Comprehensive characterization of Epimedium-Rhizoma drynariae herb pair in rat plasma, urine, and feces metabolic profiles by UHPLC-Q-Orbitrap HRMS combined with diagnostic extraction strategy and multicomponent pharmacokinetic study by UHPLC-MS/MS.

Epimedium-Rhizoma drynariae (EP-RD) was a well-known herb commonly used to treat bone diseases in traditional Chinese medicine. Nevertheless, there was incomplete pharmacokinetic behavior, metabolic conversion and chemical characterization of EP-RD in vivo. Therefore, this study aimed to establish metabolic profiles combined with multicomponent pharmacokinetics to reveal the in vivo behavior of EP-RD. Firstly, the diagnostic product ions (DPIs) and neutral losses (NLs) filtering strategy combined with UHPLC-Q-Orbitrap HRMS for the in vitro chemical composition of EP-RD and metabolic profiles of plasma, urine, and feces after oral administration of EP-RD to rats were proposed to comprehensively characterize the 47 chemical compounds and the 97 exogenous in vivo (35 prototypes and 62 metabolites), and possible biotransformation pathways of EP-RD were proposed, which included phase I reactions such as hydrolysis, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, isomerization, and demethylation and phase II reactions such as glucuronidation, acetylation, methylation, and sulfation. Moreover, a UHPLC-MS/MS quantitative approach was established for the pharmacokinetic analysis of seven active components: magnoflorine, epimedin A, epimedin B, epimedin C, icariin, baohuoside II, and icariin II. Results indicated that the established method was reliably used for the quantitative study of plasma active ingredients after oral administration of EP-RD in rats. Compared to oral EP alone, the increase in area under curves and maximum plasma drug concentration (P < 0.05). This study increased the understanding of the material basis and biotransformation profiles of EP-RD in vivo, which was of great significance in exploring the pharmacological effects of EP-RD.

Supramolecular self-assembly strategies of natural-based ß-lactoglobulin modulating bitter perception of goat milk-derived bioactive peptides.

Complete self-assembly and reassembly behavior of bitter peptide-protein necessitates multilevel theories that encompass phenomena ranging from the self-assembly of recombinant complex to atomic trajectories. An extension to the level of mechanism method was put forth, involves limited enzymatic digestion and bottom-up proteomics to dissect inherent heterogeneity within ß-LG and ß-LG-PPGLPDKY complex and uncover conformational and dynamic alterations occurring in specific local regions of the model protein. Bitter peptide PPGLPDKY spontaneously bound to IIAEKTK, IDALNENK, and YLLFCMENSAEPEQSLACQCLVR regions of ß-LG in a 1:1 stoichiometric ratio to mask bitterness perception. Molecular dynamic simulation and free energy calculation provided time-varying atomic trajectories of the recombinant complex and found that a peptide was stabilized in the upper region of the hydrophobic cavity with the binding free energy of -30.56 kJ mol-1 through 4 hydrogen bonds (Glu74, Glu55, Lys69, and Ser116) and hydrophobic interactions (Asn88, Asn90, and Glu112). Current research aims to provide valuable physical insights into the macroscopic self-assembly behavior between proteins and bitter peptides, and the meticulous design of highly acceptable taste characteristics in goat milk products.

Systematic qualitative analysis of terpenes in mastic (Pistacia lentiscus L.) extract and their fragmentations by UHPLC-Q-Orbitrap-HRMS.

INTRODUCTION: Mastic is a natural resin produced by Pistacia lentiscus L. (Anacardiaceae). The beneficial properties of this resin are attributed to its triterpenes and volatile compounds. OBJECTIVE: This study was conducted to screen and characterize the terpenes in mastic ethyl acetate extract (M-Ex). METHODS: An ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method was developed for the qualitative analysis of terpenes in M-Ex. We utilized in-house-isolated compounds as reference substance (Rs), including monoterpenes (A) with α-pinane structures, tetracyclic triterpene (B) containing tirucallane skeletons, and pentacyclic triterpene (C) belonging to olean, moronic, amyrone, and lupane types. Based on the mass spectrometric characteristics of the above compounds, and the difference in characteristic diagnostic fragment ions (DFIs) in isomeric compounds, the terpene compounds were further identified in M-Ex. RESULTS: Out of a total of 70 compounds, including monoterpenes and tetra-, and pentacyclic triterpenes, 20 were accurately determined by Rs, retention time (RT), and DFIs. Based on the cleavage patterns summarized from the above 20 compounds and with reference to the reported literature, another 50 compounds were putatively identified. Based on our discovery, six terpenic acids with A-seco-tirucallane types and one monoterpene dimer were identified for the first time in mastic. CONCLUSION: Our research serves not only as a foundation for the rapid identification and screening of terpene compounds in mastic but also as a supplementary basis for the identification of such compounds in other types of resins.

Untargeted Metabolomics Based on Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Differential Metabolite Analysis of Pinelliae Rhizoma and Its Adulterants.

The present study investigates the chemical composition variances among Pinelliae Rhizoma, a widely used Chinese herbal medicine, and its common adulterants including Typhonium flagelliforme, Arisaema erubescens, and Pinellia pedatisecta. Utilizing the non-targeted metabolomics technique of employing UHPLC-Q-Orbitrap HRMS, this research aims to comprehensively delineate the metabolic profiles of Pinelliae Rhizoma and its adulterants. Multivariate statistical methods including PCA and OPLS-DA are employed for the identification of differential metabolites. Volcano plot analysis is utilized to discern upregulated and downregulated compounds. KEGG pathway analysis is conducted to elucidate the differences in metabolic pathways associated with these compounds, and significant pathway enrichment analysis is performed. A total of 769 compounds are identified through metabolomics analysis, with alkaloids being predominant, followed by lipids and lipid molecules. Significant differential metabolites were screened out based on VIP > 1 and p-value < 0.05 criteria, followed by KEGG enrichment analysis of these differential metabolites. Differential metabolites between Pinelliae Rhizoma and Typhonium flagelliforme, as well as between Pinelliae Rhizoma and Pinellia pedatisecta, are significantly enriched in the biosynthesis of amino acids and protein digestion and absorption pathways. Differential metabolites between Pinelliae Rhizoma and Arisaema erubescens are mainly enriched in tyrosine metabolism and phenylalanine metabolism pathways. These findings aim to provide valuable data support and theoretical references for further research on the pharmacological substances, resource development and utilization, and quality control of Pinelliae Rhizoma.

The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

Identification of in vivo metabolites of Citri Sarcodactylis Fructus by UHPLC-Q/Orbitrap HRMS.

INTRODUCTION: Citri Sarcodactylis Fructus has the effects of relieving cough, removing phlegm, and reducing asthma, but little is known about the metabolic and distribution of its chemical constituents in vivo. Therefore, it is necessary to study the metabolism of Citri Sarcodactylis Fructus in vivo. OBJECTIVE: We aimed to (1) analyze the distribution of prototype compounds and metabolites of the chemical constituents of Citri Sarcodactylis Fructus in rat and (2) infer the metabolites and metabolic pathways of the chemical constituents. MATERIALS AND METHODS: A C18 column (3 × 100 mm, 2.6 µm) was used. The mobile phase was water containing 0.1% formic acid (eluent A) and acetonitrile containing 0.1% formic acid (eluent B) at a discharge rate of 0.3 mL/min. Mass spectra of biological samples were collected in electrospray ionization (ESI) positive ion mode in the m/z 100-1500 scan range. The obtained biological samples were then subjected to chemical analysis, including plasma, urine, feces, and heart, liver, spleen, lungs, kidneys, stomach, and small intestine tissues. Prototype compounds and metabolites were identified. RESULTS: In all, 40 prototype compounds and 78 metabolites, including 26 phase I metabolites and 52 phase II metabolites, were identified using UHPLC-Q/Orbitrap HRMS. Eight possible metabolic pathways (reduction, hydrolysis, dehydration, methylation, hydroxylation, sulfation, glucuronidation, and demethylation) were proposed. The prototype compounds were predominantly distributed in lung tissues. The metabolites were mainly distributed in plasma and kidney tissues. CONCLUSION: We systematically investigated the metabolites of Citri Sarcodactylis Fructus in vivo. We suggest metabolic pathways that might be relevant for further metabolic studies and screening of active ingredients of Citrus Sarcodactylis Fructus in vivo.

Analysis of Lutein Content in Microencapsulated Marigold Flower Extract (Tagetes erecta L.) Using UHPLC-Q-Orbitrap-HRMS and Its Cytotoxicity in ARPE-19 Cells.

Organic solvents are commonly used to extract lutein. However, they are toxic and are not environmental-friendly. There are only a few reports on the quantification of lutein. Therefore, this study aimed to determine a suitable extraction method by which to obtain lutein from marigold flower (Tagetes erecta L.), using coconut oil to evaluate the cytotoxicity of extract in ARPE-19 cells, to optimize the encapsulation process for the development of microencapsulated marigold flower extract, and to develop the method for analysis of lutein by using UHPLC-Q-Orbitrap-HRMS. Coconut oil was used for the extraction of marigold flowers with two different extraction methods: ultrasonication and microwave-assisted extraction. The UHPLC-Q-Orbitrap-HRMS condition for the analysis of lutein was successfully developed and validated. Marigold flower extract obtained using the microwave method had the highest lutein content of 27.22 ± 1.17 mg/g. A cytotoxicity study revealed that 16 µM of lutein from marigold extract was non-toxic to ARPE-19 cells. For the development of microencapsulated marigold extract, the ratio of oil to wall at 1:5 had the highest encapsulation efficiency and the highest lutein content. Extraction of lutein using coconut oil and the microwave method was the suitable method. The microencapsulated marigold extract can be applied for the development of functional ingredients.

Study on Pharmacokinetics and Metabolic Profiles of Novel Potential PLK-1 Inhibitors by UHPLC-MS/MS Combined with UHPLC-Q-Orbitrap/HRMS.

PLK-1 (Polo-like kinase-1) plays an essential role in cytokinesis, and its aberrant expression is considered to be keenly associated with a wide range of cancers. It has been selected as an appealing target and small-molecule inhibitors have been developed and studied in clinical trials. Unfortunately, most have been declared as failures due to the poor therapeutic response and off-target toxicity. In the present study, a novel potent PLK-1 inhibitor, compound 7a, was designed and synthetized. 1H NMR, 13C NMR, 19F NMR and mass spectrum were comprehensively used for the compound characterization. The compound exhibited higher potency against PLK-1 kinase, HCT-116 and NCI-H2030 cell lines than the positive control. Molecular docking indicated that the binding mode that the ATP binding site of PLK-1 was occupied by the compound. Then, a UHPLC-MS/MS method was established and validated to explore the pharmacokinetic behavior of the drug candidate. The method had good selectivity, high sensitivity and wide linearity. The exposure increased linearly with the dose, but the oral bioavailability was not satisfactory enough. Then, the metabolism was studied using liver microsomes by UHPLC-Q-Orbitrap/HRMS. Our research first studied the pharmacokinetic metabolic characteristics of 7a and may serve as a novel lead compound for the development of PLK-1 inhibitors.

[Comparison of chemical compositions of different parts of Xanthoceras sorbifolium seeds based on UHPLC-Q-Orbitrap HRMS].

Xanthoceras sorbifolium seeds have a wide range of applications in the food and pharmaceutical industries. To compare and analyze the chemical compositions of different parts of X. sorbifolium seeds and explore the potential value and research prospects of non-medicinal parts, this study used ultra-high-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) to detect the chemical composition of various parts of the seeds. A total of 82 components were preliminary identified from X. sorbifolium seeds, including 5 amino acids, 4 polyphenols, 3 phenylpropionic acids, 7 organic acids, 15 flavonoids, 6 glycosides, and 23 saponins. Mass spectrometry molecular networking(MN) analysis was conducted on the results from different parts of the seeds, revealing significant differences in the components of the seed kernel, seed coat, and seed shell. The saponins and flavonoids in the seed kernel were superior in terms of variety and content to those in the seed coat and shell. Based on the chromatographic peaks of different parts from multiple batches of samples, multivariate statistical analysis was carried out. Four differential components were determined using HPLC, and the average content of these components in the seed kernel, seed coat, and seed shell were as follows: 0.183 6, 0.887 4, and 1.440 1 mg·g~(-1) for fraxin; 0.035 8, 0.124 1, and 0.044 5 mg·g~(-1) for catechin; 0.032 9, 0.072 0, and 0.221 5 mg·g~(-1) for fraxetin; 0.435 9, 2.114 7, and 0.259 7 mg·g~(-1) for epicatechin. The results showed that catechin and fraxetin had relatively low content in all parts, while fraxin had higher content in the seed coat and seed shell, and epicatechin had higher content in the seed kernel and seed coat. Therefore, the seed coat and seed shell possess certain development value. This study provides rapid analysis and comparison of the chemical compositions of different parts of X. sorbifolium seeds, which offers an experimental basis for the research and clinical application of medicinal substances in X. sorbifolium seeds.

Determination of rivaroxaban in rat plasma by ultra-high-performance liquid chromatography-Q-Orbitrap high-resolution mass spectrometry and its application to a pharmacokinetic study.

Therapeutic drug monitoring is critical to decrease the incidence rate of bleeding and thrombosis for personalized treatment with rivaroxaban, especially for drug interaction treatment, patients with renal dysfunction, elderly patients, patients with cardiovascular problems, and so on. In addition, an accurate analytical method is necessary for therapeutic drug monitoring. This study developed a ultra-HPLC-tandem Orbitrap high-resolution MS (UHPLC-Q-Orbitrap HRMS) method to accurately identify and quantify rivaroxaban in rat plasma. The isotope internal standard method was applied for accurate quantification. Rivaroxaban-d4 was selected as the isotope internal standard substance. The m/z 436.07263 ([M + H]+ ) was selected as the precursor ion and m/z 144.95085 and m/z 231.11259 were selected as the main product ions for rivaroxaban. The lower limit of quantification of rivaroxaban in plasma was 0.01 mg/L. The intra- and inter-day precisions were ≤3.65% and ≤8.16%, while the recoveries ranged from 87.4% to 95.2%. This analysis method was simple, low cost, and easy to operate. The developed and validated method was subsequently applied to successfully investigate the pharmacokinetic parameters of rivaroxaban in rats after its oral administration. These results could be helpful to promote further research regarding the mechanisms of rivaroxaban and drug interaction, which can avoid false positives due to high-precision identification of the proposed method.

Multiclass and multi-residue screening of mycotoxins, pharmacologically active substances, and pesticides in infant milk formulas through ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry analysis.

Infant milk formulas are designed to substitute human milk when breastfeeding is unavailable. In addition to human milk and milk-derived products, these formulas can be a vehicle of contaminants. In this work, a multiclass method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach was developed for the simultaneous determination of contaminants (n = 45), including mycotoxins and veterinary drug residues, occurring in infant milk formulas. By using an ultra-high-performance liquid chromatography quadrupole-Orbitrap coupled with high-resolution mass spectrometry analysis (UHPLC-Q-Orbitrap HRMS; Thermo Fisher Scientific), further retrospective analysis of 337 contaminants, including pesticides, was achieved. The method was validated in accordance with European regulations and applied for the analysis of 54 infant milk samples. Risk assessment was also performed. Dexamethasone was detected in 16.6% of samples (range: 0.905-1.131 ng/mL), and procaine benzyl penicillin in 1 sample at a concentration of 0.295 ng/mL. Zearalenone was found in 55.5% of samples (range: 0.133-0.638 ng/mL) and α-zearalenol in 16.6% of samples (range: 1.534-10.408 ng/mL). Up to 49 pesticides, 11 veterinary drug residues, and 5 mycotoxins were tentatively identified via retrospective analysis based on the mass spectral library. These findings highlight the necessity of careful evaluation of contaminants in infant formulas, considering that they are intended for a vulnerable part of the population.

Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Ranunculus sceleratus L.(RS) has shown various pharmacological effects in traditional Chinese medicine. In our previous study, the positive therapeutic effect on α-naphthylisothiocyanate induced intrahepatic cholestasis in rats was obtained using TianJiu treatment with fresh RS. However, the chemical profile of RS has not been clearly clarified, which impedes the research progress on the therapeutic effect of RS. Herein, an ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed to rapidly separate and identify multiple constituents in the 80% methanol extract of RS. A total of sixty-nine compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) were successfully characterized. A total of 12 of these compounds were unambiguously identified by standard samples. Their mass spectrometric fragmentation pathways were investigated. It is worth noting that flavonoids and lignans were identified for the first time in RS. In this study, we successfully provide the first comprehensive report on identifying major chemical constituents in RS by UHPLC-Q-Orbitrap HRMS. The obtained results enrich the RS chemical profile, paving the way for further phytochemical study, quality control, and pharmacological investigation of RS.

Systematically Exploring the Chemical Ingredients and Absorbed Constituents of Polygonum capitatum in Hyperuricemia Rat Plasma Using UHPLC-Q-Orbitrap HRMS.

Polygonum capitatum as an ethnic medicine has been used to treat urinary tract infections, pyelonephritis and urinary calculi. In our previous study, P. capitatum was found to have anti-hyperuricemia effects. Nevertheless, the active constituents of P. capitatum for treating hyperuricemia were still unclear. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to comprehensively detect the chemical ingredients of P. capitatum and its absorbed constituents in the plasma of hyperuricemia rats for the first time. Xcalibur 3.0 and Compound Discoverer 2.0 software coupled to mzCloud and ChemSpider databases were utilized for qualitative analysis. A total of 114 chemical components including phenolics, flavonoids, tannins, phenylpropanoids, amino acids, amides and others were identified or tentatively characterized based on the exact mass, retention time and structural information. Compared to the previous P. capitatum study, an additional 66 different components were detected. Moreover, 68 related xenobiotics including 16 prototype components and 52 metabolites were found in the plasma of hyperuricemia rats. The metabolic pathways included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This work may provide important information for further investigation on the active constituents of P. capitatum and their action mechanisms for anti-hyperuricemia effects.

Integrated Solid-Phase Extraction, Ultra-High-Performance Liquid Chromatography-Quadrupole-Orbitrap High-Resolution Mass Spectrometry, and Multidimensional Data-Mining Techniques to Unravel the Metabolic Network of Dehydrocostus Lactone in Rats.

Dehydrocostus lactone (DL) is among the representative ingredients of traditional Chinese medicine (TCM), with excellent anticancer, antibacterial, and anti-inflammatory activities. In this study, an advanced strategy based on ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was integrated to comprehensively explore the metabolic fate of DL in rats. First, prior to data collection, all biological samples (plasma, urine, and feces) were concentrated and purified using solid-phase extraction (SPE) pre-treatment technology. Then, during data collection, in the full-scan (FS) data-dependent acquisition mode, FS-ddMS2 was intelligently combined with FS-parent ion list (PIL)-dynamic exclusion (DE) means for targeted monitoring and deeper capture of more low-abundance ions of interest. After data acquisition, data-mining techniques such as high-resolution extracted ion chromatograms (HREICs), multiple mass defect filters (MMDFs), diagnostic product ions (DPIs), and neutral loss fragments (NLFs) were incorporated to extensively screen and profile all the metabolites in multiple dimensions. As a result, a total of 71 metabolites of DL (parent drug included) were positively or tentatively identified. The results suggested that DL in vivo mainly underwent hydration, hydroxylation, dihydrodiolation, sulfonation, methylation, dehydrogenation, dehydration, N-acetylcysteine conjugation, cysteine conjugation, glutathione conjugation, glycine conjugation, taurine conjugation, etc. With these inferences, we successfully mapped the "stepwise radiation" metabolic network of DL in rats, where several drug metabolism clusters (DMCs) were discovered. In conclusion, not only did we provide a refined strategy for inhibiting matrix effects and fully screening major-to-trace metabolites, but also give substantial data reference for mechanism investigation, in vivo distribution visualization, and safety evaluation of DL.

Integrated metabolomic analysis for intervention effects of Gushudan on glucocorticoid-induced osteoporostic rat plasma based on RP/HILIC-UHPLC-Q-Orbitrap HRMS.

An integrated metabolomic strategy based on both RP-UHPLC-Q-Orbitrap HRMS and HILIC-UHPLC-Q-Orbitrap HRMS in rat plasma was developed and validated, to further understand the anti-osteoporosis effect of Gushudan (GSD) and its mechanism on glucocorticoid-induced osteoporosis (GIOP) rats in this study. The metabolites were separated and identified on C18 column (100 mm × 2.1 mm, 1.7 µm) and Amide column (100 mm × 2.1 mm, 1.7 µm) using the UHPLC-Q-Orbitrap system (Thermo Fisher Scientific, USA). As a result, a total of 40 differential metabolites were identified, which were mainly related to lipid metabolism, amino acid metabolism, energy metabolism and intestinal flora metabolism. It's worth mentioning that some new potential biomarkers associated with osteoporosis such as 3-hdroxybutyric acid and glycocholic acid were discovered in this study for the first time. With pattern recognition analysis of metabolite profile, a clear separation of the model group and the control group was acquired for plasma samples. The GSD group showed a predisposition towards recovery mimicking the control group, which was in agreement with the behavioral and biochemical results. The present study suggested that GSD had significant anti-osteoporotic effects on glucocorticoid-induced osteoporosis rat plasma, which might be attributed to regulating multiple metabolic pathways. Thus, metabolomics would be a useful tool in the evaluation of the efficacy and elucidation of the mechanism underlying the complex traditional Chinese medicine prescriptions.

Target analysis and retrospective screening of mycotoxins and pharmacologically active substances in milk using an ultra-high-performance liquid chromatography/high-resolution mass spectrometry approach.

Milk is a nutritious food suitable for infants and adults, and it plays an important role in the human diet. However, it may also be a vehicle for food contaminants. In this report, we developed a method using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS; Thermo Fisher Scientific, Waltham, MA) for simultaneous identification of target pharmacologically active substances and mycotoxins in milk. We also used the Q-Orbitrap operating in full scan mode to identify other possible drugs and microbial metabolites that occurred in samples. Fifty-six commercially available milk samples from the Italian market were analyzed. Investigated analytes were extracted using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach. Method detection and quantification limits and performance criteria set by European regulations were fulfilled. Pharmacologically active substances were detected in 49% of samples (range 0.007-4.53 ng/mL), including nontarget mycotoxins. Retrospective analysis allowed us to identify other antibiotics and pharmacologically active substances, as well as nonregulated fungal/bacterial metabolites at a relatively high incidence. From the obtained values, the need for continuous monitoring of contaminants in the milk production chain is clear. This is the first study to assess the presence of pharmacologically active substances, mycotoxins, and other microbial metabolites in Italian milk samples using the UHPLC-Q-Orbitrap HRMS system.

Analysis of Phenolic Compounds in Commercial Cannabis sativa L. Inflorescences Using UHPLC-Q-Orbitrap HRMS.

Industrial hemp (Cannabis sativa L. Family Cannabaceae) contains a vast number of bioactive relevant compounds, namely polyphenols including flavonoids, phenolic acids, phenol amides, and lignanamides, well known for their therapeutic properties. Nowadays, many polyphenols-containing products made of herbal extracts are marketed, claiming to exert health-promoting effects. In this context, industrial hemp inflorescence may represent an innovative source of bioactive compounds to be used in nutraceutical formulations. The aim of this work was to provide a comprehensive analysis of the polyphenolic fraction contained in polar extracts of four different commercial cultivars (Kompoti, Tiborszallasi, Antal, and Carmagnola Cs) of hemp inflorescences through spectrophotometric (TPC, DPPH tests) and spectrometry measurement (UHPLC-Q-Orbitrap HRMS). Results highlighted a high content of cannflavin A and B in inflorescence analyzed samples, which appear to be cannabis-specific, with a mean value of 61.8 and 84.5 mg/kg, meaning a ten-to-hundred times increase compared to other parts of the plant. Among flavonols, quercetin-3-glucoside reached up to 285.9 mg/kg in the Carmagnola CS cultivar. Catechin and epicatechin were the most representative flavanols, with a mean concentration of 53.3 and 66.2 mg/kg, respectively, for all cultivars. Total polyphenolic content in inflorescence samples was quantified in the range of 10.51 to 52.58 mg GAE/g and free radical-scavenging included in the range from 27.5 to 77.6 mmol trolox/kg. Therefore, C. sativa inflorescence could be considered as a potential novel source of polyphenols intended for nutraceutical formulations.

[Identification of chemical constituents in Qizhi Tongluo Capsules by UPLC-Q-Orbitrap HRMS].

Qizhi Tongluo Capsules, which is composed of 26 herbal drugs, is mainly used as the assistant therapy for apoplexy sequelae. The chemical composition of Qizhi Tongluo Capsules was complex,but its chemical constituents and the pharmacodynamic material basis remain unreported. The ultrahigh performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry(UPLC-Q-Orbitrap HRMS) was applied to recognize the chemical constituents of Qizhi Tongluo Capsules. The analysis was performed on a Waters Acquity UHPLC HSS T3 column with acetonitrile-water as the mobile phase for gradient elution. Heated electrospray ionization(HESI) in both positive and negative ion modes was adopted to collect the data. The chemical constituents were identified and confirmed by analyzing the accurate molecular weight, the mass fragmentation pattern, and comparing with the mass data from the reference substances and literature. A total of 119 components were identified, including 22 flavones, 12 saponins, 10 salvianolic acids, 5 butylphthalides, 4 anthraquinones, 4 monoterpenoid glycosides, 2 caffeoyl-quinic acids, 2 coumarins, 2 alkaloids, and 1 stilbene, as well as the common constituents in the herb, such as amino acids and organic acids. The chemical constituents of Qizhi Tongluo Capsules were characterized rapidly for the first time in this study, laying a foundation for the further analysis of active compounds and quality control.

Tissue distribution profiles of multiple major bioactive components in rats after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS.

Xuebijing injection (XBJI) is a traditional Chinese medicine prescription extracted from five Chinese herbs. Hydroxysafflor yellow A, oxypaeoniflorin, ferulic acid and benzoylpaeoniflorin are the main bioactive ingredients of XBJI. This paper presents an application of ultra-high-performance liquid chromatography-Q-exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) to quantify four compounds of XBJI in rats various tissues for tissue distribution studies. The analytes were separated on a Waters Acquity UHPLC® BEH C18 column with a gradient mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed by parallel reaction monitoring via a heated electrospray ionization source under the negative ionization mode. The method was validated in various tissue samples, and has demonstrated great performance for rapidity, accuracy, high sensitivity and selectivity. It was successfully applied to the tissue distribution studies of XBJI after intravenous administration to rats. It was also the first study to investigate the tissue distribution of XBJI in rats and we found that the concentrations of four compounds were high in kidney, liver, stomach and intestine. The clinical use of XBJI should focus on its pharmacodynamics and safety studies in these tissues.

A novel liquid chromatography Orbitrap mass spectrometry method with full scan for simultaneous determination of multiple bioactive constituents of Shenkang injection in rat tissues: Application to tissue distribution and pharmacokinetic studies.

Shenkang injection is a traditional Chinese formula with good curative effect on chronic renal failure. In this paper, a novel, rapid and sensitive ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole Orbitrap high-resolution accurate mass spectrometry was developed and validated for simultaneous determination of seven bioactive constituents of Shenkang injection in rat plasma and tissues after intravenous administration. Acetonitrile was used as a protein precipitation agent in biological samples disposal with carbamazepine as internal standard. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). The MS analysis was performed in the full-scan positive and negative ion mode. The lower limits of quantification for the seven analytes in rat plasma and tissues were 0.1-10 ng/mL. The validated method was successfully applied to tissue distribution and pharmacokinetic studies of Shenkang injection after intravenous administration. The results of the tissue distribution study showed that the high concentrations of seven constituents were primarily in the kidney tract. This is the first report of the application of Q-Orbitrap with full-scan mass spectrometry in tissue distribution and pharmacokinetic studies of Shenkang injection.