Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography.

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30 MPa for 30 min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20 mm internal diameter, 10 µm) within 8 min by gradient elution increasing from 5% to 20% modifier (methanol) at 55 ml/min and 15 MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19 mm internal diameter, 5 µm) at 50 ml/min and 13.5 MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa.

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.

Bioactivity-guided separation of antifungal compounds by preparative high-performance liquid chromatography.

Bioactivity-guided chromatographic methods are of great significance for the isolation of the active compounds in complex samples. In this study, four anti-fungal compounds were located by activity screening and successfully isolated from a microbial fermentation sample by preparative high-performance liquid chromatography. Separation performance of columns including C18, positively charged C18, negatively charged C18 and C8 were firstly investigated. And it showed a better capacity of mixed-mode stationary phases for retention and separation. Therefore, the positively charged C18 column was used to separate the sample into several fractions, among which the active one was identified by the antifungal test. And then the active fraction was enriched and separated again by successively using the negatively charged C18 and C8 columns to obtain four compounds, which were identified as polyoxins A, K, F and H. With activity verification, four polyoxins were found to have good inhibitory effects against the three fungal plant diseases including rice sheath blight, tomato grey mould disease, and apple spot leaf disease.

Separation and characterization of phenylamides from Piper kadsura using preparative supercritical fluid chromatography and ultra-high-performance supercritical fluid chromatography-tandem mass spectrometry.

A preparative supercritical fluid chromatography method for the separation of Piper kadsura obtained five phenylamide compounds, which had the same structural skeleton, but changed in the number and position of methoxyl substituents. To improve the separation selectivity of these structural analogues, silica, phenyl, and chiral stationary phases were screened. Only through the combination of Chiral C and phenyl columns could the separation of the five phenylamides be solved. The two-step strategy using preparative supercritical fluid chromatography presented good orthogonality that ensured the purity of the phenylamides. Then, an ultra-high-performance supercritical fluid chromatography hyphened tandem mass spectrometry method was developed, and the fragmentation pattern of phenylamides was summarized. It mainly cleaved in the amide bond to produce the fragment ion, which could help to judge the substituent positions. Twenty-eight possible molecular weights of hydroxyl and methoxyl substituted phenylamides were calculated and screened. Nine compounds were extracted in three [M + H]+ ions at m/z 284.13, 314.13, and 344.13, including five purified compounds and the other four positional or trans-cis phenylamide isomers in low content. The methods developed in this research were useful in the separation and characterization of phenylamide analogues.

Isolation of three polyoxins by reversed-phase liquid chromatography with pure aqueous mobile phase.

Developing methods for the isolation of highly polar compounds from complex samples is of great significance. In this study, three polyoxins were successfully isolated from a complex sample (PN1-1# ) by preparative high-performance liquid chromatography. Separation was carried out on five polar reversed-phase stationary phases, using pure aqueous as mobile phase, where the C18HC column can provide the best performance for PN1-1# . Next, the effects of the mobile phase composition were studied. It was found that adding NaClO4 can enhance the retention and resolution, and adding NaH2 PO4 was beneficial to maintain good peak shapes when the sample loading increased. Therefore, the optimized mobile phase consisting of 20 mmol NaH2 PO4 and 20 mmol NaClO4 (adding H3 PO4 to adjust pH 2) was used to separate PN1-1# . This method of using 100% aqueous phase can effectively improve both the retention and the solubility of polar samples. Eventually, through further purification, three compounds, namely, polyoxins B, D, and G, were obtained. This paper provided an effective and eco-friendly strategy for the preparative-scale separation of polar samples.

Comprehensive HPLC fingerprint analysis based on a two-step extraction method for quality evaluation of Perilla frutescens (L.) Britt.

Abundant chemical components are key to ensure the evaluation accuracy of fingerprint analysis of traditional Chinese medicines (TCMs). A two-step extraction method combining supercritical fluid extraction (SFE) and water ultrasonic extraction was established for the quality evaluation of Perilla frutescens (L.) Britt. Weakly polar components were extracted under optimal SFE conditions (15% co-solvent (EtOH : n-hexane = 1 : 14, (v/v)), 40 °C, 250 bar, and 30 min), and polar components were subsequently extracted by an ultrasonic step (100% water as solvent, 40 °C, and 45 min). Then, HPLC methods were established, which were validated to be accurate, stable, and reliable. In this work, 25 batches of samples were evaluated and the data were analysed by similarity analysis (SA) and hierarchical cluster analysis (HCA). The similarity values of SFE extracts and aqueous extracts were respectively 0.616-0.999, and 0.252-0.997, proving the importance of the extraction method for the accuracy of the subsequent fingerprint analysis results. For the HCA, 25 samples were divided into two categories (leaves and stems), among which four batches of leaves with less similarity were considered as stems, indicating that quality differences of P. frutescens depending on medicinal parts and origin exist. The two-step extraction method developed in this work has been proved to be suitable for the quality evaluation of TCMs with complex compositions.

An in-depth investigation of supercritical fluid chromatography retention mechanisms by evaluation of a series of specially designed alkylsiloxane-bonded stationary phases based on linear solvation energy relationship.

Fundamental research on supercritical fluid chromatography (SFC) has gained considerable interest, with many studies focusing on its retention mechanism based on the linear solvation energy relationship (LSER) model. In this paper, a series of alkylsiloxane-bonded stationary phases were specifically designed and synthesized, then evaluated using the mobile phase composed of CO2 with 10% (v/v) methanol. The study demonstrated the close relationship between the interactions (manner and magnitude) of stationary phases and the C-chain length, bonding density and the endcapping treatment. All C8 phases provide positive e, v and negative s, whose magnitude was regularly affected by bonding density. It was worth mentioning the non-endcapped C8 phases could provide H-bonding (positive a and b) by reducing the bonding density of the alkyl chain. Once it was endcapped, the interaction manner did not vary with bonding density adjustment. The non-endcapped C4 phases with higher bonding density could establish additional dispersion interaction (positive v). It can be seen that two synthesis strategies, 1) non-endcapped, long C-chain (C8) combined with low bonding density, and 2) non-endcapped, short C-chain (C4) combined with high bonding density, can obtain the alkylsiloxane-bonded stationary phases (C8-1 and C4-3) to provide both polar and dispersion interactions, showing different separation selectivity. Furthermore, the LSER model with ionic terms was applied to evaluate partial C8 columns, and its rationality was verified. The non-endcapped C8 showed great d+ values, which originated from the silanol groups. C8SCX also possessed a great d+ value due to the benzenesulfonic acid groups. A remarkable result showed that C8SAX exhibited prominent d- and d+ values simultaneously due to the combined effect of silanol and quaternary ammonium groups, which indicates the unique selectivity when separating ionic compounds. This study provides in-depth insights into the retention mechanism of alkylsiloxane-bonded stationary phases in SFC, as well as a reference for the design of SFC stationary phases.

Rapid fingerprint analysis based on supercritical fluid chromatography for quality evaluation of Hedyotis diffusa.

In this study, quality evaluations of Hedyotis diffusa (H. diffusa) batches by rapid fingerprint analysis based on supercritical fluid chromatography (SFC) were accomplished. Abundant chemical components of H. diffusa were effectively extracted by optimal supercritical fluid extraction conditions (20 % MeOH as modifier, 45 °C, 300 bar and 60 min). Then, the extract was separated by SFC on a Torus 1-AA column (100 × 3.0 mm i.d., 1.7 µm) within 10 min by gradient elution increasing from 5 % to 45 % modifier (MeOH containing 0.05 % TFA) at 1.2 mL/min, 30 °C and 2000 psi. The SFC approach exhibited short analysis time, while maintaining good peak shape and resolution. Seven major compounds were further identified by SFC coupled with tandem mass spectrometer to be phenylpropanoid, iridoids and anthraquinones. Finally, fingerprint analysis of 10 batches of H. diffusa by the developed SFC method was accomplished. The similarity values were between 0.894 and 0.968, indicating quality differences of H. diffusa from depending on origin and harvest year exist. The result demonstrates the feasibility of the SFC in batch quality evaluation of H. diffusa.

Isolation of achiral aliphatic acid derivatives from Piper kadsura using preparative two-dimensional chiral supercritical fluid chromatography.

The separation of structural analogues in natural products has always been one of the challenges in separation science, where supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an unconventional but potential solution. In this study, a preparative two-dimensional chiral SFC (2D cSFC) method that was established with two kinds of CSPs was applied in the isolation of the aliphatic acid derivatives in Piper kadsura (P. kadsura). The RPLC unseparated peaks of two samples A and B of P. kadsura were evenly scattered on the CSP-1 column while they clustered into two groups on the CSP-2 column by SFC. There was impressively complementary selectivity between CSP-1 and CSP-2, which were used for construction of 2D cSFC. The first dimension (1D) separation with CSP-1 fractionated the sample A into six parts by a heart-cutting method and the sample B into nine parts for a comprehensive 2D analysis; then 29 and 71 peaks were respectively found in these parts in the second dimension (2D) separation with CSP-2. Further through 2D preparative separation, 19 high purity components were obtained, and the chemical structures of two of them were confirmed, including a novel unsaturated aliphatic acid compound (8Z,10Z)-12-methoxyheptadeca-8,10-dienoic acid and a known octadecadienoic acid lactone Lactariolide. The 2D cSFC method presented the superiority of separating the achiral compounds of complex samples.

Highly sensitive microcantilever-based immunosensor for the detection of carbofuran in soil and vegetable samples.

Microcantilever-based immunosensor is a next-generation electromechanical technique with broad application in biological detection. In this paper, we reported a microcantilever-based immunosensor that quantitatively detect the carbofuran, by using monoclonal antibodies to carbofuran as the receptor molecules. The surface of gold-coated microcantilever was chemically modified by the crosslinking of l-cysteine (l-cys)/glutaraldehyde (GA). The monoclonal antibodies to carbofuran were then immobilized on the side of the microcantilever to fabricate the immunosensor, the mechanical bending induced by antigen-antibody specific binding under an experimental environment. Under the optimized conditions, immunosensor detected carbofuran showed a good linear relationship over the range from 1.0×10-7 to 1.0×10-3g/L (R=0.998), with a detection limit of 0.1ng/mL. Moreover, the proposed immunosensor exhibited high sensitivity, specificity and good stability and can be successfully applied in the carbofuran determination in soil and vegetable samples with satisfactory results.