Bufadienolides possess various bioactivities especially antitumor. Due to the high structural diversity, the separation of bufadienolides often suffers from coelution problem on conventional RP columns. In this work, an off-line two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D-NPLC × RPLC) method was developed to separate and characterize bufadienolides in toad skin. Several RP and NP columns were evaluated with five reference bufadienlides. The XUnion C18 and XAmide columns exhibited superior chromatographic performances for bufadienlide separation, and were selected in RPLC and NPLC, respectively. RPLC was used in the second-dimension for the good compatibility with MS, while NPLC was adopted in the first-dimension. The orthogonality of the 2D-NPLC × RPLC system was investigated by the geometric approach using fifteen bufadienolide mixtures. The result was 49.6%, demonstrating reasonable orthogonality of this 2D-LC system. By combining the 2D-LC system with MS, 64 bufadienlides including 33 minor ones and 11 pairs of isomers in toad skin were identified. This off-line 2D-NPLC × RPLC allowed to solve the coelution problem of bufadienlides in one-dimension RPLC, and thus facilitated the identification significantly.

The preparative separation of anthocyanins by HPLC often suffers from insufficient separation selectivity. In this work, a two-dimensional liquid (LC-LC) method was established to efficiently purify a challenging anthocyanin in Lycium ruthenicum Murray. Reversed phase liquid chromatography (RPLC) was used in the first-dimension preparation to fractionate the sample for its high separation efficiency. After the optimization of second-dimension methods, hydrophilic interaction chromatography (HILIC) was applied to further isolate the anthocyanin for the good orthogonality to RPLC. To improve HILIC separation for anthocyanins, stationary phases and mobile phases were investigated systematically. A satisfactory result was obtained on a zwitterionic Click XIon column with 1% phosphoric acid as an acidic additive. Using the above method, the anthocyanin and three new alkaloids were isolated from L. ruthenicum for the first time. This RPLC/HILIC method solved the coelution problem of anthocyanin and basic non-anthocyanins in one-dimensional HPLC, benefiting from the significantly improved separation resolution.

The cis–trans isomerism is a common phenomenon for acylated anthocyanins. Nevertheless, few studies reported effective methods for the preparation of isomeric anthocyanins from natural products. In this work, a high-performance liquid chromatography (HPLC) method was developed to efficiently purify anthocyanin isomers from Lycium ruthenicum Murr. based on a mixed-mode reversed-phase/strong anion-exchange column (named XCharge C8SAX). Four commercially available columns were evaluated with a pair of isomeric anthocyanins, and the results demonstrated that the XCharge C8SAX column exhibited improved selectivity and column efficiency for the isomers. The chromatographic parameters, including pH, organic content, and ionic strength, were investigated. Optimal separation quality for the anthocyanin isomers was achieved on the XCharge C8SAX column. Six pure anthocyanins, including two pairs of cis–trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum and identified. All of the results indicated that this method is an effective way to separate anthocyanins, especially for cis–trans isomers.

L. ruthenicum, which grows mainly in the salinized desert of the Qinghai-Tibet plateau, contains abundant anthocyanins. To date, information on its anthocyanin composition is still limited. In this work, the systematic characterization of anthocyanins in L. ruthenicum was carried out by HPLC-DAD/QTOF-MS/MS. Six mono-acylated anthocyanins isolated from L. ruthenicum were analyzed by ESI-QTOF-MS/MS, and their fragmentation behaviors were investigated and summarized. Different anthocyanins could be identified through the characteristic product ions and their relative intensities. In addition, various rules were applied to cope with the difficulty of rapidly differentiating cis–trans anthocyanin isomers. A spectral rule was first proposed from the UV/Vis spectra of two pairs of cis–trans anthocyanin isomers. This rule provided important information for the identification of the isomers. Based on these rules, sixteen anthocyanins in L. ruthenicum were tentatively characterized, and seven of them were reported in this plant for the first time. In order to validate the method, an anthocyanin was isolated from L. ruthenicum and identified by NMR and MS.

•It is a new way to quickly separate and purify bufadienolides from toad skin.•A distinct separation selectivity was obtained by the positively charged C18 column.•The peak tailing problem was resolved and two kinds of bufadienolides were purified.As a kind of promising anticancer compounds, the preparation of bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of bufadienolides from toad skin.

•Two classes of bufadienolides were separated by HILIC-SPE.•This prevented their co-elution in RP-HPLC.•A total of 12 compounds from the two classes were purified by this method.Traditional Chinese medicines (TCMs) have played a significant role in the process of discovering natural bioactive compounds, especially in anticancer therapeutics. However, the components of TCMs are complex mixtures with wide variation in polarity and content, which leads to inefficiency in the process of active compound discovery from TCMs. In this paper, the popular strategy of utilizing “pre-fractionated natural product libraries” has been improved by a new class separation approach to accelerate the process. As an example, the skin of Bufo bufo gargarizans Cantor, a well-known TCM, mainly contains two distinct bufadienolide classes: amino acid-conjugated bufadienolides (AACBs) and free form bufadienolides (AAUBs). We utilized hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE) to resolve the two types of bufadienolides, which co-eluted on C18 columns. By this strategy, twelve bufadienolides of the two types were purified via prep-HPLC from one active fraction, and eight of them were identified by 1H NMR and 13C NMR. These results indicated that the class separation method not only overcame the limited orthogonality in a 2D-RPLC × RPLC system but also accelerated the process of active compound discovery.

Cinobufacini (Huachansu) injections have been widely used for the treatment of various cancers in clinical applications in China. As an aqueous extract of the skin of Bufo bufo gargarizans Cantor (toad skin), cinobufacini contains many polar compounds; however the chemical profile of these polar compounds is still unclear, which increases the risk of pharmacological safety. There are very few studies on the separation and purification of polar components from toad skin, especially in the preparative scale. In this study, a unique method based on a homemade polar-modified C18 stationary phase was developed and successfully applied to separate polar compounds from toad skin. Compared with previous purification systems based on the C18 stationary phase, the poor retention problem of polar compounds was well resolved through the use of the polar-modified C18 column and good resolutions were also achieved. Seven compounds were purified and five of them (uracil, hypoxanthine, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one, thymine and bufothionine) were identified by MS, 1H NMR and 13C NMR. In addition, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one was identified as a new compound and hypoxanthine was found from the skin of Bufo bufo gargarizans Cantor for the first time. These results indicate that this method shows promise for the separation of polar compounds in toad skin.

•It is a new protocol to efficiently purify high-purity glycyrrhizin from licorice.•A distinct separation selectivity of saponins and flavonoid compounds was obtained by the Click XIon material.•The retention mechanism for glycyrrhizin in HILIC mode has been discussed.•A HILIC-SPE method was developed for the enrichment of glycyrrhizin.Glycyrrhizin (GA), a major bioactive compound in licorice, has been extensively used throughout the world as a medicine to treat chronic viral hepatitis and allergic dermatitis. In this study, a new method based on hydrophilic interaction solid phase extraction (HILIC-SPE) and preparative reversed-phase liquid chromatography (prep-RPLC) was developed to purify GA with high purity from the complex licorice extract. Via evaluation of retention behavior of GA and flavonoids in different commercially available columns, a hydrophilic column——Click XIon was finally chosen for the purification due to its excellent resolution toward GA and flavonoids under HILIC mode. To optimize the SPE elution conditions, relative factors including water content, pH and ionic strength had been investigated in chromatographic condition. The result indicated that the most appropriate water content was 30% and pH at 4.00, as well as salt concentration should be controlled at 5 mM. In addition, the optimization revealed that GA experiences both hydrophilic interaction and ion-exchange interaction on the Click XIon material. According to the chromatographic evaluation, the optimized conditions were applied to HILIC-SPE to enrich GA from licorice, which leads to an increased content of GA from 13.67% to 64.22%. Finally, prep-RPLC was performed to obtain GA with purity higher than 99.00%，which demonstrating great prospect in large-scale preparation of GA.

L. ruthenicum, which grows mainly in the salinized desert of the Qinghai-Tibet plateau, contains abundant anthocyanins. To date, information on its anthocyanin composition is still limited. In this work, the systematic characterization of anthocyanins in L. ruthenicum was carried out by HPLC-DAD/QTOF-MS/MS. Six mono-acylated anthocyanins isolated from L. ruthenicum were analyzed by ESI-QTOF-MS/MS, and their fragmentation behaviors were investigated and summarized. Different anthocyanins could be identified through the characteristic product ions and their relative intensities. In addition, various rules were applied to cope with the difficulty of rapidly differentiating cis–trans anthocyanin isomers. A spectral rule was first proposed from the UV/Vis spectra of two pairs of cis–trans anthocyanin isomers. This rule provided important information for the identification of the isomers. Based on these rules, sixteen anthocyanins in L. ruthenicum were tentatively characterized, and seven of them were reported in this plant for the first time. In order to validate the method, an anthocyanin was isolated from L. ruthenicum and identified by NMR and MS.

Cinobufacini (Huachansu) injections have been widely used for the treatment of various cancers in clinical applications in China. As an aqueous extract of the skin of Bufo bufo gargarizans Cantor (toad skin), cinobufacini contains many polar compounds; however the chemical profile of these polar compounds is still unclear, which increases the risk of pharmacological safety. There are very few studies on the separation and purification of polar components from toad skin, especially in the preparative scale. In this study, a unique method based on a homemade polar-modified C18 stationary phase was developed and successfully applied to separate polar compounds from toad skin. Compared with previous purification systems based on the C18 stationary phase, the poor retention problem of polar compounds was well resolved through the use of the polar-modified C18 column and good resolutions were also achieved. Seven compounds were purified and five of them (uracil, hypoxanthine, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one, thymine and bufothionine) were identified by MS, 1H NMR and 13C NMR. In addition, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one was identified as a new compound and hypoxanthine was found from the skin of Bufo bufo gargarizans Cantor for the first time. These results indicate that this method shows promise for the separation of polar compounds in toad skin.