Zhimou Guo

Comprehensive characterization of Stevia Rebaudiana using two‐dimensional reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography

Two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography (2D-RPLC/HILIC) system was successfully applied for comprehensive characterization of steviol glycosides from Stevia rebaudiana. The experiments were performed in offline mode using an XCharge C18 column in first dimension and an XAmide column in second dimension. In first dimension, preliminary separation of Stevia aqueous extract was accomplished and 30 fractions were collected. Then fractions 1-20 were selected for further purification and 13 compounds with high purity were obtained in second dimension. Comprehensive characterization of these compounds was completed by determination of their retention time, accurate molecular weight, diagnostic fragmentation ions, and nuclear magnetic resonance spectroscopy. As a result, all nine known steviol glycosides, as well as other four steviol glycosides were fully purified. The result demonstrated that this procedure is an effective approach for the preparative separation and comprehensive characterization of steviol glycosides in Stevia. This 2D-RPLC/HILIC method will be a promising tool for the purification of low-abundance compounds from natural products.

Identification of prenyl flavonoid glycosides and phenolic acids in Epimedium koreanum Nakai by Q-TOF-MS combined with selective enrichment on "click oligo (ethylene glycol)" column

Prenyl flavonoid glycosides and phenolic acids are constituents of the medicinal plant Epimedium koreanum Nakai (EK). An efficient method was developed to enrich these compounds and identify them, using ultra performance liquid chromatography combined with Q-TOF-MS, and a click oligo (ethylene glycol) (Click OEG) column. Using this method, 51 prenyl flavonoid glycosides and 18 phenolic acids were identified or tentatively identified. Of these, 11 prenyl flavonoid glycosides and 4 phenolic acids were new compounds, and 7 phenolic acids were newly identified in EK. Therefore, MS combined with selective enrichment provided a powerful means for analyzing prenyl flavonoid glycosides and phenolic acids.

Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix

AbstractSelective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α1-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis.n FigureA reversed-phase depletion coupled with HILIC glycopeptide enrichment strategy by using Click OEG-CD matrix was developed in the work

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

AbstractSaponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.n FigureSeparation of saponins using 2-D RPLC/HILIC

Two-dimensional strong cation exchange/positively charged reversed-phase liquid chromatography for alkaloid analysis and purification

Peak tailing and nonalkaloid coelution usually hinder alkaloid purification. In this study, a 2DLC, strong cation exchange (SCX) coupled with positively charged RP (PGRP) LC, was developed to overcome these problems. Ten compounds including basic and nonbasic compounds were analyzed. Nonbasic compounds, which are coeluted with basic compounds on RP or PGRP columns, were weakly retained on the SCX column. In addition, a symmetrical peak shape (tailing factors <1.2) of basic compounds can be obtained in the current system. Compared to two other 2D systems, the current system provided the highest orthogonality (R(2) = 0.045). Furthermore, the SCX coupled with PGRP system was applied for alkaloid purification from a traditional Chinese medicine. Nineteen alkaloids were obtained and one of them was identified as a novel compound. The overall results demonstrate that the proposed system is a powerful tool for alkaloid purification.

Selective enrichment with "click oligo (ethylene glycol)" column and TOF-MS characterization of simple phenylpropanoids in the fruits of Forsythia suspensa.

To separate samples of complex natural products, highly efficient and selective separation methods should be developed. Herein, a selective enrichment method was developed to separate Forsythia suspensa components with click oligo (ethylene glycol) (OEG) column in reversed phase (RP) mode. In this method, F. suspensa aqueous extract was successfully separated. And three fractions with structure-related compounds were obtained. Fraction I mainly consisted of C(6)-C(2) natural alcohols and glycosides, fraction II mostly consisted of lignans, and fraction III mainly consisted of simple phenylpropanoids (SP). Then, the three fractions were separated with ultra-performance liquid chromatography (UPLC). Four more lignans were observed in fraction II, and eight more SP were observed in fraction III than that without OEG column. Fraction III was successively characterized by TOF-MS, 2 acids (caffeic acid and chlorogenetic acid), 26 SP with caffeoyl, and 2 SP with coumaroyl were characterized. The results prove that a valid method has been developed to selectively enrich SP and lignans. And the method combined with UPLC can efficiently separate SP and lignans. Furthermore, the TOF-MS method is effective to confirm the substituents of SP.

[Quantitative analysis of five antiviral drugs by hydrophilic interaction liquid chromatography-charged aerosol detection].

Antiviral drugs are widely used for human and animals. However, the analysis of the mixture of antiviral drugs is a challenge for high performance liquid chromatography, since some of the antiviral drugs have weak UV absorbance and poor retention in reversed phase liq- uid chromatography. A method of hydrophilic interaction liquid chromatography-charged aerosol detection (HILIC-CAD) was optimized for the qualitative and quantitative analysis of five antiviral drugs. In this study, Click TE-Cys was used as the stationary phase and CAD was used as the detector. Various chromatographic conditions including the kind of detector, chromatographic mode, column and mobile phase composition were investigated. Compared to UV-Vis, more antiviral drugs could be detected by CAD, since it is a universal detector. HILIC mode is an alternative to reversed phase liquid chromatography mode. HILIC provides higher sensitivity and unique selectivity to target compounds. After the optimized parameters were obtained, the developed method was used for the quantitative analysis of the five antiviral drugs. As a result, the current method has good repeatability, a wide linear range (0.07-2.28 mg/mL) and good sensitivity (LOQ ≤ 0.04 mg/mL). The RSDs of intra-day and inter-day peak areas were less than 3. 06% and 5. 38% respectively. The above results demonstrated that the current method is sensitive, robust and effective for the separation and determination of these five antiviral drugs.

Enhanced multi-phosphopeptide enrichment and Nano LC-ESI-qTOF-MS detection strategy using click OEG-CD matrix

Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio(ethylene glycol)(OEG) linked β-cyclodextrin(CD)] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry(MS) detection of multi-phosphopeptide was developed via optimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qTOF-MS). The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestion α-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 α-casein phosphopeptide signals(including 15 multi-phosphopeptides) were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non- or mono-phosphopeptides and the stronger interactions between multi-phosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.

[Detection of drug-human serum albumin binding ratios of two Chinese medicinal ingredients by high performance affinity chromatography].

The interaction of two Chinese medicinal ingredients and human serum albumin (HSA) has been investigated by high performance affinity chromatography (HPAC). HSA bounded silica-based stationary phase was prepared based on the click chemistry strategy, and packed in a column (named as HSA column). The drug-HSA binding ratio was calculated from the difference of the drugs retention times on the HSA column and silica column (blank column). The warfarin-HSA binding ratio determined by this method was similar to the reference reported value by ultrafiltration method. The results indicated that the new HSA column and the HPAC method can be used for the detection of binding ratio of drug and HSA. The binding ratios of puerarin and goitrin determined by the HPAC method were 10.26% and 10.20%, respectively. And the binding ratio of puerarin determined by ultrafiltration was 14.25%. All these results showed that HPAC is a useful method to investigate the interaction between drugs and protein.