Mengzhe Guo

Two-dimensional liquid chromatography and its application in traditional Chinese medicine analysis and metabonomic investigation

Two-dimensional liquid chromatography has become an attractive analytical tool for the separation of complex samples due to its enhanced selectivity, peak capacity, and resolution compared with one-dimensional liquid chromatography. Recently, more attention has been drawn on the application of this separation technique in studies concerning traditional Chinese medicines, metabonomes, proteomes, and other complex mixtures. In this review, we aim to examine the application of two-dimensional liquid chromatography in traditional Chinese medicine analysis and metabonomic investigation. The classification and evaluation indexes were first introduced. Then, various switching methods were summarized when used in an on-line two-dimensional liquid chromatography system. Finally, the applications of this separation technique in traditional Chinese medicine analysis and metabonomic investigation were discussed on the basis of specific studies.

Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with quadrupole-time of flight mass spectrometry

HIGHLIGHTSHILIC × RP 2D‐LC was used to separate Ginkgo biloba extract (GBE) for the first time.The off‐line 2D‐LC was established with high orthogonality and peak capacity.125 peaks were detected and structures of 104 compounds were characterized. ABSTRACT Ginkgo biloba extract (GBE), derived from the leaves of Ginkgo biloba L., is one of the most widely used traditional Chinese medicines worldwide. Due to high structural diversity and low abundance of chemical constituents in GBE, conventional reversed‐phase liquid chromatography has limited power to meet the needs of its quality control. In this study, an off‐line hydrophilic interaction × reversed‐phase two‐dimensional liquid chromatography (HILIC × RP 2D‐LC) system coupled with diode array detection (DAD) and quadrupole time‐of‐flight mass spectrometry (qTOF‐MS) was established to comprehensively analyze the chemical constituents of GBE. After optimizing the chromatographic columns and mobile phase of 2D‐LC, a Waters XBridge Amide column using acetonitrile/water/formic acid as the mobile phase was selected as the first dimension to fractionate GBE, and the obtained fractions were further separated on an Agilent Zorbax XDB‐C18 column with methanol/water/formic acid as the mobile phase. As a result, a total of 125 compounds were detected in GBE. The orthogonality of the 2D‐LC system was 69.5%, and the practical peak capacity was 3864 and 2994, respectively, calculated by two different methods. The structures of 104 compounds were tentatively characterized by qTOF‐MS analysis, and 21 of them were further confirmed by comparing with reference standards. This established HILIC × RP 2D‐LC‐qTOF/MS system can greatly improve the separation and characterization of natural products in GBE or other complicated herbal extracts.

Accurate quantification of 5-Methylcytosine, 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine in genomic DNA from breast cancer by chemical derivatization coupled with ultra performance liquid chromatography- electrospray quadrupole time of flight mass spectrometry analysis

The DNA demethylation pathway has been discovered to play a significant role in DNA epigenetics. This pathway removes the methyl group from cytosine, which is involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) proteins. Then, 5-hmC can be iteratively oxidized to generate 5-formylcytosine (5-foC) and 5-carboxylcytosine (5-caC). However, 5-hmC, 5-foC, and 5-caC are hardly detected due to their low content. In this study, we have developed a LC-HRMS method coupled with derivatization to accurately and simultaneously quantify 5-mC levels, along with its oxidation products in genomic DNA. Derivatization was carried out using 4-dimethylamino benzoic anhydride, which has been shown to improve separation and enhance the detection sensitivity. Finally, we successfully applied this method towards the quantification of 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA isolated from both human breast cancer tissue and tumor-adjacent normal tissue. We show that 5-foC and 5-caC are increased in tumor tissue. In addition, the levels of 5-mC, 5-hmC, 5-foC, and 5-caC measured in tumor tissue versus tumor-adjacent tissue were found to be distinct among different classifications. This suggests that cytosine modifiers could be used as potential biomarkers for determining the stage of development of breast cancer, as well as prognosis.

Optimization and Assessment of Three Different High Performance Liquid Chromatographic Systems for the Combinative Fingerprint Analysis and Multi-Ingredients Quantification of Sangju Ganmao Tablet

Chromatographic separation is still a critical subject for the quality control of traditional Chinese medicine. In this study, three different high performance liquid chromatographic (HPLC) systems employing commercially available columns packed with 1.8, 3.5 and 5.0 μm particles were respectively developed and optimized for the combinative fingerprint analysis and multi-ingredients quantification of Sangju Ganmao tablet (SGT). Chromatographic parameters including the repeatability of retention time and peak area, symmetry factor, resolution, number of theoretical plates and peak capacity were used to assess the chromatographic performance of different HPLC systems. The optimal chromatographic system using Agilent ZORBAX SB-C18 column (2.1 mm × 100 mm, 3.5 μm) as stationary phase was respectively coupled with diode array detector or mass spectrometry detector for the chromatographic fingerprint analysis and simultaneous quantification or identification of nine compounds of SGT. All the validation data conformed to the acceptable requirements. For the fingerprint analysis, 31 peaks were selected as the common peaks to evaluate the similarities of SGT from 10 different manufacturers using heatmap, hierarchical cluster analysis and principal component analysis. The results demonstrated that the combinations of the quantitative and chromatographic fingerprint analysis offer an efficient way to evaluate the quality consistency of SGT.

Succinic Acid Enhanced Quantitative Determination of Blood Modified Nucleosides in the Development of Diabetic Nephropathy Based on Hydrophilic Interaction Liquid Chromatography Mass Spectrometry

HighlightsWe have designed a new LC–MS method to analyze the modified nucleosides.The succinic acid was chosen as the extra mobile phase additives.We applied this method into the quantitative analysis of 189 serum samples.Modified nucleosides can act as the potential biomarkers through the process of DN. Abstract The epigenetic modification of DNA or RNA may have relationship with the development of diabetic nephropathy (DN). We designed to develop a liquid chromatography mass spectrometry (LC–MS) coupled with mobile phase additive sensitization method for the analysis of the dissociative epigenetic modified nucleosides in serum of patients from different periods of DN with the health comparison. Serum samples were collected from 43 healthy volunteers and 156 patients, which divided into four groups. A succinic acid enhanced LC–MS/MS strategy was developed for the quantitative analysis of seven nucleosides in serum samples. The signal intensity of seven nucleosides increased three to seven times, respectively. A series of statistical analysis were carried out to demonstrate the dynamic changes of modified nucleosides in four groups. This method showed good specific, accuracy, and stability. The results were calibrated and presented through the formation of m6A/C, I/C, 5‐mdC/C, 5‐mC/C, pseU/U to eliminate the individual and age differences. The m6A/C, I/C, and pseU/U were found having the dynamic changes in four groups (p < 0.05). And these five modified nucleosides can also used as one parameter to distinguish these four groups by ROC and LDA analysis. Additionally, they were found to be connected with several clinical biochemical indexes through multiple linear regression analysis. Our LC–MS/MS method assay successfully quantifies the modified nucleosides in serum. And three modified nucleosides can act as the potential biomarkers to describe the development of DN and help to evaluate the diagnosis, treatment, and prognosis of DN in clinical.

Chemical Profiling and Comparison of Sangju Ganmao Tablet and Its Component Herbs Using Two-Dimensional Liquid Chromatography to Explore Compatibility Mechanism of Herbs

Sangju Ganmao tablet (SGT), a well-known Chinese patent medicine used to treat cold symptoms, is made from eight herbal medicines. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) method was developed to comprehensively separate the chemical constituents of SGT. Through optimization of the experimental conditions, a total of 465 peaks were finally detected in SGT, and the structures of 54 selected compounds were fully identified or tentatively characterized by quadrupole time-of-flight mass spectrometry (qTOF-MS) analysis. The established 2D-LC analysis showed high orthogonality (63.62%) and approximate 11-fold improvement in peak capacity (2399 and 1099, obtained by two calculation methods), in contrast to conventional one-dimensional RPLC separation. The eight component herbs of SGT were also respectively separated by using the 2D-LC system, and we found that a total of 12 peaks detected in SGT were not discovered in any component herbs. These newly generated chemical constituents would benefit better understanding of the compatibility mechanism of the component herbs. The strategy established in this study could be used for systematic chemical comparison of SGT and its component herbs, which contributes to exploration of herbal compatibility mechanism.

Enhanced detection of amino acids in hydrophilic interaction chromatography electrospray tandem mass spectrometry with carboxylic acids as mobile phase additives

Liquid chromatography coupled with mass spectrometry technique has been widely used in the analysis of biological targets such as amino acids, peptides, and proteins. In this work, eight common single carboxylic acids or diacids, which contain different pKa have been investigated as the additives to the analysis of amino acids. As the results, carboxylic acid additive can improve the signal intensity of acidity amino acids such as Asp and Glu and the chromatographic separation of basic amino acids such as Arg, His, and Lys. In particular, the diacids have better performance than single acids. The proposed mechanism is that the diacid has hydrogen bond interaction with amino acids to reduce their polarity/amphiprotic characteristics. Besides, oxalic acid has been found having better enhancement than phthalic acid by overall consideration. Therefore, we successfully quantified the 15 amino acids in Sepia bulk pharmaceutical chemical by using oxalic acid as the additive.