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An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.

Current China Pharmacopoeia (ChP) standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines (CPMs) that contain Panax notoginseng as the monarch drug. These conventional, HPLC-based approaches, utilizing a complex sample preparation procedure, can easily result in low analytical efficiency and possible component loss. Here, a "monomethod-heterotrait matrix" (MHM) strategy is proposed, that is, developing a universal multi heart-cutting two-dimensional liquid chromatography (MHC-2D-LC) approach that facilitates the simultaneous quantitation of five P. notoginseng saponins (noto-R1, Re, Rg1, Rb1, and Rd) in eight different CPMs. The MHC-2D-LC system was constructed on a dual-gradient liquid chromatography instrument equipped with a Poroshell SB C18 column and a Zorbax SB-Aq column for respective (1)D and (2)D separation. Method validation was performed in terms of specificity, linearity (r(2) and F-test), intra-/inter-day precision (0.4-7.9%), stability (1.2-3.9%), and recovery (90.2-108.7%), and the LODs and LOQs (loaded masses) of the five analytes varied between 4.0-11.0ng and 6.0-33.0ng, respectively. The validated MHC-2D-LC approach was subsequently applied to quantify the five saponins in thirty batches of different CPMs. The method demonstrated superiority over the current ChP assay methods in respect of specificity (avoiding co-elution), resolution (Rs>1.5), sample preparation (easy-to-implement ultrasonic extraction without repeated re-extraction), and transfer rate (minimum component loss). This is the first application of an MHC-2D-LC method for the quantitative assessment of the constituents of CPMs. The MHM approach represents a new, strategically significant methodology for the quality control of CPMs that involve complex chemical matrix.

Single standard to determine multi-components (SSDMC) is a novel and rational method for quality control of botanical products and traditional Chinese medicines (TCMs). However, it is restricted to wide application due to unknown fluctuation in conversion factors when it is performed in different laboratories. To evaluate the fluctuations of conversion factors, we selected Salvia miltiorrhiza as an example to determine three components of tanshinones by SSDMC method. Then ruggedness and robustness test were adopted to comprehensively investigate three kinds of factors that may influence stability of conversion factors, which were related with environmental parametric variables, operational parametric variables and peak measurement parametric variables. Nested-factorial-design was used to perform ruggedness tests. One-variable-at-a-time (OVAT) procedure and Plackett-Burman (PB) design were both used in robustness test. The results showed that stability of conversion factors was principally related with accuracy of wavelength of UV detector, peak measurement parameters and concentration of standard solution. The acceptable range of conversion factors was obtained from robustness test. Our results showed that conversion factors were inevitable to change, but when key parameters were well controlled, the range of its fluctuation was acceptable and the SSDMC method could be used widely in different laboratories.

Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control. The present review mainly focuses on the phytochemical analysis of TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonly used ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) and time-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible and hence be applied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the identification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is not a universal detector, high-performance liquid chromatography coupled with multistage mass spectrometry (HPLC-MS(n)) technique was still proved to be a rapid and sensitive method to analyze the majority of the many constituents in herbal medicines, particularly for the detection of those present in minor or trace amounts. The methods established using HPLC-MS techniques facilitate the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. However, the quantitative analysis is not the topic of this review.