High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase.

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15 mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R2  > 0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87-110.99%), limits of detection (5.35-5.76 µg/mL), and limits of quantification (20 µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents of three batches of Ephedrae Herba from three pharmaceutical companies.

Computer-Aided Rapid Establishment of Fingerprint of Xiaojin Capsule by HPLC.

Traditional Chinese medicine (TCM) formulas have a significant clinical efficacy, and the fingerprint technology has been widely accepted to fully reveal the quality of TCM. Whereas, it is a great challenge to establish the fingerprint chromatogram which can fully reflect every single herb material in a short time. In this study, we used Xiaojin capsule (XJC) as a case and developed a rapid fingerprint method based on increasing the column temperature and flow rate simultaneously combined with computer-aided. First, the elution gradient was optimized based on the retention parameters and peak shape parameters of the four linear gradients, and then, the column temperature and flow rate were increased simultaneously to shorten the analysis time. Next, the standard fingerprint chromatogram of XJC, which can reflect every herb material, was generated. Finally, quality markers were screened through unsupervised cluster analysis and supervised orthogonal partial least squares discrimination analysis. Combining computer-aided with increasing column temperature and flow rate simultaneously can develop the rapid method for establishing HPLC fingerprint of XJC, which can fully reflect every single herb material and provide comprehensive quality control. The strategy for establishing HPLC fingerprint of TCM formula could be applied to other traditional Chinese medicine formulas and herbal medicine.

Combination of multi-model statistical analysis and quantitative fingerprinting in quality evaluation of Shuang-huang-lian oral liquid.

A model consisting of quantitative fingerprinting integrated with fundamental statistical analysis was established to carry out quality control analysis of Shuang-huang-lian (SHL) oral liquid. The quantitative fingerprinting approach was developed by systematic investigation of the chromatographic condition and optimization of a gradient using a complex sample analysis software system (CSASS). Five pivotal components from three traditional Chinese medicines of SHL oral liquid were determined at dual wavelengths, including phillyrin, baicalin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid. Among them, neochlorogenic acid and cryptochlorogenic acid were quantified by quantitative analysis of multi-components with a single marker (QAMS) method. Further, the developed quantitative fingerprinting approach was validated. Good linearity with correlation coefficients (R2) higher than 0.9999 were achieved for phillyrin, baicalin and chlorogenic acid. Recoveries of the three analytes were between 96% and 108%. Relative standard deviation (RSD) values were below 3% regarding the stability and intra-day and inter-day precision. Besides, the feasibility of the QAMS method was verified by an external standard method (ESM) using 18 batches of SHL oral liquid. Fifty-nine batches of SHL oral liquid from nine manufacturers were then analyzed. Effective distinction was realized based on a linear principal component analysis (linear-PCA) model by the combination of the quantitative data and chromatographic fingerprinting. The linear-PCA model based on quantitative chromatographic fingerprinting exhibited great advantage over conventional similarity analysis to distinguish between different samples. The strategy provided a particularly simple and effective approach for quality evaluation of SHL oral liquid from various manufacturers. Graphical abstract.

[Determination of five protopanaxadiol ginsenosides in ginseng by solid-phase extraction-ultra performance liquid chromatography].

A new method based on solid-phase extraction-ultraperformance liquid chromatography (SPE-UPLC) was developed for the determination of five protopanaxadiol ginsenosides in ginseng. The ginsenosides were extracted from ground ginseng samples using water-saturated n-butanol and purified on a hydrophilic solid-phase extraction column. Chromatographic separation was achieved on an ACQUITY UPLC BEH Shield RP18 column (100 mm×2.1 mm, 1.7 µm) by linear gradient elution using an acetonitrile/water mobile phase. Five protopanaxadiol ginsenosides were detected by a photodiode array detector, and they showed a strong positive linear correlation (r2>0.999) in the range of 5-500 µg/mL. In addition, the instrument precision ranged between 0.95% and 2.62% (n=6), with the sample stability between 0.90% and 2.15% (n=8) within 22 h. Intra- and inter-day repeatabilities were 5.35%-6.47% (n=6) and 5.56%-6.34% (n=8), respectively. Sample recoveries and the corresponding relative standard deviations (RSDs) were 87.16%-101.92% and 1.54%-4.01% (n=6), respectively. Hydrophilic chromatography materials were used in SPE, and the extract was directly loaded and purified without pretreatment. Besides, with the use of UPLC, the analysis time was greatly shortened. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the five protopanaxadiol ginsenosides in ginsengs.

[An analytical method for alkaloids of Coptis chinensis based on mixed-mode surface electrostatic exclusion/reversed-phase chromatography].

A mixed-mode chromatographic method based on surface electrostatic exclusion and reversed-phase chromatography was established for the determination of alkaloids present in Coptis chinensis. The effects of two mobile phase additives, formic acid and acetic acid, on retention, peak shape and selectivity of the alkaloids in Coptis chinensis were investigated using the self-made C18HCE column. Acetic acid (0.1%, v/v) used as the additive was found to be optimum for effective separation of the main alkaloids present in Coptis chinensis. The main chromatographic peaks of Coptis chinensis were recognized by the established method and references, which were coptisine, epiberberine, columbamine, jatrorrhizine, berberine and palmatine, respectively. With reference to the content determination method of Coptis chinensis in the 2015 edition of pharmacopoeia, the linear relationship of berberine in the range of 0.5-100 mg/L was good, the correlation coefficient was 0.9996, and the average recovery was 93.74%. The contents of alkaloids in Coptis chinensis in different batches of Hubei and Chongqing were determined. The method is simple, reliable and accurate, and can be used as reference for separation and analysis of other basic compounds.

On-line comprehensive two-dimensional liquid chromatography tandem mass spectrometry for the analysis of Curcuma kwangsiensis.

A novel online comprehensive two-dimensional liquid chromatography (2DLC) coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) method is developed for the analysis of Curcuma kwangsiensis (C. kwangsiensis) extract. In this system, a newly developed phenyl/tetrazole sulfoether (PTAS) bonded stationary phase was introduced to construct RPLC×RPLC combined with C18. The unique structure endowed PTAS with very different selectivity from C18, reaching a high orthogonality of 93.2%. Moreover, such a combination settled compatibility issues because of the weaker hydrophobic retaining property of PTAS, thus allowing direct interfacing in online configuration. As a result of coupling with the mass spectrometry, a four-dimensional (4D) data plot was presented, in which 439 peaks (containing positive mode and negative mode) were counted, and 105 compounds were grouped and tentatively identified in C. kwangsiensis extract, including 73 unreported ones. Some novel types of compounds with masses exceeding 500 were discovered for the first time. Besides, compared to one-dimensional liquid chromatography (1DLC), the great resolution power of this system allowed separation of more isomers. These results provide supplementary to the material basis of C. Kwangsiensis and in-depth research should be conducted. The configuration of RPLC×RPLC-Q-TOF MS can be a powerful and efficient tool for separation and characterization of chemical substances in complicated herbal extracts.

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol-epoxy ring opening reaction.

A silica-based reversed-phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol-epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π-π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid-state 13 C cross-polarization magic-angle-spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed-phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed-phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.

2-D RP/RPLC method to separate components in Fructus schisandrae chinensis.

An off-line 2-D RP/RPLC method was developed to separate components in extracts of Fructus schisandrae chinensis, which is a traditional Chinese medicine. In this method, Click beta-CD stationary phase was proposed for constructing 2-D RP/RPLC system combined with C18 column. Beta-CD column showed great potential to separate compounds in traditional Chinese medicines under RP mode. Collected fractions from Click beta-CD column used in first dimension were resolved into many additional components, indicating that the 2-D LC system based on Click beta-CD and C18 phases is orthogonal. The mobile phase was same on both columns. This 2-D LC method facilitates us not only to separate components that cannot be resolved by unidimensional chromatography, but also to achieve much more efficient detection of minor sample components.

[Optimization of high performance liquid chromatographic method for analysis of extract of Fructus schisandrae chinensis].

For the separation of the complex system of alcohol fraction of Fructus schisandrae chinensis, complex sample analysis system software (CSASS) was utilized to optimize the high performance liquid chromatographic (HPLC) conditions. The HPLC retention parameters a and c, and the peak shape parameters sigma and W1/2 were obtained accurately and rapidly by analyzing 4 linear gradient elutions. Based on the 4 parameters, the chromatogram in any gradient condition could be simulated and predicted precisely. The HPLC optimization method was developed using moved overlapping separation ranging map and simulated chromatogram technology. The HPLC analysis of the alcohol fraction of Fructus schisandrae chinensis could be finished in 40 min, and good resolution for components with general or low abundance was achieved. This method could be used to predict retention times and peak shapes of components in Fructus schisandrae chinensis. And based on the precise predicted results, the optimized separation conditions can be obtained.

Computer-aided target optimization for traditional Chinese medicine by ultra-performance liquid chromatography.

A new strategy of target optimization was developed for resolving the separation problems of complex samples. Computer-aided target optimization for separation of complex samples by ultra-performance liquid chromatography (UPLC) was studied. Taking traditional Chinese medicine (TCM) (Rhizoma Coptidis) as an example, separation conditions of target compounds - overlapping peaks and all components - were optimized based on moved overlapping separation ranging map (OSRM) and artificially intervention. After calculating retention parameters and peak shape parameters of the target peaks, the optimization was operated in an emulational picture through the computer without further experiments. The emulational chromatogram was proved to be authentic by the estimated values, which were almost identical to experimental results. The method was very helpful for obtaining the satisfied separation conditions of target compounds rapidly and efficiently.

Qualitative and quantitative analysis in quality control of traditional Chinese medicines.

Separation techniques with high efficiency and sensitive detection have been widely used for quality control of traditional Chinese medicines (TCMs). High-performance liquid chromatography, gas chromatography, and capillary electrophoresis are commonly used to separate various components in TCMs. Ultraviolet detection, fluorescence detection, evaporative light-scattering detection, mass spectrometry and nuclear magnetic resonance can be applied to separation techniques for qualitative and quantitative analysis of TCMs. The development of quality control for TCMs based on quantitative and qualitative analysis from 2000 to 2007 are reviewed; the fingerprint technique is also discussed due to its broad application in the quality control of TCMs. Prospects for further research based on our primary results are also discussed.

Study on the retention equation in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) is an effective technique for separating polar compounds. But its retention equation has not been studied systematically yet. In this study, an appropriate retention equation was established by using eight nucleosides as model analytes and by comparing four retention models on six different HILIC columns. As a result, retention equation Ink = a + b x InC(B) + cxC(B) could be quantitatively described the retention factors with good accuracy in HILIC mode. Based on this equation, the retention times of eight nucleosides under the conditions by other mobile phase can be predicted on each column. All of the predicted relative errors of retention times were smaller than 5%. The established retention model was also successfully applied to predict retention times of the real Traditional Chinese Medicine-Carthaus tinctorius L. sample.