Ultra-performance Liquid Chromatography-high Resolution Research Articles

Elevated Temperature Effects on Protein Turnover Dynamics in Arabidopsis thaliana Seedlings Revealed by 15N-Stable Isotope Labeling and ProteinTurnover Algorithm

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for the cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ultra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22 °C and 30 °C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adaptations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mitochondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis highlights the intricate balance between proteome stability and adaptability, advancing our understanding of plant responses to heat stress and supporting the development of improved thermotolerant crops.

Surface multi-walled carbon nanotube modified quaternary amine-functionalized polymers for purification and determination of glyphosate and its four metabolites in plasma samples

The present study focused on the pretreatment and detection of GLY and its four metabolites AMPA, N-acetyl AMPA, N-methyl GLY and N-acetyl GLY in plasma samples. Multi-walled carbon nanotube-modified quaternary amine-functionalized polymers (QA-PDNV@MWCNTs) were synthesized in a controlled manner by self-assembly, and its morphology and composition were extensively characterized. The QA-PDNV@MWCNTs microspheres were then used as an SPE adsorbent for the preparation and rapid determination of GLY and its four metabolites in plasma samples combined with ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The SPE conditions based on QA-PDNV@MWCNTs were optimized for GLY and its metabolites to obtain the best purification efficiency. The experimental results show that when the adsorbent contains 8% MWCNTs, it can balance the adsorption of target analytes and the purification performance of the adsorbent for impurities. In addition, this study compared the QA-PDNV@MWCNTs based SPE method with the commercial Waters Oasis MAX SPE cartridge and the results showed that the developed method in this study has better resistance to matrix interference. Under optimal conditions, the recoveries of GLY and its metabolites spiked in plasma were 82.6-99.4% with relative standard deviations (RSDs) of 1.0-7.8%. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥9) of the method were 0.05-0.33 μg/L and 0.15-1.00 μg/L, respectively. Finally, the developed QA-PDNV@MWCNTs based SPE-UPLC-HRMS method was used to confirm GLY poisoning not only on the basis of the detection of the GLY prototype, but also on the basis of its four metabolites.

In vivo pharmacokinetic study of vanillic acid in monocrotaline-induced pulmonary arterial hypertension rats and its tissue distribution.

Vanillic acid (VA) is a bioactive chemical present in many food plants and fruits. It has been shown to have a protective effect on pulmonary tissues in monocrotaline-induced pulmonary arterial hypertension, as well as an intervention effect on right ventricular remodeling. The purpose of this study was to develop and test a reliable method for assessing VA utilizing ultra-performance liquid chromatography-high resolution mass spectrometry using caffeic acid as the internal standard. Across diverse substrates, the correlation coefficient for VA ranged from 0.9992 to 0.9995. The method's intraday precision was <13.53% (RSD), and its accuracy (RE) ranged from -9.88 to 4.35%. The precision across days was <13.69% (RSD), while the accuracy ranged from 2.16 to 10.94% (RE). The extraction recoveries ranged from 80.30 to 118.81%, with a lower limit of quantification of 20 ng/mL. The approach was successfully applied to pharmacokinetic and tissue distribution studies of VA in rat plasma after gavage administration, and the pharmacokinetic parameters of VA in the plasma of the monocrotaline-induced pulmonary arterial hypertension were significantly different from those of the control group.

Detection of three metabolites of xylene in urine samples by solid-phase extraction coupled with liquid chromatography-mass spectrometry

Objective: To establish a method for the rapid determination of the three metabolites of xylene, 2-methylmarmaluronic acid, 3-methylmarmaluronic acid and 4-methylmarmaluronic acid, in urine of occupationally exposed workers by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) . Methods: In July 2022, urine samples were diluted and extracted with pH=6.86 phosphate cache solution, cleaned up by a MAX solid-phase extraction (SPE) column and separated by an Accucore Ph/Hexyl column (100 mm×2.1 mm, 2.6 μm) with a gradient of 5 mmol/L ammonium formate-0.1% formic acid aqueous solution and methanol as mobile phases. The analysis was carried out in electrospray ionization mode and full mass-data dependent secondary mass spectrometry mode, and quantified by external standard method. The characteristics of each index of this method were analyzed. Results: A good linearity was obtained in the concentration range of 1.0-200.0 μg/L for 2-methylmuramic acid, 3-methylmuramic acid and 4-methylmuramic acid with the correlation coefficients of 0.9979-0.9993. The limits of detection of the method were 0.18-0.24 μg/L. While the spiked recoveries at the three concentrations (1.0 μg/L, 100.0 μg/L, and 180.0 μg/L) were in the range of 83.0%-93.7%, with the relative standard deviations of 2.2%-7.9%. Conclusion: The UPLC-HRMS method is simple, rapid, sensitive and accurate, and is suitable for the simultaneous determination of the three metabolites of xylene in the urine of occupationally exposed workers.

LC-MS-based serum metabolomics analysis for the screening and monitoring of colorectal cancer.

Colorectal Cancer (CRC) is a prevalent digestive system tumour with significant mortality and recurrence rates. Serum metabolomics, with its high sensitivity and high throughput, has shown potential as a tool to discover biomarkers for clinical screening and monitoring of the CRC patients. Serum metabolites of 61 sex and age-matched healthy controls and 62 CRC patients (before and after surgical intervention) were analyzed using a ultra-performance liquid chromatography-high resolution mass spectrometer (UPLC-MS). Statistical methods and pathway enrichment analysis were used to identify potential biomarkers and altered metabolic pathways. Our analysis revealed a clear distinction in the serum metabolic profile between CRC patients and healthy controls (HCs). Pathway analysis indicated a significant association with arginine biosynthesis, pyrimidine metabolism, pantothenate, and CoA biosynthesis. Univariate and multivariate statistical analysis showed that 9 metabolites had significant diagnostic value for CRC, among them, Guanosine with Area Under the Curve (AUC) values of 0.951 for the training group and0.998 for the validation group. Furthermore, analysis of four specific metabolites (N-Phenylacetylasparticacid, Tyrosyl-Gamma-glutamate, Tyr-Ser and Sphingosine) in serum samples of CRC patients before and after surgery indicated a return to healthy levels after an intervention. Our results suggest that serum metabolomics may be a valuable tool for the screening and monitoring of CRC patients.

Characterization of a novel endo-1,3-fucanase from marine bacterium Wenyingzhuangia fucanilytica reveals the presence of diversity within glycoside hydrolase family 168

Sulfated fucans attract increasing research interests in recent decades for their various physiological activities. Fucanases are indispensable tools for the investigation of sulfated fucans. Herein, a novel GH168 family endo-1,3-fucanase was cloned from the genome of marine bacterium Wenyingzhuangia fucanilytica. The expressed protein Fun168D was a processive endo-acting enzyme. Ultra performance liquid chromatography-high resolution mass spectrum and NMR analyses revealed that the enzyme cleaved the α-1 → 3 bonds between α-l-Fucp(2OSO3−) and α-l-Fucp(2OSO3−) in sulfated fucan from Isostichopus badionotus, and α-1 → 3 bonds between α-l-Fucp(2OSO3−) and α-l-Fucp(2,4OSO3−) in sulfated fucan from Holothuria tubulosa. Fun168D would prefer to accept α-l-Fucp(2,4OSO3−) than α-l-Fucp(2OSO3−) at subsite +1, and could tolerate the absence of fucose residue at subsite +2. The novel cleavage specificity and hydrolysis pattern revealed the presence of diversity within the GH168 family, which would facilitate the development of diverse biotechnological tools for the molecule tailoring of sulfated fucan.

106 Glucocorticoid and progestogen profiling in bovine skimmed milk, saliva and plasma using ultra-performance liquid chromatography high resolution mass spectrometry (UHPLC-HR-MS)

106 Glucocorticoid and progestogen profiling in bovine skimmed milk, saliva and plasma using ultra-performance liquid chromatography high resolution mass spectrometry (UHPLC-HR-MS)

Sulfated fucan could serve as a species marker of sea cucumber with endo-1,3-fucanase as the essential tool

In the past few decades, sulfated fucan from sea cucumber had attracted considerable interest owing to its abundant physiological activities. Nevertheless, its potential for species discrimination had not been investigated. Herein, particular attention was given to sea cucumber Apostichopus japonicus, Acaudina molpadioides, Holothuria hilla, Holothuria tubulosa, Isostichopus badionotus and Thelenota ananas to examine the feasibility of sulfated fucan as a species marker of sea cucumber. The enzymatic fingerprint suggested that sulfated fucan exhibited significant interspecific discrepancy and intraspecific stability, which revealed that sulfated fucan could serve as the species marker of sea cucumber, by utilizing the overexpressed endo-1,3-fucanase Fun168A and the ultra-performance liquid chromatography-high resolution mass spectrum. Moreover, oligosaccharide profile of sulfated fucan was determined. The oligosaccharide profile combined with hierarchical clustering analysis and principal components analysis further confirmed that sulfated fucan could serve as a marker with a satisfying performance. Besides, load factor analysis showed that the minor structure of sulfated fucan also contributed to the sea cucumber discrimination, besides the major structure. The overexpressed fucanase played an indispensable role in the discrimination, due to its specificity and high activity. The study would lead to a new strategy for species discrimination of sea cucumber based on sulfated fucan.

Characterization and anti-aging effects of Opuntia ficus-indica (L.) Miller extracts in a D-galactose-induced skin aging model.

Opuntia ficus-indica (L.) Miller (OFI), belonging to the family Cactaceae, is widely cultivated not only for its delicious fruits but also for its health-promoting effects, which enhance the role of OFI as a potential functional food. In this study, the in vitro collagenase and elastase enzyme inhibitory effects of extracts from different parts of OFI were evaluated. The most promising extracts were formulated as creams at two concentrations (3 and 5%) to investigate their effects on a D-galactose (D-gal)-induced skin-aging mouse model. The ethanolic extracts of the peel and cladodes exhibited the highest enzyme inhibitory effects. Cream made from the extract of OFI peel (OP) (5%) and cream from OFI cladodes extract (OC) (5%) significantly decreased the macroscopic aging of skin scores. Only a higher concentration (5%) of OC showed the normalization of superoxide dismutase (SOD) and malondialdehyde (MDA) skin levels and achieved significant improvements as compared to the vitamin E group. Both OC and OP (5%) showed complete restoration of the normal skin structure and nearly normal collagen fibres upon histopathological examination. The Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-ESI-TOF-MS) metabolite profiles revealed the presence of organic acids, phenolic acids, flavonoids, betalains, and fatty acids. Flavonoids were the predominant phytochemical class (23 and 22 compounds), followed by phenolic acids (14 and 17 compounds) in the ethanolic extracts from the peel and cladodes, respectively. The anti-skin-aging effects could be attributed to the synergism of different phytochemicals in both extracts. From these findings, the OFI peel and cladodes as agro-waste products are good candidates for anti-skin-aging phytocosmetics.

Determination of Haloacetic Acids, Disinfection Byproducts, in Tap Water with Reversed-Phase Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry

To establish a method for quantitative analysis of haloacetic acids (HAAs), disinfection byproducts, in tap water with reversed-phase ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry. Tap water samples were collected and 0.70 g/L ascorbic acid was added to eliminate residual chlorine. Then, the water samples were directly injected into the instrument for analysis after filtration. After separation on a pentafluorobenzene (PFP) column with an inner diameter of 1.0 mm at a higher linear velocity and a lower volume flow rate compared with those of a narrow-bore column, nine HAAs, namely, monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), trichloroacetic acid (TCAA), bromodichloroacetic acid（BDCAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were examined by negative electrospray ionization and full MS/dd-MS 2 acquisition mode. In order to adjust for the matrix effect, matrix matching calibration curves were used to quantitate the nine HAAs. Good linearity was obtained for each of the nine HAAs within their respective linear ranges. The detection limits and quantification limits of the method were 0.020-1.0 μg/L and 0.060-3.0 μg/L. The recoveries were 69.8%-119%. The proposed method showed strengths in separation speed and qualitative accuracy. It did not require for complicated pretreatment procedures and can meet the need of tap water sample analysis.

Rapid microwave synthesis of PCN-134-2D for singlet oxygen based-oxidative degradation of ranitidine under visible light: Mechanism and toxicity assessment

Two-dimensional porphyrin metal–organic frameworks (2D-PMOFs) showed potential applications in the field of photocatalysis. In this study, PCN-134-2D was rapidly prepared by the microwave-assisted solvothermal (MS) method for photocatalytic degradation of ranitidine (RAN), and exhibited enhanced photocatalytic performance due to the augmentation of the TCPP/BTB ratio (0.55). 93.1% of RAN could be effectively photodegraded in 120 min through PCN-134-2D under visible light. During the photosensitizing process, singlet oxygen (1O2) was proved to be the major reactive oxygen species (ROS) and showed the unique ability of non-radical resistance to conquer the inhibiting effects of inorganic anions (Cl−, HCO3−, NO3−, SO42−, H2PO4−) and natural organic matter (humic acid [HA]). The effects of practical reaction parameters were studied in detail. Light intensity and oxygen content were demonstrated as the key factors to photosensitization. The final removal rates of RAN in lake waters and river waters were 84.7% and 84.1% under the solar light. Meanwhile, the total organic carbon (TOC) removal rates were 30.4% and 31.5%, respectively. Ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to analyze the photodegradation products of the target RAN, and 17 intermediate compounds were obtained in ESI positive mode. The degradation pathways of RAN by 1O2 were proposed as C–N bond and C–S bond cleavage. Ecotoxicity assessment by the Ecological Structure Activity Relationships (ECOSAR) program indicated that most of the intermediate products were “not harmful” to fish, daphnid and green algae. The inhibition rate of Vibrio fischeri bioluminescence was approximately reduced 62% after RAN was treated by PCN-134-2D in 120 min.

Comprehensive analysis of chemical constituents of tea flowers by ultra-performance liquid chromatography-high resolution mass spectrometry combined with integrated filtering strategy

茶树花与茶鲜叶同为茶树的生物产出,但茶树花往往被视为茶叶生产过程中的废物被舍弃,造成了茶树花资源的极大浪费。目前对于茶树花中化学成分的分析主要集中在氨基酸、茶多酚等单一类型化学成分上,对于茶树花中多类化学成分的同时分析仍鲜见报道。研究者对于茶树花中所含化学成分的种类和含量不完全清楚,成为制约茶树花深度开发与利用的重要原因。该研究采用超高效液相色谱-高分辨质谱联用技术(UPLC-HRMS)对茶树花中的化学成分进行检测,结合氮规则过滤(NRF)、质量亏损过滤(MDF)和诊断碎片离子过滤(DFIF)的整合过滤策略(IFS)筛选目标化学成分的特征质谱,并利用化学成分的色谱保留时间、一级质谱、二级质谱等信息对化学成分进行定性分析。共定性出茶树花中6大类共137种化学成分,包括3种生物碱、38种黄酮、31种酚酸及其衍生物、37种儿茶素及其衍生物、18种氨基酸以及10种其他类成分。采用内标法对所有定性出的137种化学成分进行定量分析,结果表明,茶树花中6类化学成分含量从高到低依次为氨基酸(9371.42 μg/g)、儿茶素及其衍生物(9068.43 μg/g)、酚酸及其衍生物(8696.92 μg/g)、生物碱(4392.52 μg/g)、黄酮(1192.88 μg/g)、其他类成分(139.94 μg/g)。该研究采用质量控制样本评价仪器的稳定性和检测数据的重复性,9种代表性化学成分的相对标准偏差在2.11%~12.17%范围内,表明仪器的稳定性和检测数据重复性良好。同时,选取绿原酸类成分以及糖基化槲皮素类成分作为代表性成分,说明了整合过滤策略提取目标类型化学成分的全过程。该研究全面揭示了茶树花中化学成分的种类和含量,可为茶树花的深度开发和利用提供有价值的信息和数据参考。

Analysis of glycan ratio of Chinese hamster ovary cell-cetuximab antigen-binding segment via rapid enzyme digestion with endo-β-N-acetylglucosaminidase F

西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min, Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。

Effects of fruit bagging on the physiochemical changes of grapefruit (Citrus paradisi)

Abstract Fruit bagging is a commonly used cultivation measure to protect citrus fruit from insects and adverse environments. The present study aimed to comprehensively investigate the effects of bagging on the physiochemical characteristics of grapefruit. The grapefruit were bagged at approximately 110–120 d after anthesis with a one-layer kraft paper bag with black coating inside (SL), a double-layer kraft paper bag with one black paper as the inner layer (DL), and a three-layer kraft paper bag with two black papers as inner layers (TL), respectively. Ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) technique was used to identify a total of 19 flavonoids, 2 phenylpropanoids, 9 coumarins, and 5 limonoids. By using UPLC, 50 carotenoids were identified. Gas chromatography–mass spectrometry was used to identify 3 soluble sugars, 3 organic acids, and 11 amino acids. In the quantitated components in the peel (albedo and flavedo), the chlorophylls and the carotenoids components (such as luteoxanthin, violaxanthin, 9-cis-violaxanthin, xanthophyll, zeaxanthin and β-carotene) were significantly downregulated by bagging, causing the surface color of bagged fruit to turn yellow earlier but paler than that of the unbagged control, particularly in the three-layer kraft bag treatment. Unlike the peel, the color and the carotenoid content of the juice sacs were less affected. The physiochemical compounds other than pigments, including soluble sugars, organic acids, amino acids, flavonoids, coumarins and limonoids, were minimally affected by bagging treatments. Our results indicated that bagging at approximately 110–120 d after anthesis exerted influence mainly on peel color, but less on sugars, acids, amino acids, flavonoids, limonoids and coumarins of grapefruit.

Placental metabolic profiling in gestational diabetes mellitus: An important role of fatty acids.

AimGestational diabetes mellitus (GDM) is the most common metabolic disorder during pregnancy. Accumulating studies have reported metabolites that are significantly associated with the development of GDM. However, studies on the metabolism of placenta, the most important organ of maternal‐fetal energy and material transport, are extremely rare. This study aimed to identify and discuss the relationship between differentially expressed metabolites (DEM) and clinical parameters of the mothers and newborns.MethodsIn this study, metabolites from 63 placenta tissues (32 GDM and 31 normal controls) were assayed by ultra‐performance liquid chromatography‐high resolution mass spectrometry (UPLC‐HRMS).ResultsA total of 1297 annotated metabolites were detected, of which 87 significantly different in GDM placenta. Lipids and lipid‐like molecules accounted for 62.1% of DEM as they were significantly enriched via the “biosynthesis of unsaturated fatty acids” and “fatty acid biosynthesis” pathways. Linoleic acid and α‐linolenic acid appeared to be good biomarkers for the prediction and diagnosis of GDM. In addition, the level of PC(14:0/18:0) was negatively correlated with neonatal weight. 14 metabolites significantly different in male and female offspring, with the most increase in female newborns.ConclusionEven if maternal blood glucose level is well controlled, there are still metabolic abnormalities in GDM. Lipids and lipid‐like molecules were the main differential metabolites, especially unsaturated fatty acids.

Effect of red light on the composition of metabolites in tea leaves during the withering process using untargeted metabolomics.

Red light withering significantly improves the sensory flavor qualities of tea, although changes in metabolites during this process have not been systematically studied until now. The present study comprehensively analyzes metabolites in withered tea leaves at 2-h intervals up to 12 h under red light (630 nm) and dark conditions using ultra performance liquid chromatography-high resolution mass spectrometry (untargeted metabolomics). Ninety-four non-volatile compounds are identified and relatively quantified, including amino acids, catechins, dimeric catechins, flavonol glycosides, glycosidically-bound volatiles, phenolic acids and nucleosides. The results show that amino acids, catechins and dimeric catechins are most affected by red light treatment. Ten free amino acids, theaflavins and theasinensin A increase after red light irradiation, whereas epigallocatechin gallate and catechin fall. The present study provides a comprehensive and systematic profile of the dynamic effects of red light on withering tea and a rationale for its use in tea processing quality control. © 2021 Society of Chemical Industry.

Transcriptomic and Weighted Gene Co-expression Correlation Network Analysis Reveal Resveratrol Biosynthesis Mechanisms Caused by Bud Sport in Grape Berry

Resveratrol is a natural polyphenol compound produced in response to biotic and abiotic stresses in grape berries. However, changes in resveratrol caused by bud sport in grapes are scarcely reported. In this study, trans-resveratrol and cis-resveratrol were identified and quantified in the grape berries of ‘Summer Black’ and its bud sport ‘Nantaihutezao’ from the veraison to ripening stages using ultra performance liquid chromatography-high resolution tandem mass spectrometry (UPLC-HRMS). We found that bud sport accumulates the trans-resveratrol earlier and increases the contents of cis-resveratrol in the earlier stages but decreases its contents in the later stages. Simultaneously, we used RNA-Seq to identify 51 transcripts involved in the stilbene pathways. In particular, we further identified 124 and 19 transcripts that negatively correlated with the contents of trans-resveratrol and cis-resveratrol, respectively, and four transcripts encoding F3'5'H that positively correlated with the contents of trans-resveratrol by weighted gene co-expression network analysis (WGCNA). These transcripts may play important roles in relation to the synergistic regulation of metabolisms of resveratrol. The results of this study can provide a theoretical basis for the genetic improvement of grapes.

Simultaneous determination of 29 pesticides residues in bayberry by pass-through solid-phase extraction and ultra-performance liquid chromatography-high resolution mass spectrometry

建立了基于PRiME HLB通过型固相萃取净化-超高效液相色谱-高分辨质谱法(UPLC-HRMS)快速准确测定杨梅中29种常见农药残留的检测方法。杨梅样品经乙腈涡旋提取、盐析和PRiME HLB固相萃取净化后,以5 mmol/L乙酸铵水溶液和乙腈溶液作为流动相在Waters ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)上进行色谱分离,采用正离子电喷雾离子化模式(ESI+)和一级全扫描-数据依赖二级质谱扫描模式(Full mass-ddMS2),基质匹配外标法定量分析。该研究首先优化了液相色谱条件,重点考察了Waters ACQUITY UPLC HSS T3色谱柱和Waters ACQUITY UPLC BEH C18色谱柱对29种农药色谱行为的影响,结果表明Waters ACQUITY UPLC HSS T3色谱柱相比后者具有更强的色谱保留能力;流动相优化结果显示,相比于乙腈-甲酸水溶液体系和乙腈-甲酸-乙酸铵水溶液体系,乙腈-乙酸铵水溶液体系作为流动相时29种农药普遍具有更佳的色谱保留,部分农药的质谱响应有了显著的提高。此外,该研究通过考察3种不同净化方法的基质效应以优化杨梅中29种农药残留检测,实验结果表明,相比于GCB SPE和QuEChERS法两种净化方式,PRiME HLB法对于杨梅提取液具有较好的基质净化能力。在最佳实验条件下,29种农药在1.0~200.0 μg/L范围内呈现良好的线性关系(线性相关系数R2>0.999),方法检出限为2.0 μg/kg;低(6 μg/kg)、中(200 μg/kg)、高(400 μg/kg)3个加标水平下,29种农药的加标回收率为69.2%~135.6%,相对标准偏差为0.7%~14.6%。该方法具有快速、简便、灵敏和准确等优点,适用于理化实验室大批量样品的日常监测。

Characterization of phase I and phase II metabolites of hop (Humulus lupulus L.) bitter acids: In vitro and in vivo metabolic profiling by UHPLC-Q-Orbitrap

Bitter acids are a class of prenylated phloroglucinol derivatives present in Humulus lupulus L., known for their multiple healthy properties, nevertheless, research regarding their metabolism and stability is lacking. This study was aimed to elucidate the metabolic stability of hop α- and β-acids and characterize I and II phase metabolites in vitro and in vivo. For this purpose, an ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method was developed and validated. Mice liver microsomes were used to assess metabolic stability; in vitro t1/2 and clearance values were calculated, showing a moderate metabolism for α-acids (avgt1/2: 120.01 min, avgCLint 11.96 μL/min/mg), while β-acids were metabolized faster (avgt1/2: 103.01 min, avgCLint: 13.83 μL/min/mg). I and II phase metabolites were characterized both in in vitro, and in vivo, in mouse plasma and urine after oral administration. A combined full scan/data dependent/precursor ion list-triggered neutral loss (FS/dd-MS2/PIL-tNL) strategy was used to detect unknown and expected metabolites. As a result, 33 compounds were detected, including novel metabolites, such as 9 potential oxidized metabolites of humulones (M6-M14), and 10 glucuronide conjugates of α-acids, comprising 7 glucuronide derivatives of oxidized phase I metabolites (M26-M32). The proposed method extends the current knowledge regarding metabolization of hop α- and β-acids and could be applied for the comprehension of the metabolic fate of this class of compounds in different species, as well as for in vivo pharmacokinetic studies.

Urine metabolomics analysis based on ultra performance liquid chromatography-high resolution mass spectrometry combined with osmolality calibration sample concentration variability

尿液是代谢组学研究中主要关注的体液样本之一。尿液样本中的代谢物浓度受饮食、疾病等因素影响变异较大,这极大阻碍了高质量组学数据的获取和可靠生物标志物的鉴定。研究为克服尿液样本的浓度变异性,在原始数据采集前,根据样本渗透压的大小,针对性地调整进样量或者稀释样本,从而确保代谢组学分析样本的渗透压与进样量的乘积相当,再经超高效液相色谱-高分辨质谱技术(UPLC-HRMS)分析,采用总离子丰度或总有用峰面积(MSTUS)对数据集进行归一化处理。研究利用临床样本及其梯度稀释的溶液,对该方法与现有研究普遍使用的方法进行了比较,随后通过先天性肾积水患者及健康志愿者的尿液样本做了进一步的方法学验证。数据集经校正后,峰面积RSD<30%的提取峰数量增加,主成分分析结果较校正前有更高的组内聚集和组间分群效应,正交偏最小二乘判别分析的统计模型更不易过拟合。与肌酐比较,渗透压值与质谱信号间呈现了更好的线性关系。以上结果表明,数据采集前通过样本渗透压进行校正,能有效消除因样本本身代谢物浓度变化引起的组内差异,提高方法的重复性和统计模型的可靠度。以渗透压为基准的校正策略,比肌酐校正法适用范围更广,结果也更准确。研究可对后续各类来源的尿液代谢组学研究提供数据归一化的指导和参考。