Liquid Chromatography Detection Research Articles

Preconcentration of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Clinical Samples by Various Hollow Fiber–Liquid Phase Microextraction (HF-LPME) Configurations With High-Performance Liquid Chromatography (HPLC) Detection

Hollow fiber liquid-phase microextraction is commonly used in analytical chemistry, providing high clean-up and pre-concentration factors. However, a major issue with this method is the long extraction time. The objective of this project is to reduce the extraction time by inducing different types of fluid motion in the sample solution. Three methods were employed: agitation using a magnetic stir bar in the horizontal position (conventional method), utilization of an air pump, and agitation using a magnetic stir bar in the vertical position for the pre-concentration and determination of non-steroidal anti-inflammatory drugs (naproxen, ibuprofen, and mefenamic acid) in biological samples via high-performance liquid chromatography with ultraviolet absorbance detection. The results demonstrated a significant reduction in extraction time and limit of detection when the air pump was employed compared to the conventional method of stirring with a magnetic stir bar in the horizontal position. Moreover, the mentioned parameters were even lower when stirring solutions were accomplished using a vertically-positioned magnetic stir bar, surpassing the method utilizing the air pump for agitation. Furthermore, the relative standard deviations were also lower when a vertically-positioned magnetic stir bar was used compared to the conventional method with a horizontally-positioned magnetic stir bar, while those of the air pump-assisted agitation method fell between the values of the aforementioned methods.

Preparation of branched polyethyleneimine-assisted boronic acid-functionalized magnetic MXene for the enrichment of catecholamines in urine samples.

Herein, a magnetic borate-functionalized MXene composite with multiple boronic affinity sites was fabricated by embedding Fe3 O4 nanoparticles with 4-formylphenylboronic acid functionalized Ti3 C2 Tx nanosheets and served as sorbent for the simultaneous extraction of catecholamines (CAs) in urine samples. The morphology and structure of the magnetic materials were investigated using scanning microscopy, vibrating sample magnetometer, X-ray photoelectron spectrometer, and X-ray diffraction. The introduction of polyethyleneimine can amplify the bonded boronic acid groups, thereby effectively improving the adsorption capacities for CAs based on the multiple interactions of boronic affinity, hydrogen bonding, and metal coordination. The adsorption performance was investigated using the kinetics and isotherms models, and the main parameters that influence the extraction efficiency were optimized. Under the most favorable magnetic solid-phase extraction condition, a sensitive method for the analysis of CAs in urine samples was developed by combining magnetic solid-phase extraction conditions with high-performance liquid chromatography detection. The findings illustrated that the proposed approach possessed a wide linearity range of 0.05-250ng/mL with an acceptable correlation coefficient (R2 ≥0.9984) and detection limits of 0.010-0.015ng/mL for the target CAs. The research not only provides a notable composite with multiple boronic affinity sites but also offers an effective and feasible measure for the detection of CAs in biological samples.

Hyphenated liquid electrode glow discharge-dielectric barrier discharge molecular emission spectrometry for determination of dithiocarbamates.

A hyphenated liquid electrode glow discharge (LEGD)-dielectric barrier discharge (DBD) molecular emission spectrometer was constructed and used as a novel liquid chromatography (LC) detector for dithiocarbamates (DTC) determination. The LEGD was used as an acidolysis reactor for the in-situ transformation of DTCs into CS2 with high efficiencies of 74.11-97.98%. The DBD was used to excite CS2 gas to generate a specific molecular emission at 257.94nm. The linear correlation coefficient of the method was>0.99 from 1 to 200μgmL-1. The detection limits ranged from 0.1 to 0.3μgmL-1 with 76-119% recovery and relative standard deviations of 0.2-8.5%. Moreover, the hyphenated microplasma spectrometer achieved low power consumption, low temperature, immediate acidolysis, and high transformational efficiency, and can detect each DTC when combined with LC.

Establishment and Optimization of Flavonoid Extraction and Detection System for Hemerocallis

Hemerocallis is a characteristic vegetable with outstanding edible and medicinal value. Flavonoids are important bioactive components of Hemerocallis. To improve the extraction efficiency and detection accuracy of flavonoids from Hemerocallis, we established a high-performance liquid chromatography (HPLC) detection system, which can simultaneously detect multiple flavonoids. In addition to the previously developed organic solvent extraction method, an ultrasonic-assisted extraction technique that uses fewer samples was established to extract flavonoids from Hemerocallis. The extraction conditions of the ultrasonic-assisted extraction were optimized via a single-factor experiment and a response surface experiment. The HPLC system detected and determined the contents of rutin, isoquercetin, myricetin, quercetin, apigenin, and diosmetin from 70 Hemerocallis germplasm resources. In addition, we evaluated the antioxidant activity of flavonoids in Hemerocallis using DPPH free radical scavenging capacity with ascorbic acid (Vc) as a positive control. The results showed that the optimum conditions for the ultrasonic extraction process were as follows: sample weight of 0.25 g, ethanol volume fraction of 72%, ethanol volume of 2.5 mL, and ultrasonic extraction time of 17 min. Under these conditions, flavonoid extraction had a strong scavenging effect on DPPH. With the increase in the sample solutions’ concentrations, its antioxidant capacity was gradually enhanced, and the DPPH scavenging rate reached 70.2%. The optimized ultrasonic-assisted extraction technology can increase the total content of six flavonoids in day lily bud by 59.01%, especially the content of rutin (increased by 64.41%) in Hemerocallis flower buds. Among 70 Hemerocallis plant resources, we selected materials H0087 and H0059 with high and stable flavonoid content, with the total content of six substances being 4390.54 ug/g and 3777.13 ug/g. Thus, this study provides a reference for extracting and determining flavonoid contents in Hemerocallis materials. It also provides a theoretical basis for high-quality individual plant breeding.

Molecularly imprinted polymer membranes based on single template for recognition and extraction of carbamazepine and its structurally analogues

In this work, a novel molecularly imprinted polymer membrane (MIPM) using oxcarbazepine (OXC) as the single template and polyvinylidene difluoride membrane as the subtract was fabricated for recognition and extraction of carbamazepine (CBZ) and its analogues including OXC, carbamazepine-10,11-epoxide (CBZE), and monohydroxy derivative 10, 11-dihydro-10-hydroxy carbamazepine (MHD). The prepared MIPMs were characterized by scanning electron microscope, Fourier transform infrared spectroscopy, Brunaner-emmet-teller measurements and X-ray photoelectron spectroscopy, showing the successful construction of uniform and porous polymers on the surface of membranes. A series of adsorption affinity tests were investigated, indicating the prepared materials had specific recognition capacity and excellent stability as novel sorbents. The maximum static adsorption capacities of MIPMs towards OXC, CBZ, MHD or CBZE was 4.246, 3.621, 2.316, or 4.877 mg·g−1, respectively. The MIPMs showed reusability and reproducibility. The usability of MIPMs was evaluated by combining MIPMs extraction and high performance liquid chromatography detection for the determination of CBZ, CBZE, OXC and MHD in different matrices including methanol, normal saline, phosphate buffer solution, and plasma. The mean recovery of four target compounds in methanol, normal saline and phosphate buffer solution was more than 75 %, while the mean recovery of CBZ and CBZE in plasma was more than 80 %. The results demonstrated that the proposed MIPMs is very suitable for the determination of OXC, CBZ, MHD and/or CBZE in different matrices.

Smartphone as a fluorescence detector for high-performance liquid chromatography

BackgroundFluorescence detection is employed in high-performance liquid chromatography (HPLC) due to its high specificity and sensitivity. However, it is often limited by expensive components and bulkiness. Recently, advances in technology and electronics have led to the development of smartphones that can serve as portable recording, analysis, and monitoring tools. Smartphone-based detection provides advantages of cost effectiveness, rapid signal/data processing, and the display of results on a handhold monitor. The combination of smartphone-based detection with HPLC can offer unique features that are beneficial in overcoming limitations of commercial fluorescence detectors. (90) ResultsA miniaturized and low-cost HPLC fluorescence detector based on a smartphone is introduced for the detection of six fluorescent molecules. The smartphone is able to capture emitted fluorescence in video format while MATLAB code is used for data processing to provide chromatograms based on different detection channels. A custom designed double-channel flow cell was utilized to enable simultaneous detection of fluorescent compounds with different excitation wavelengths. The detector consists of a lab-made flow cell, monochromatic LEDs as the light source, 3D printed housing and connector box, fiber optic cables, and a smartphone. The effects of flow cell geometry, channel width and light slit diameter, as well as a comparison of different flow cell manufacturing techniques, are studied and discussed. The validated system was successfully applied to samples from diverse water sources, yielding spiking recoveries within the range of 91.7% and 109.7%. (141) SignificanceThis study introduces the first smartphone-based fluorescence detector for HPLC with cost-effective and customizable flow cells, allowing for the simultaneous detection of fluorescent compounds with different excitation wavelengths and offering a potential solution for the analysis of co-eluting compounds. Beyond its user-friendly interface and low-cost, smartphone detection in HPLC provides tremendous opportunities in further miniaturizing chromatographic instrumentation while offering high sensitivity and can be expanded to other mechanisms of detection. (70)

Advances in Extraction Techniques for Beer Flavor Compounds

Owing to the unique chemical properties exhibited by beer flavor compounds, different extraction methods have been utilized to extract these compounds from the sample matrix. Carbonyl compounds, which significantly contribute to flavor instability in beer, pose challenges in detection due to their low concentrations and reactivity. Consequently, the analysis of beer flavor compounds has focused on improving sensitivity and specificity through techniques that minimize sample preparation requirements and reduce interactions between factors involved in the analysis. Notably, extraction techniques such as headspace solid-phase microextraction (HS-SPME), stir bar sorptive extraction (SBSE), and gas diffusion microextraction (GDME) have been successfully applied to the analysis of carbonyl compounds in alcoholic beverages, including beer. Derivatization agents like 2,4-dinitrophenylhydrazine (DNPH) and O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) enhance the volatility and stability of analytes, facilitating their separation and detection in gas chromatography and high-performance liquid chromatography. Chromatographic separation methods, particularly gas chromatography and liquid chromatography, are extensively employed to identify and quantify aroma/flavor compounds in various foodstuffs, including beer. This review provides a comprehensive overview of extraction techniques and chromatographic methods used in the analysis of beer compounds.

Synthesis of novel hydroxyl-functionalized hyper-crosslinked polymer for extraction of phenylurea herbicides from environmental waters

Water resources are frequently contaminated by organic micropollutants (OMPs), which present a significant peril to ecosystem and well-being of the people. Phenylurea herbicides (PUHs) are an important class of OMPs since they are widely used in modern agriculture. However, efficient extraction of PUHs from water is of challenge because of their high water affinity. Therefore, the synthesis of novel adsorbent capable of effectively extracting PUHs from water is urgently needed. Here, three novel hydroxyl-functional hyper-crosslinked polymers (HCPs) were synthesized through Friedel-Crafts reaction by using the brominated β-cyclodextrin, i.e., heptakis(6-bromo-6-deoxy)-β-CD) [(CD-(Br)7)], as the building unit. Among them, PQ-CD-HCP, which was synthesized by using p-quaterphenyl (PQ) and CD-(Br)7, had the best adsorption capability for the PUHs. Finally, the quantitative determination of the PUHs for environmental waters was realized by using the PQ-CD-HCP as the solid-phase adsorbent in conjunction with high-performance liquid chromatographic detection. Under the optimal conditions, the limits of detection for environmental water samples were 0.02–0.05 ng mL−1. The method recoveries were 86.0%-116.5% with the relative standard deviations ≤ 7.8%. The PQ-CD-HCP also showed good adsorption performance for other OMPs such as triazine herbicides, chlorophenols, polycyclic aromatic hydrocarbons, endocrine disruptors, nitroimidazoles and benzoylureas insecticides, indicating its good application prospect. The adsorption experiments show that the PQ-CD-HCP extracts the analytes by encapsulation, π-stacking, hydrophobic and hydrogen bonding interactions.

Dispersive solid-phase extraction of alkaloids and polyphenols using borate hypercrosslinked polymers.

In this study, a borate hyper-crosslinked polymer was synthesized by crosslinking 1-naphthalene boric acid and dimethoxymethane via the Friedel-Crafts reaction. The prepared polymer exhibits excellent adsorption performance toward alkaloids and polyphenols with maximum adsorption capacities ranging from 25.07 to 39.60mg/g. Adsorption kinetics and isotherms model results indicated the adsorption was a monolayer and chemical process. Under the optimal extraction conditions, a sensitive method was established for the simultaneous quantification of alkaloids and polyphenols in green tea and Coptis chinensis by coupling with the proposed sorbent and ultra-high performance liquid chromatography detection. The proposed method exhibited a wide linear range of 5.0-5000.0ng/ml with R2 ≥ 0.99, a low limit of detection (0.66-11.25ng/ml), and satisfactory recoveries (81.2%-117.4%). This work provides a simple and convenient candidate for the sensitive determination of alkaloids and polyphenols in green tea and complex herbal products.

Green construction of hydroxyl-functionalized magnetic porous organic framework for effective extraction of triazine herbicides from environmental water and watermelon juice samples

Triazine herbicides have been widely detected in water resources and food, which poses a potential hazard to both ecosystem and human health. Due to their high polarity, conventional adsorbents have limitations for their extractions. Herein, for the effective magnetic extraction of triazine herbicides, a novel and effective magnetic adsorbent was prepared with a satisfactory extraction performance. In the experiments, five porous organic frameworks (POFs) with hydroxyl functional groups were synthesized by diazo-coupling reactions in aqueous solution with β-cyclodextrin (β-CD) as a green monomer. After evaluation of the five POFs, the DDM-CD-POF, which was synthesized with 4′4-diaminodiphenylmethane (DDM) and β-CD, showed the largest specific surface area and the best adsorption capacity for the five triazine herbicides. Then, it was magnetized by introducing Fe3O4@SiO2 into it to prepare a magnetic adsorbent (M-DDM-CD-POF) to facilitate separation and recycling. Finally, the M-DDM-CD-POF-based magnetic solid-phase extraction in combination with high performance liquid chromatographic detection method was established for the quantitative determination of the triazine herbicides in environmental water and watermelon juice samples. The current strategy showed low limits of detection of 0.03–0.11 ng mL−1 for environmental water and 0.07–0.22 ng mL−1 for watermelon juice sample. The method recoveries for spiked samples ranged from 84.0% to 113.0% with the relative standard deviations ≤8.8%. This work provides a new approach for the detection of the triazine herbicides with good application prospect.

Preparation and evaluation of molecularly imprinted polymers based on magnetic graphene oxide for selective extraction and determination of quercetin in red wine

In this work, a high selectivity molecularly imprinted polymer (Fe3O4/GO@MIP) for quercetin was successfully synthesized by combination of surface molecular imprinting technology and magnetic graphene oxide (Fe3O4/GO). Several characterization methods were adopted to verify the successful synthesis of Fe3O4/GO@MIPs. The resultant Fe3O4/GO@MIPs exhibited outstanding adsorption capacity (18.24 mg g−1), remarkable regeneration ability and excellent selectivity toward quercetin. The results indicated that the Fe3O4/GO@MIPs had higher adsorption capacity and better selectivity for quercetin compared with non-imprinted polymers (Fe3O4/GO@NIPs). The wide linearity of the proposed method was achieved within the concentration of quercetin from 0.1 to 200 μg mL−1 with R2 > 0.999, and the limit of detection (LOD) was calculated as 0.03 μg mL−1. Under the optimum conditions, the satisfactory recoveries for quercetin in the spiked red wine samples were in the range of 96.8%–101.4% with the relative standard deviation (RSD) less than 5%. Furthermore, the obtained Fe3O4/GO@MIPs were further employed as promising adsorbents for the separation and detection of quercetin in red wine combined with high performance liquid chromatography (HPLC) detection.

Thoughtful design of a covalent organic framework with tailor-made polarity and pore size for the enrichment of bisphenols and their derivatives: Extraction performance, adsorption mechanism and toxicity evaluation

A stable, reusable and cost-effective covalent organic framework (COF) with medium polarity was successfully decorated on Fe3O4. The Fe3O4@COF contained tailor-made polarity and pore size that fitted well with bisphenols and their derivatives (BPs). When coupling magnetic solid-phase extraction (MSPE) with high-performance liquid chromatography (HPLC) detection, the Fe3O4@COF featured efficient recognition and enrichment for BPs due to π-π stacking, C–H⋯π interactions, pore-filling effect, dispersion force and hydrophobic interactions. Under optimized conditions, calibration plots exhibited good linearity (5–1000 ng mL−1), and limits of detection (LOD) ranged from 0.15 to 0.39 ng mL−1. The method was successfully employed in quantifying BPs in authentic lake and river water samples with satisfactory recoveries ranging from 81.4% to 120%. Molecular dynamics simulation revealed extraction mechanisms, and a microscopic behavior related to the clustering property of the emerging brominated compounds was first discovered. Ecotoxicological assessments of target pollutants were conducted from multiple aspects, highlighting the harmfulness of the chemicals and the significance of the analytical method. The proposed methodology offered sensitive detection and quantification, which was beneficial for the timely tracking of the concentration, transportation and distribution of BPs to better explore their environmental behavior and tackle contamination problems in complex environmental matrices.

Synthesis of spherical amine-functionalized silica molecular sieve and application as selective adsorbents for aromatic hydrocarbons analysis

In this work, spherical amine-functionalized mesoporous silica (Sp-Amino-Si) molecular sieve was fabricated by an acid hydrothermal method using 3-aminopropyltriethoxysilane as a modifier and toluene as the solvent. The results of scanning electron microscopy, Barrett-Joyner-Halenda pore size distribution, Fourier transform-infrared spectroscopy, and low-angle X-ray diffraction patterns revealed that the Sp-Amino-Si was monodispersed microspheres with uniform and controllable pore sizes sharing two-dimensional hexagonal P6mm symmetry and a large number of amino groups. The Sp-Amino-Si molecular sieve was verified to be suitable for the selective separation of aromatic hydrocarbons (AMHs) containing two fused benzene rings among other compounds from environmental water matrix. Under the optimized conditions, the Sp-Amino-Si exhibited much higher adsorption efficiency for AMHs (above 88.19%), comparing to the original SBA-15 (below 44.52%). The maximum adsorption capacities for naphthalene, methyl naphthalene, 2-methyl naphthalene, and acenaphthene were 477.1, 1006.5, 893.3, and 1253.2 µg·g−1, respectively. The high selectivity of Sp-Amino-Si for the four AMHs attributed to the size sieving and p-π interactions between the lone pair electrons of amine groups and the π-electrons in AMHs molecules. The Sp-Amino-Si based solid-phase extraction coupled with high performance liquid chromatography detection method was constructed for the analysis of the four AMHs, and the recoveries of spiked AMHs ranged from 89.67% to 112.74% with a relative standard deviation lower than 5.75%. This study provides some inspiration and a reliable method for AMHs separation, removal, and analysis.

One-Step Synthesis of Polydopamine-Coated Dummy Molecularly Imprinted Polymers on the Surface of Fe3O4 for Selective Extraction and Enrichment of Aflatoxin B in Foods Prior to High-Performance Liquid Chromatography Detection

One-Step Synthesis of Polydopamine-Coated Dummy Molecularly Imprinted Polymers on the Surface of Fe3O4 for Selective Extraction and Enrichment of Aflatoxin B in Foods Prior to High-Performance Liquid Chromatography Detection

Preparation and anti-tumor effects of mesoporous silica nanoparticles loaded with trifluoperazine.

We have developed a targeted nano-drug delivery system that effectively harnesses the anti-tumor properties of trifluoperazine (TFP), while concurrently mitigating its side effects on the central nervous system. The manufacturing process entailed the preparation of mesoporous silica nanoparticles (MSN-NH2), followed by the loading of trifluoperazine into the pores of MSN-NH2 and then surface modification with polyethylene glycol (PEG) and anisamide (AA), resulting in the formation of TFP@MSN@PEG-AA (abbreviated as TMPA) nanoparticles. In vitro and in vivo anti-tumor activity and hemolysis experiments showed that TMPA had an excellent safety profile and a good anti-tumor effect. Importantly, the drug content of the TMPA nanoparticle group was found to be significantly lower than that of the TFP group in the mouse brain tissue as determined by High Performance Liquid Chromatography (HPLC) detection. Therefore, the developed drug delivery system achieved the goal of maintaining TFP's anti-tumor action while avoiding its negative effects on the central nervous system.

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

An improved design of the fused silica capillary flow cell for absorbance detection in microcolumn liquid chromatography

Efficient absorbance detection of a low-volume chromatography peak is a difficult task. In this work, an improved design of the fused silica capillary flow cell for absorbance detection in microcolumn liquid chromatography is described. The cell was fabricated from 0.15 mm I. D. fused silica capillary and silica optical fibres. Optical fibres were fully integrated into the cell design and enabled a convenient and effective connection of the cell with the light source and light detector (265 nm UV LED and photodiode in this work). Manufactured cells covered the range of physical lengths 3.1–9.9 mm (55–175 nL) and were used without any focusing optics and slits. Baseline noise was typically below 0.05 mAU and the effective optical path determined in the experiments was 83–97% of the cell's physical length. The level of stray (parasitic) light indicated by a 1% deviation from linearity at 1.7 AU was 0.08% only. The proposed cell design was found to be moderately susceptible to the refractive index change (20–35 mAU baseline change in 5–95% (v/v) gradient of acetonitrile or methanol in a mixture with water, G index up to 4 AU·s/RIU). Manufactured cells were finally applied for absorbance detection of components of test the mixture eluted off 0.3 mm I. D. microcolumn. 9.9 mm cell (175 nL) with an effective optical path of 8.9 mm exhibited contribution to the broadening of chromatography peak comparable with commercial 6 mm (80 nL) rectangular flow cell.

Agrobacterium tumefaciens mediated genetic transformation of Tripterygium wilfordii and its application to enhance the accumulation of triptolide

Triptolide, an important representative active compound of Tripterygium wilfordii, is hopeful to be developed into modern drug. However, the content of triptolide in natural plants is very low, which seriously restricts its development in the pharmaceutical industry. As a perennial woody vine, research on the genetic transformation of T. wilfordii is challenging and slow, which limits the process of improving triptolide content through biotechnology. In this study, the concentrations and combinations of growth regulators for T. wilfordii callus induction and regeneration were firstly optimized. The 2,4-dichlorophenoxyacetic acid (1 mg/L) was efficient to induce callus formation from leaf explants, while 2.0 mg/L 6-benzylaminopurine with 0.1 mg/L α-naphthaleneacetic acid had the best effect on regeneration from callus. Subsequently, the effect of different concentrations of screening agent (kanamycin or hygromycin B) on callus induction rate was determined. Then, Agrobacterium tumefaciens mediated genetic transformation was established and validated by two different plant expression vectors, pCambia1301 and pJCV53, corresponding to β-glucuronidase histochemical assay and monomeric red fluorescent protein fluorescence detection, respectively. It took 3–4 months from A. tumefaciens infection to complete transgenic plant regeneration. Based on this transformation system of T. wilfordii, the overexpression of miltiradiene synthase gene (TwMS) was performed. Real-time quantitative PCR analysis indicated that the expression levels of TwMS were highly up regulated in the transgenic plants compared with control line. The detection of high-performance liquid chromatography showed that overexpression of TwMS resulted in a 1.20–1.51-fold increase of triptolide in the transgenic lines. These results indicated that this efficient A. tumefaciens mediated genetic transformation provides a useful tool for future T. wilfordii genetic engineering studies to enhance the accumulation of triptolide in this economically important medicinal plant.

Unpruning improvement the quality of tea through increasing the levels of amino acids and reducing contents of flavonoids and caffeine.

Tea tree [Camellia sinensis var. sinensis or assamica (L.) O. Kuntze], an important crop worldwide, is usually pruned to heights of 70 to 80 cm, forming pruned tea tree (PTT) plantations. Currently, PTTs are transformed into unpruned tea tree (UPTT) plantations in Yunnan, China. This has improved the quality of tea products, but the underlying reasons have not been evaluated scientifically. Here, 12 samples of sun-dried green teas were manufactured using fresh leaves from an UPTT and the corresponding PTT. Using sensory evaluation, it was found that the change reduced the bitterness and astringency, while increasing sweetness and umami. Using high performance liquid chromatography detection showed that the contents of free amino acids (theanine, histidine, isoleucine and phenylalanine) and catechin gallate increased significantly (P < 0.05), whereas the content of alanine decreased significantly (P < 0.05). A liquid chromatography–mass spectrometry-based metabolomics analysis showed that the transformation to UPTT significantly decreased the relative levels of the majority of flavonols and tannins (P < 0.05), as well as γ-aminobutyric acid, caffeine and catechin (epigallocatechin, catechin, epigallocatechin gallate, gallocatechin gallate), while it significantly increased the relative contents of catechins (gallocatechin, epicatechin, epicatechin gallate and catechin gallate), phenolic acids and some amino acids (serine, oxidized glutathione, histidine, aspartic acid, glutamine, lysine, tryptophan, tyramine, pipecolic acid, and theanine) (P < 0.05). In summary, after transforming to UPTT, levels of amino acids, such as theanine increased significantly (P < 0.05), which enhanced the umami and sweetness of tea infusions, while the flavonoids (such as kaempferol, myricetin and glycosylated quercetin), and caffeine contents decreased significantly (P < 0.05), resulting in a reduction in the bitterness and astringency of tea infusions and an increase in tea quality.

Study on microbial community of "green-covering" Tuqu and the effect of fortified autochthonous Monascus purpureus on the flavor components of light-aroma-type Baijiu.

"Green-covering" Tuqu (TQ), as one of Xiaoqu, is a special fermentative starter (also known as Jiuqu in Chinese) that originated in southern China and is characterized by a layer of green mold covering (Aspergillus clavatus) the surface and (sometimes) with a red heart. It plays a vital role in producing light-aroma-type Baijiu (LATB). However, to date, the microbiota that causes red heart of TQ remain largely unexplored, and it is still unclear how these microbiota influence on the quality of LATB. In this study, two types of TQ, one with a red heart (RH) and another with a non-red heart (NRH), were investigated by high throughput sequencing (HTS) and directional screening of culture-dependent methods. The obtained results revealed the differences in the microbial communities of different TQ and led to the isolation of two species of Monascus. Interestingly, the results of high performance liquid chromatography (HPLC) detection showed that citrinin was not detected, indicating that Monascus isolated from TQ was no safety risk, and the contents of gamma-aminobutyric acid in the fermented grains of RH were higher than that of NRH during the fermentation. Selecting the superior autochthonous Monascus (M1) isolated from the TQ to reinoculate into the TQ-making process, established a stable method for producing the experimental "red heart" Tuqu (ERH), which confirmed that the cause of "red heart" was the growth of Monascus strains. After the lab-scale production test, ERH increased ethyl ester production and reduced higher alcohols production. In addition, Monascus had an inhibitory effect on the growth of Saccharomyces and Aspergillus. This study provides the safe, health-beneficial, and superior fermentation strains and strategies for improving the quality of TQ and LATB.