) and an applied voltage of 15 kV (room temperature). Samples were introduced by hydrodynamic injections (10 cm x 20 s)and determined with on-column UV monitoring at 210 nm. Phenobarbital was chosen as the internal standard. RESULT: Ephedrine and pseudoephedrine are separated successfully within 8 min. The linear responses covered the ranges from 21.3 to 213 mg x L(-1) (r = 0.9996) for ephedrine and from 8.4 to 84 mg x L(-1) (r = 0.9995) for pseudoephedrine. The detection limits (S/N = 3) of ephedrine and pseudoephedrine were shown to be 1.45 and 1.48 microg x mL(-1), respectively, The quantitation limits (S/N = 10) of ephedrine and pseudoephedrine were shown to be 4.81 and 4.93 mg x L(-1), respectively. The average recoveries for ephedrine and pseudoephedrine were 97.5% and 98.6% with RSD less than 5.0%. CONCLUSION: The method is simple, rapid, cost-effective and precise with satisfactory results.

[Determination of cytidine and adenosine in cordyceps by monolithiccapillary electrochromatography].

OBJECTIVE: To develop a capillary electrochromatography method for determination of cytidine and adenosine in cordyceps with monolithic column. METHOD: The total length of the home-made ploy-butyl methacrylate (PBMA) monolithic capillary electrochromatographic column was 34.5 cm with the effective length of 26.0 cm. The mobile phase was 20 mmol x L(-1) borax solution (adjusted pH to 3.5 using acetic acid); the operation voltage was 15 kV; sample injection pressure was 6 bar x 0.1 min; column temperature was 30 degrees C and the detection wavelength was set at 214 nm. The internal standard solution was 100 mg x L(-1) trimethoprim solution [ethanol-mobile phase (1 : 1) was used as the solvent]. RESULT: The results indicated that the concentrations of cytidine and adenosine within the range of 12.5-125 mg x L(-1) were linearly correlated with the relative peak areas, and the correlative coefficients (r) were 0.999 8 and 0.999 3, respectively. The LOD (S/N = 3) and LOQ (S/N = 10) of cytidine were 2.14 and 7.14 mg x L(-1), and those of adenosine were 1.88 and 6.25 mg x L(-1). The average recoveries of the two nucleosides were from 97.2% to 103.5% with relative standard deviation (RSD) within 0.9%-2.6% in three levels. CONCLUSION: The method is effective and credible. It can be used to determine the contents of cytidine and adenosine in cordyceps.

[Determination of the binding constant of bovine serum albumin and gatifloxacin using affinity capillary electrophoresis].

The interaction between bovine serum albumin (BSA) and gatifloxacin (GT) was investigated using affinity capillary electrophoresis (ACE) method, mobility-shift method. The mobility ratio of the internal standard and the sample was used to calculate the binding constant K(b) of bovine serum albumin and gatifloxacin. At first, 20 mmol/L, pH 7.4 sodium phosphate buffer solution (PBS) added with 0 - 1 000 micromol/L GT as running buffer was used, BSA as the sample was used. K(b) calculated from this experiment was 4.4 x 10(4) L/mol. Then 0 - 12.5 micromol/L BSA was used as an additive to do the ACE experiment, the K(b) obtained was 4.2 x 10(4) L/mol. The two values matched well with each other. In order to improve the shape of the peak and restrain the adsorption of BSA to the internal wall of the quartz column, in the second method, 0.25 mol/L Gly and 0.5 mmol/L ethylenediamine tetracetate (EDTA) were added to the running buffer, and 0.5% sodium dodecyl sulfate (SDS) solution was used to rinse the column. Satisfactory effect was obtained. The K(b) from fluorescence method was also calculated with a value of 2.7 x 10(4) L/mol. This work demonstrated that the determination of the binding constant by ACE is simple with high performance.

[Discussion on the expression of retention factor in capillary electrochromatography].

On the assumption that the component electrophoresis factor does not have parametric interaction with its chromatographic behavior in capillary electrochromatography, two new retention factor expressions k*CEC = k' - microep/microeo + microep (I) and k**CEC = k'microep - micro(0)ep/microeo + microep (II) are deduced, and they are complementary to each other. Two expressions of component retention factor in the literature, kCEC = k' + k' (microep + microep/microeo + microeo (III) and kCEC = k' microep/microeo/1 + microep/microeo (IV), are discussed. Wrong citation of the component electrophoresis migration distance expression in the deduction of expression (III) is pointed out. But the expressions (I) and (IV) also can't reflect the influences of microep and k' on kCEC under some conditions. The expression (II) can make up the limitation of the above expressions, especially when microeo = 0. Expressions (I) and (II) can reflect the integrated effect of component chromatographic and electrophoretic behaviors.