Linear Ion trap-Orbitrap Mass Research Articles

Multi-omics reveals bufadienolide Q-markers of Bufonis Venenum based on antitumor activity and cardiovascular toxicity in zebrafish

BackgroundBufonis Venenum (BV) is a traditional animal-based Chinese medicine with therapeutic effects against cancer. However, its clinical use is significantly restricted due to associated cardiovascular risks. BV's value in China's market is typically assessed based on “content priority,” focusing on indicator components. However, these components of BV possess both antitumor activity and toxicity, and the correlation between the antitumor activity and toxicity of BV has not yet been elucidated. PurposeThis study employs an integrated multi-omics approach to identify bufadienolide Q-markers and explore the correlation between BV's antitumor activity and toxicity. The aim is to establish a more comprehensive method for BV's quality. MethodsNormal zebrafish and HepG2 xenograft zebrafish were chosen as activity and toxicity evaluation models. Ultra-high performance liquid chromatography (UHPLC) coupled with a linear ion trap orbitrap (LTQ-Orbitrap) mass spectrometry was used to quantify eight batches of BV and key “toxic and effective” components were screened out. Transcriptomic and metabolomic analyses were performed to elucidate the regulatory mechanisms underlying the antitumor activity and cardiovascular toxicity of the key components in BV. ResultsEight key “toxic and effective” compounds were identified: resibufogenin, cinobufagin, arenobufagin, bufotalin, bufalin, gamabufotalin, desacetylcinobufagin, and telocinobufagin. The findings showed that bufalin and cinobufagin interfered with calcium homeostasis through CaV and CaSR, induced cardiotoxicity, and upregulated CASP9 to activate myocardial cell apoptosis. However, desacetylcinobufagin exhibited greater potential in terms of anti-tumor effects. Combining the results of untargeted and targeted metabolomics revealed that desacetylcinobufagin could have a callback effect on differential lipids and correct abnormal energy and amino acid metabolism caused by cancer, similar to cinobufagin and bufalin. Microscale thermophoresis (MST) ligand binding measurements also showed that the binding of desacetylcinobufagin to GPX4 has a more potent ability to induce ferroptosis in tumor cells compared to cinobufagin. ConclusionAn innovative evaluation method based on the zebrafish was developed to investigate the relationship between the toxicity and efficacy of BV. This study identified toxicity and activity Q-markers and explored the mechanism between the two effects of BV. The research data could offer valuable insights into the efficacy of BV. Additionally, desacetylcinobufagin, an active ingredient with low toxicity, was found to enhance the quality of BV.

A comprehensive strategy based on ultra-high performance liquid chromatography with diode array detector fingerprinting and multi-component ultra-performance liquid chromatography with tandem mass spectrometry technology for quality control of Jiawei Huoxiang Zhengqi Pill.

Jiawei Huoxiang Zhengqi Pill (JHZP) is a commonly used Chinese patent medicine for the clinical treatment of headache, dizziness, chest tightness as well as abdominal distension, and pain caused by wind-cold flu. In this study, a comprehensive strategy combining ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD) fingerprinting and multi-component quantitative analysis was established and validated for quality evaluation of JHZP. A total of 49 characteristic common peaks were selected in a chromatographic fingerprinting study to assess the similarity of 15 batches of JHZP. Furthermore, 109 compounds were identified or preliminarily identified from JHZP by coupling with an advanced hybrid linear ion trap-Orbitrap mass spectrometer. For quantification, the optimized ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was employed for the simultaneous determination of 13 target compounds within 12min. The sensitivity, precision, reproducibility, and accuracy of the method were satisfactory. This validated UPLC-MS/MS method was successfully applied to analyzing 15 batches of JHZP. The proposed comprehensive strategy combining UHPLC-DAD fingerprinting and multi-component UPLC-MS/MS analysis proved to be highly efficient, accurate, and reliable for the quality evaluation of JHZP, which can be considered as a reference for the overall quality evaluation of other Chinese herbal formulations.

Dissecting new lipids and their composition in herbal tea using untargeted LC/MS

Herbal teas and beverages have gained global attention because they are rich in natural bioactive compounds, which are known to have diverse biological effects, including antioxidant and anticarcinogenic properties. However, the lipidomic profiles of herbal teas remain unclear. In this study, we applied an untargeted lipidomics approach using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry to comprehensively profile, compare, and identify unknown lipids in four herbal teas: dokudami, kumazasa, sugina, and yomogi. A total of 341 molecular species from five major classes of lipids were identified. Multivariate principal component analysis revealed distinct lipid compositions for each of the herbs. The fatty acid α-linolenic acid (FA 18:3) was found to be abundant in kumazasa, whereas arachidonic acid (FA 20:4) was the most abundant in sugina. Interestingly, novel lipids were discovered for the first time in plants; specifically, short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) with 4-hydroxy phenyl nonanoic acid as the structural core. This study provides insight into the lipidomic diversity and potential bioactive lipid components of herbal teas, offering a foundation for further research into their health-promoting properties and biological significance.

Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

A comprehensive quality evaluation for Huangqi Guizhi Wuwu decoction by integrating UPLC-DAD/MS chemical profile and pharmacodynamics combined with chemometric analysis

Ethnopharmacological relevanceHuangqi Guizhi Wuwu Decoction (HGWD), a classical Chinese formula originally recorded in Jin Kui Yao Lue, was used for the treatment of human “blood impediment” (a type of “Bi” syndrome). In clinical practice, HGWD has been applied to treat rheumatoid arthritis (RA). Aim of the studyThe characterization of chemical markers reflecting both efficacy and chemical characteristics is of great significance for TCM quality control. With the anti-RA effects of HGWD as an example, the aim of this study was to develop a comprehensive strategy combining the overall chemical profile and biological activity data to identify chemical markers. Materials and methodsFirst, an ultra-performance liquid chromatography-diode array detector (UPLC-DAD) fingerprint was established and validated to evaluate the holistic quality of HGWD of different origins. Characteristic markers associated with HGWD from different geographical origins were screened by a combination of UPLC-DAD fingerprint and chemometrics methods. Second, the chemical profiles of the 15 batches of HGWD samples were characterized by UPLC coupled tohybrid linear ion trap-Orbitrap mass spectrometry (UPLC-HRMS). The in vitro anti-RA activities of the 15 HGWD samples were then evaluated. Third, spectrum-effect relationship analysis was performed to identify bioactive compounds that could potentially be used as quality markers. Finally, a UPLC-triple quadrupole tandem mass spectrometry approach was optimized and established for quantitative analysis of the characteristic and quality markers in 15 batches of HGWD. ResultsIn total, 30 common peaks were assigned in the UPLC-DAD fingerprint. Nine peaks were recognized and considered characteristic markers: protocatechuic acid, coumarin, cinnamic acid, oxypaeoniflorin, paeoniflorin, calycosin, formononetin, catechin, and albiflorin. Furthermore, ninety-five common compounds were identified in the UPLC-HRMS chemical profile. The pharmacological analysis indicated that the anti-RA activities of the 15 HGWD samples were vastly different. The spectrum-effect relationship analysis revealed 30 potential bioactive constituents positively correlated with anti-RA activity. Among them, five compounds with relative amounts >1%, paeoniflorin, astragaloside IV, hexahydrocurcumin, formononetin and calycosin-7-glucoside, were selected as quality markers, and their activity was verified in LPS-induced RAW264.7 macrophages. Finally, the above 12 representative components were simultaneously quantified in the 15 batches of HGWD samples. ConclusionCombining a holistic chemical profile with representative component evaluation, this systematic strategy could be a reliable and effective method to improve quality evaluations of HGWD.

Comprehensive online two-dimensional liquid chromatography coupled to hybrid linear ion trap Orbitrap mass spectrometry with optimized dilution and modulation for analysis of Huangqi Guizhi Wuwu decoction.

Huangqi Guizhi Wuwu decoction (HGWD), as a classical formula, has been used for thousands of years in China. In this work, a comprehensive strategy was proposed for characterizing the chemical profile of HGWD based on online two-dimensional hydrophilic interaction and reversed-phase liquid chromatography coupled with hybrid linear ion trap-Orbitrap mass spectrometry (online HILIC × RP-ESI/HRMS/MSn). The compounds in HGWD were first separated by the combined use of an XBridge amide column (150 × 4.6mm, 3.5μm) and Accucore C18 column (50mm × 4.6mm, 2.6μm). Modulation with assistant technology, including trap columns and online dilution, was optimized and developed to decrease potential analyte loss and improve the resolution of the system. Subsequently, the accurate mass was determined by high-resolution Orbitrap and MSn fragment data by a hybrid linear ion trap (LTQ). In total, 170 chemical constituents were unambiguously identified or tentatively characterized in both positive and negative ion modes. Our study demonstrated that the proposed online HILIC × RP system coupled to the LTQ-Orbitrap MS platform is an efficient analytical technique for characterizing the chemical profile of multicomponent systems.

Lipid Composition Analysis and Characterization of Acyl Sterol Glycosides in Adzuki and Soybean Cultivars by Non-Targeted LC-MS.

Beans, a globally significant economic and nutritional food crop, are rich in polyphenolic chemicals with potential health advantages, providing high protein, fiber, minerals, and vitamins. However, studies on the global profiling of lipids in beans are limited. We applied a non-targeted lipidomic approach based on high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (HPLC/LTQ-Orbitrap-MS) to comprehensively profile and compare the lipids in six distinct bean cultivars, namely, adzuki red beans-adzuki cultivar (ARB-AC), adzuki red beans-Benidainagon cultivar (ARB-BC), adzuki red beans-Erimoshouzu cultivar (ARB-EC), soybean-Fukuyutaka cultivar 2021 (SB-FC21), soybean-Fukuyutaka cultivar 2022 (SB-FC22), and soybean-Oosuzu cultivar (SB-OC). MS/MS analysis defined 144 molecular species from four main lipid groups. Multivariate principal component analysis indicated unique lipid compositions in the cultivars except for ARB-BC and ARB-EC. Evaluation of the concentrations of polyunsaturated fatty acid to saturated fatty acid ratio among all the cultivars showed that SB-FC21 and SB-FC22 had the highest value, suggesting they are the most beneficial for health. Furthermore, lipids such as acyl sterol glycosides were detected and characterized for the first time in these bean cultivars. Hierarchical cluster correlations revealed the predominance of ceramides in ARB-EC, lysophospholipids in SB-FC21, and glycerophospholipids in SB-OC. This study comprehensively investigated lipids and their compositions in beans, indicating their potential utility in the nutritional evaluation of beans as functional foods.

Comparison of APCI orbitrap MS and GCxGC/EI TOF MS for the hydrocarbon analysis of heavy base oils

The performance of positive-ion mode atmospheric pressure chemical ionization linear quadrupole ion trap orbitrap mass spectrometry (APCI LQIT Orbitrap MS) has been compared to that of two-dimensional gas chromatography coupled with positive-ion mode electron ionization time-of-flight mass spectrometry (GCxGC/EI TOF MS) for the analysis of group III heavy base oils. Upon APCI, all compounds were predominantly ionized via protonation. However, protonated alkanes are not stable and predominantly generated [M−H]+ ions via elimination of a hydrogen molecule from the protonated molecule. The high-resolution APCI mass spectra provided elemental composition information for the compounds while low-resolution APCI mass spectra were used for semiquantitative evaluation of the relative amounts of the compounds. Ions corresponding to compounds in seven hydrocarbon classes, i.e., (1) linear and branched alkanes, (2) monocycloalkanes, (3) dicycloalkanes, (4) tricycloalkanes and alkylbenzenes, (5) tetracycloalkanes, (6) pentacycloalkanes, and (7) hexacycloalkanes were identified. On the other hand, the GCxGC/EI TOF MS method enabled the chromatographic separation of most analytes. The measured EI mass spectra were used for the identification of the compounds while the GC peak areas were used for semiquantitative evaluation. The detected compounds were classified into three different hydrocarbon classes, (1) linear and branched alkanes, (2) cycloalkanes, and (3) aromatic hydrocarbons. The (+)APCI LQIT Orbitrap MS method was found to be faster and to provide more information for the larger compounds in the samples. For example, polycycloalkanes were only detected by using (+)APCI MS. However, no aromatic compounds were detected. Data processing was more straightforward than when using the GCxGC/EI TOF MS method. However, the latter method provided more accurate results for aromatic hydrocarbons and small alkanes as the analysis of small alkanes by using (+)APCI MS is limited due to the fact that [M−H]+ ions of small alkanes overlap with alkane fragment ions. Both light and heavy aliphatic and aromatic hydrocarbons were detected by using the GCxGC/EI TOF MS method but not polycycloalkanes. Therefore, these two methods complement each other, and each has different strengths and weaknesses.

Effect of processing method on chemical constituents of Rehmanniae Radix: based on UHPLC-LTQ-Orbitrap MS

This study aims to explore the chemical composition of Rehmanniae Radix braised with mild fire and compare the effect of processing method on the chemical composition of Rehmanniae Radix. To be specific, ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was used to screen the chemical constituents of Rehmanniae Radix. The chemical constituents were identified based on the relative molecular weight and fragment ions, literature information, and Human Metabolome Database(HMDB). The ion peak area ratio of each component before and after processing was used as the index for the variation. SIMCA was employed to establish principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) models of different processed products. According to the PCA plot, OPLS-DA plot, and VIP value, the differential components before and after the processing were screened out. The changes of the content of differential components with the processing method were analyzed. A total of 66 chemical components were identified: 57 of raw Rehmanniae Radix, 55 of steamed Rehmanniae Radix, 55 of wine-stewed Rehmanniae Radix, 51 of repeatedly steamed and sundried Rehmanniae Radix Praeparata, 62 of traditional bran-braised Rehmanniae Radix, and 63 of electric pot-braised Rehmanniae Radix. Among them, the 9 flavonoids of braised Rehmanniae Radix were from Citri Reticulatae Pericarpium. PCA suggested significant differences in the chemical composition of Rehmanniae Radix Praeparata prepared with different processing methods. OPLS-DA screened out 32 chemical components with VIP value >1 as the main differential components. Among the differential components, 9 were unique to braised Rehmanniae Radix(traditional bran-braised, electric pot-braised) and the degradation rate of the rest in braised(traditional bran-braised, electric pot-braised) or repeatedly steamed and sundried Rehmanniae Radix was higher than that in the steamed or wine-stewed products. The results indicated the chemical species and component content of Rehmanniae Radix changed significantly after the processing. The 32 components, such as rehmapicrogenin, martynoside, jionoside D, aeginetic acid, hesperidin, and naringin, were the most important compounds to distinguish different processed products of Rehmanniae Radix. The flavonoids introduced by Citri Reticulatae Pericarpium as excipient may be the important material basis for the effectiveness of braised Rehmanniae Radix compared with other processed products.

Analysis of absorbed constituents and network pharmacology research of Xiaoer Fupi Granules

Xiaoer Fupi Granules, a refined version of the classical prescription Shenling Baizhu Powder, has the effect of invigora-ting spleen, replenishing Qi, harmonizing stomach and resolving accumulation and is commonly used to treat Qi deficiency in spleen and stomach, disordered transportation and transformation, and indigestion of children. However, its medicinal constituents and mechanism remain unclear. We studied the main active constituents and action mechanism of Xiaoer Fupi Granules by integrating network pharmacology and prototype constituent analysis in vivo. This study will help to increase the reliability of database analysis results and lay a foundation for precise medication and mining of quality control markers. On the basis of Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine(BATMAN-TCM), the "key chemical constituents-target" network was constructed. Ultra-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS) was employed to analyze the absorbed constituents in rat urine and plasma, so as to validate the network. Further, we used BATMAN-TCM to construct the "absorbed constituents-target-pathway" network and explore the functioning mechanism of Xiaoer Fupi Granules. A total of 86 chemical constituents of Xiaoer Fupi Granules were predicted via BATMAN-TCM, among which only 18.6% were detected in rat plasma and urine. Accor-ding to the "absorbed constituents-target-pathway" network, 8 chemical constituents such as stearic acid and caprylic acid capable of regulating gastric acid and insulin secretion may be the critical constituents of Xiaoer Fupi Granules in invigorating spleen and harmonizing stomach. This study identified the critical active constituents and predicted the action mechanism of Xiaoer Fupi Granules, providing the reference for the research on the material basis of Xiaoer Fupi Granules.

Differential metabolic profiles of ginsenosides in artificial gastric juice using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry.

Ginsenosides have poor oral bioavailability and undergo rapid biological transformation in the complex gastrointestinal environment. Most studies on the metabolism of ginsenosides have focused on gut bacteria, yet gastric juice remains a nonnegligible factor. Metabolic profiles of ginsenoside monomers formed in artificial gastric juice were separately investigated and qualitatively identified using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MSn ). A common pattern of their metabolic pathways was established, showing that ginsenosides were transformed via deglycosylation, hydration, and dehydration pathways. Two major structure types, 20(S), 20(R)-protopanaxatriols and 20(S), 20(R)-protopanaxadiols, basically shared similar transformation pathways and yielded deglycosylated, hydrated, and dehydrated products. Fragmentation patterns of major ginsenosides were also discussed. Consequently, gastric juice, as the primary link in ginsenoside metabolism and as important as the intestinal flora, produces considerable amounts of degraded ginsenosides, providing a partial explanation for the low bioavailabilities of primary ginsenosides.

Determination of pharmaceuticals and metabolites in sludge and hydrochar after hydrothermal carbonization using sonication-QuEChERS extraction method and UHPLC LTQ/Orbitrap MS.

Pharmaceuticals (PhACs) are an important group of emerging contaminants that are released continuously in the environment from wastewater treatments plants (WWTPs). They can produce biological effects even though at very low concentrations. Conventional WWTPs are not able to remove or degrade completely emerging pollutants resulting in the presence of PhACs in sewage sludge after wastewater treatment. PhACs are found in sludge at low ppb-ppt levels, and their analysis and detection is a difficult task due to the complexity of sewage sludge matrices. Hydrothermal carbonization is currently being proposed as a suitable conversion technology for sewage sludge management to recover valuable products and to be used for soil amendment. In this work, a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based methodology with a dispersive solid-phase extraction (d-SPE) clean-up followed by ultra-high-performance liquid chromatography coupled with high-resolution linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ/Orbitrap MS), operated in positive ionization mode, was adopted to investigate 33 multiclass pharmaceuticals in sewage sludge and in hydrochar produced after hydrothermal carbonization. The analytical method was first optimized studying various extraction parameters and finally validated in terms of linearity, recovery, intra and inter-day precisions, expanded uncertainty (%U)/Horrat ratio at three spiking levels, matrix-effects (ME), process efficiency (PE), and limits of detection and quantification. The developed methodology fulfilled all analytical requirements and was finally applied to sludge samples from the WWTP of Ioannina city where a group of antibiotics was detected at concentrations up to 15ngg-1 and psychiatric drugs such as amisulpride, clozapine, and citalopram were detected at higher concentration levels up to 205, 87.4 and 63.2ngg-1, respectively. The method was also applied to hydrothermally treated sludge sample under different reaction conditions. Most of the antibiotic compounds were not detected, and several psychiatric drugs such as mirtazapine, bupropion, valsartan, diazepam, and caffeine were found at concentrations below the LOQ.

Deep chemical identification of phytoecdysteroids in Achyranthes bidentata Blume by UHPLC coupled with linear ion trap-Orbitrap mass spectrometry and targeted isolation.

Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones. In this work, a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode. As a result, 47 phytoecdysteroids were unambiguously or tentatively characterized. Among them, seven known compounds were identified according to the reference standards along with molecular formula, retention time and fragmentation patterns, while others were mostly potential new compounds. Through targeted isolation, the structures of three new compounds were determined by NMR spectra, which were consistent with LC-MS characterization. The present study provides an efficient method to deeply characterize phytoecdysteroids.

Detection and characterization of lipids in eleven species of fish by non-targeted liquid chromatography/mass spectrometry

Fish is an important nutrition source because its lipids, which are rich in ω-3 fatty acids, are beneficial for human health. However, studies focusing on their detection, composition, and nutritional value are limited. In this study, we applied a non-targeted lipidomic approach based on ultra-high performance liquid chromatography coupled with linear-ion trap-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap-MS) to comprehensively profile, compare, and detect unknown lipids in eleven types of dietary fish. A total of 287 molecular species from five major lipid classes were characterized by MS/MS analysis. Multivariate principal component analysis revealed the distinct lipid composition in shishamo smelt and Japanese sardine compared to other fish types. The assessment of nutritional indices based on the levels of free fatty acid suggested that among the eleven fish types, shishamo smelt is highly beneficial for health. Further, lipids such as N-acyl lysophosphatidylethanolamine were detected and characterized for the first time in fish fillets. Hierarchical cluster correlations indicated the predominance of glycerophospholipids (GPs) and sphingolipids in sardine, whereas fatty acyls and triacylglycerols (TAGs) were predominant in shishamo smelt. The high levels of polyunsaturated fatty acid-enriched GPs and TAGs in dietary fish endow it with great potential as a health-promoting food for human consumption. This study offers a comprehensive analysis of lipids and their compositions in fish fillets, demonstrating their potential use in the nutritional assessment of functional foods.

Identification of chemical constituents in Simiao Yong'an Decoction based on UPLC-LTQ-Orbitrap-MS

This study aims to identify the chemical constituents of Simiao Yong'an Decoction based on ultra-performance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS). The elution was performed through a UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 μm) with the mobile phase of water(containing 0.1% formic acid)-acetonitrile at a flow rate of 0.4 mL·min~(-1). LTQ-Orbitrap-MS with heat electrospray ion(HESI) source was employed to collect MS fragment information in the negative ion mode. A total of 72 compounds were identified based on reference substance comparison, fragmentation rules, accurate molecular weight, related reports and databases(MassBank and HMDB), including 30 iridoid glycosides, 9 organic acids, 15 flavonoids, 10 phenylpropanoids, 7 triterpenoids, and 1 saccharide. The method established in this study is comprehensive, rapid, and accurate, which can help summarize the fragmentation rules of constituents and provide reference for revealing the active constituents and pharmacodynamic mechanism of Simiao Yong'an Decoction.

Determination of multiple active constituents in Da‐Huang‐Xiao‐Shi decoction using HPLC–LTQ–Orbitrap mass spectrometry: Application in comparing the differences in the formula and its constituent herbs

Da-Huang-Xiao-Shi decoction (DHXSD) is a traditional Chinese medicine formula and is used to treat cholestasis. In this study, we developed a reliable and comprehensive HPLC coupled with a linear ion trap-Orbitrap mass spectrometry method for the separation and determination of 21 components including six alkaloids, five anthraquinones, three tannins, three terpenes, two iridoid glycosides, one organic acid and one flavonoid in DHXSD. A C18 column was eluted using a gradient mobile phase at a flow rate of 1ml/min. Detection was operated with an electrospray ionization source in positive and negative ion modes using selective ion monitoring. The calibration curves for all analytes showed good linearity (r > 0.9901), and the inter- and intra-day precision did not exceed 4.98%. The recovery, repeatability and stability were also within the acceptable limits. The method was successfully applied to determine multiple active constituents in DHXSD and its constituent herbs. Compared with Da Huang, the total contents of the five anthraquinones were significantly higher in DHXSD. However, the changes in the components from Zhi Zi/Huang Bo were complicated in DHXSD. The study could serve as a fundamental reference for establishing comprehensive DHXSD quality control measures and be helpful to understand some compatibility laws of DHXSD.

Chemical characterization of flavonoids and alkaloids in safflower (Carthamus tinctorius L.) by comprehensive two-dimensional hydrophilic interaction chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry

Safflower (Carthamus tinctorius L.) is a famous food additive and herbal medicine in China. In the present research, an online comprehensive two-dimensional hydrophilic interaction chromatography coupled to a diode array detector and a hybrid linear ion trap-Orbitrap mass spectrometry (HILIC × HILIC-DAD-ESI/HRMS/MSn) platform was developed to analyze the flavonoids and alkaloids in safflower. By combining with an XBridge Amide column (150 mm × 4.6 mm, 3.5 μm) and an Ultimate amide column (50 mm × 4.6 mm, 5 μm), the system orthogonality reached 88% and a total of 231 peaks were detected. Altogether 93 compounds, including 75 flavonoids and their glycosides and 10 alkaloids were unambiguously or tentatively identified in both negative and positive ion modes, using accurate mass and MS fragment data. Among them, 5 compounds were discovered and reported from safflower for the first time. The established HILIC × HILIC platform should be a powerful tool for the separation and characterization of complicated matrices in natural herbs.

Determination of metabolites of phloretin in rats using UHPLC-LTQ-Orbitrap mass spectrometry

Purpose: To study the metabolites of phloretin in vivo using ultra-high performance liquid chromatography linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap).
 Methods: After administration of phloretin (50 mg/kg; oral route) to six rats, blood samples were taken from each animal. Each sample was then subjected to solid-phase extraction to prepare it for chromatographic/spectroscopic analysis. Finally, each sample was analyzed using UHPLC-LTQOrbitrap with a negative-mode electrospray ionization source.
 Results: Based on mass measurements, chromatographic retention times, and MS2 fragmentation ions, we detected and identified phloretin and 16 metabolites of the drug in vivo in rats. Metabolic reactions of phloretin included glucosylation and glucuronide conjugation, diglucuronide conjugation, glucosylation and sulfate conjugation, sulfate conjugation, glucuronide conjugation, and glucosylation and hydroxylation.
 Conclusion: The findings provide a better understanding of phloretin metabolism and metabolites, and new information about their effective forms, pharmacological actions, metabolic fate, and toxic actions in vivo.

Systematic characterization of chemical constituents in Mahuang decoction by UHPLC tandem linear ion trap‐Orbitrap mass spectrometry coupled with feature‐based molecular networking

Comprehensive characterization of traditional Chinese medicine prescriptions has long been a hurdle due to the chemical complexity and the lack of analytical tools. Mahuang decoction is a well-known traditional Chinese medicine prescription widely used for sweating and relieving the exterior, relieving cough and asthma, but it was insufficiently chemically scrutinized. In this study, the chemical component information of Mahuang decoction was investigated by ultrahigh-performance liquid chromatography tandem linear ion trap-Orbitrap mass spectrometry. A new data processing tool, feature-based molecular networking, was introduced for grouping and elucidating the compounds. In this way, 156 chemical components were identified or tentatively characterized, including alkaloids, triterpenoid saponins, flavanone-O-glycosides, flavone-C-glycosides, and procyanidins. Thus, this research provides a solid foundation for further development of Mahuang decoction, and the adopted method is expected to be applied to other traditional Chinese medicine prescriptions.