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 1 ml/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.     

A novel method for investigating the mechanism of the anti-rheumatoid arthritis activity of Angelicae pubescentis radix by integrating UHPLC–QTOF/MS and network pharmacology

The study aimed to establish a strategy to elucidate the in vivo constituents of Angelicae Pubescentis Radix (APR, also known as Duhuo) and reveal the probable mechanisms underlying its anti-rheumatoid arthritis activity. First recorded by Shennong Bencao Jing, APR is mainly used to treat Bi syndrome. Eleven absorbed components of APR were successfully identified using the rheumatoid arthritis (RA) rat model and the UHPLC–QTOF/MS technique. Two active ingredients (osthole and columbianadin) and five corresponding targets (PTGS1, PTGS2, RXRA, CCNA2 and ACHE) were found to construct a compound–protein interaction network in RA. In addition, a non-alcoholic fatty liver disease pathway, which was related to anti-RA activity, was eventually identified by KEGG analysis. Subsequently, molecular docking was performed by establishing a mixed matrix network, including the absorbed component, corresponding target and signaling pathway with two key compounds (osthole and columbianadin) and two important targets (PTGS2 and PTGS1). The result of molecular docking is in agreement with the network pharmacology.     

A novel UPLC–MS–MS method for simultaneous determination of seven uremic retention toxins with cardiovascular relevance in chronic kidney disease patients

Chronic kidney disease (CKD) is a devastating illness characterized by accumulation of uremic retention solutes in the body. The objective of this study was to develop and validate a simple, rapid, and robust UPLC–MS–MS method for simultaneous determination, in serum, of seven organic acid uremic retention toxins, namely uric acid (UA), hippuric acid (HA), indoxylsulfate (IS), p-cresylglucuronide (pCG), p-cresylsulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF). Isotopically labeled internal standards (d₅-HA; 1,3-¹⁵N₂-UA, and d₅-IAA) were used to correct for variations in sample preparation and system performance. Separation on a C18 column was followed by negative electrospray ionization and tandem mass spectrometric detection. Accuracy was below the 15 % threshold. Within-day precision varied from 0.60 to 4.54 % and between-day precision was below 13.33 % for all compounds. The applicability of the method was evaluated by analyzing 78 serum samples originating both from healthy controls and from patients at different stages of CKD. These results were compared with those obtained by use of conventional HPLC–PDA–FLD methods. A good correlation was obtained between both methods for all compounds.     

Efficient and rapid method for extraction of intact phospholipids from sediments combined with molecular structure elucidation using LC–ESI-MS–MS analysis

This paper presents the application of an efficient method for extraction and fractionation of intact phospholipids (PLs) from complex sediment matrices and elucidation of their molecular structure by normal-phase HPLC–ESI-MS–MS. Flow-blending extraction was tested with different solvent mixtures and the best recovery of all PLs classes from the sediment matrix was achieved by using methanol–dichloromethane–buffer, 2:1:0.8. The applied LC–ESI-MS system has linearity of R²=0.98 and a detection limit of 0.5 ng/PL, sufficient for reliable identification of complex mixtures of PLs. MS–MS analyses using a triple-quadrupole mass spectrometer enables detection of individual PL side-chain composition and, hence, characterization of the living organisms contributing to the sedimentary organic material. Parallel GC–MS analysis of the hydrolysed phospholipid fatty acids supports the characterized fatty acid patterns determined from intact PLs. The PL inventory of different investigated lacustrine surface sediments shows predominantly high abundance of phosphatidylglycerols and phosphatidylethanolamines and phosphatidyl-mono- and dimethyl-ethanolamines with fatty acyl side-chains typically known from bacteria. In a sample from Lake Baikal intense signals of bacterial 14:0-acyl-PGs were also identified, for the first time in sediments as far as we are aware.     

Serum metabolomics using ultra-high performance liquid chromatography–Q-Exactive tandem mass spectrometry reveals the mechanism of action of exercise training on chronic obstructive pulmonary disease rats

Exercise training is the cornerstone component of pulmonary rehabilitation, which results in symptom-reducing, psychosocial, and health economic benefits for chronic obstructive pulmonary disease (COPD) patients. However, the potential mechanisms of its action are poorly understood. This study conducted serum metabolomics using ultra-high performance liquid chromatography–Q-Exactive tandem mass spectrometry to determine the metabolic changes in COPD rats, and the effects of exercise training on improvement in COPD were further investigated. Twelve differential metabolites—which are primarily related to tryptophan metabolism, sphingolipid metabolism, glycerophospholipid metabolism, riboflavin metabolism, pantothenate and CoA biosynthesis, and lysine degradation—were identified in relation to COPD. After the intervention of exercise training, the levels of most metabolites were restored, and the changes in five metabolites were statistically significant, which suggested that exercise training provided effective protection against COPD and might play its role by rebalancing disordered metabolism pathways. This work enhanced our comprehension of the protective mechanism of exercise training on COPD.     

Application of dynamic liquid-phase microextraction and injection port derivatization combined with gas chromatography–mass spectrometry to the determination of acidic pharmaceutically active compounds in water samples

A method has been established for the determination of four pharmaceutically active compounds (ibuprofen, ketoprofen, naproxen and clofibric acid) in water samples using dynamic hollow fiber liquid-phase microextraction (HF/LPME) followed by gas chromatography (GC) injection port derivatization and GC–mass spectrometric (MS) determination. Dynamic HF/LPME is a novel approach to microextraction that involves the use of a programmable syringe pump to move the liquid phases participating in the extraction so as to facilitate the process. Trimethylanilinium hydroxide (TMAH) was used as derivatization reagent for the analytes to increase their volatility and improve chromatographic separation. Parameters that affect extraction efficiency (selection of organic solvent, volume of organic solvent, agitation in the donor phase, plunger movement and extraction time) were investigated. Under optimal conditions, the proposed method provided good enrichment factors up to 251, reproducibility ranging from 3.26% to 10.61%, and good linearity from 0.2 to 50 μg/L. The limits of detection ranged between 0.01 and 0.05 μg/L (S/N = 3) using selective ion monitoring. This method was applied to the determination of the four pharmaceutically active compounds in tap water and wastewater collected from a drain in the vicinity of a hospital.     

UPLC–MS/MS method for the determination of voriconazole plasma concentration from pediatric patients with hematologic tumors: An application toward personalized therapy

Untreated invasive fungal infection is one of the important risk factors affecting the prognosis of pediatric patients with hematologic tumors. Voriconazole (VOR) is the first-line antifungal drug for the treatment of Aspergillus infections. In order to reduce the risk of adverse drug reactions while producing an ideal antifungal effect, therapeutic drug monitoring was performed to maintain the VOR plasma concentration in a range of 1,000–5,500 ng/ml. In the present study, a reliable, accurate, sensitive and quick ultra-high performance liquid chromatograph–tandem mass spectrometry (UPLC–MS/MS) method was developed for the determination of the VOR level. Protein precipitation was performed using acetonitrile, and then the chromatographic separation was carried out by UPLC using a C₁₈ column with the gradient mobile phases comprising 0.1% methanoic acid in acetonitrile (A) and 0.1% methanoic acid in water (B). In the selective reaction monitor mode, the mass spectrometric detection was carried out using an TSQ Endura triple quadruple mass spectrometer. The performance of this UPLC–MS/MS method was validated as per the National Medical Products Administration for Bioanalytical Method Validation. Additionally, the plasma concentrations of VOR in pediatric patients with hematologic tumors were detected using this method, and the analyzed results were used for personalized therapy.     

In vivo and in vitro metabolite profiling of nirmatrelvir using LC–Q-ToF–MS/MS along with the in silico approaches for prediction of metabolites and their toxicity

Nirmatrelvir (NRV), a 3C-like protease or M^pro inhibitor of SARS-CoV-2, is used for the treatment of COVID-19 in adult and paediatric patients. The present study was accomplished to investigate the comprehensive metabolic fate of NRV using in vitro and in vivo models. The in vitro models used for the study were microsomes (human liver microsomes, rat liver microsomes, mouse liver microsomes) and S9 fractions (human liver S9 fractions and rat liver S9 fractions) with the appropriate cofactors, whereas Sprague–Dawley rats were used as the in vivo models. Nirmatrelvir was administered orally to Sprague–Dawley rats, which was followed by the collection of urine, faeces and blood at pre-determined time intervals. Protein precipitation was used as the sample preparation method for all the samples. The samples were then analysed by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC-Q-ToF-MS/MS) using an Acquity BEH C18 column with 0.1% formic acid and acetonitrile as the mobile phase. Four metabolites were found to be novel, which were formed via amide hydrolysis, oxidation and hydroxylation. Furthermore, an in silico analysis was performed using Meteor Nexus software to predict the probable metabolic changes of NRV. The toxicity and mutagenicity of NRV and its metabolites were also determined using DEREK Nexus and SARAH Nexus.     

An integrated approach of high-performance liquid chromatography–mass spectrometry-based chemical profiling,network pharmacology,and molecular docking to reveal the potential mechanisms of Qishen Gubiao granules for treating coronavirus disease 2019

Qishen Gubiao granules, a traditional Chinese medicine preparation composed of nine herbs, have been widely used to prevent and treat coronavirus disease 2019 with good clinical efficacy. In the present study, an integrated strategy based on chemical profiling followed by network pharmacology and molecular docking was employed, to explore the active components and potential molecular mechanisms of Qishen Gubiao granules in the therapy of coronavirus disease 2019. Using the ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry technique, a total of 186 ingredients corresponding to eight structure types in Qishen Gubiao preparation were identified or structurally annotated with the elucidation of the fragmentation pathways in the typical compounds. The network pharmacology analysis screened 28 key compounds including quercetin, apigenin, scutellarein, luteolin and naringenin acting on 31 key targets, which possibly modulated signal pathways associated with immune and inflammatory responses in the treatment of coronavirus disease 2019. The molecular docking results observed that the top 5 core compounds had a high affinity for angiotensin-converting enzyme 2 and 3-chymotrypsin-like protease. This study proposed a reliable and feasible approach for elucidating the multi-components, multi-targets, and multi-pathways intervention mechanism of Qishen Gubiao granules against coronavirus disease 2019, providing a scientific basis for its further quality evaluation and clinical application.     

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury,butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–inductively coupled plasma mass spectrometry (GC–ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt₄ and NaBPr₄ was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing ²⁰¹Hg-enriched methylmercury (MeHg), ¹⁹⁹Hg-enriched inorganic mercury (iHg), ¹¹⁹Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade ¹¹⁶Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12 % for all analytes, and LOQs of 0.06–1.45 pg for GC–MS and 0.02–0.27 pg for GC–ICP/MS.

A strategy for the rapid discovery and validation of active diterpenoids as quality markers in different habitats of Langdu using ultrahigh-performance liquid chromatography–tandem mass spectrometry with multivariate statistical analysis

Langdu, known as a traditional Chinese medicine, was identified as the roots of species of Euphorbia ebracteolata Hayata and Euphorbia fischeriana Steud, displaying anti-tuberculosis activity. To clarify the potent quality markers of Langdu, this research first developed a fast and sensitive ultrahigh-performance liquid chromatography–tandem mass spectrometry method for the quantification of 13 diterpenoids in Langdu. The developed method was further applied in the analyses of 12 authentic E. ebracteolata and E. fischeriana samples collected in northern and southeastern China. Then, the anti-tuberculosis evaluation of 12 batches of Langdu samples was performed in vitro. Finally, partial least squares discrimination analysis was used in the discrimination of E. ebracteolata and E. fischeriana from different origins and processing methods. Jolkinolide A ( 1 ), jolkinolide E ( 3 ), yuexiandajisu D ( 6 ), and ebractenone A ( 11 ) were identified as key, potent diterpenoids for the quality control of E. ebracteolata Hayata and E. fischeriana Steud. The present study established a qualitative chemical analysis method for Langdu (E. ebracteolata and E. fischeriana) and suggested the key bioactive components that will improve qualitative control methodology for this important medicine.