In this study, a detailed experimental characterization of the relative ion abundance bias and the ion fragmentation in an ion funnel trap were reported by using both cytochrome C and ubiquitin samples in a tandem ion funnel hybrid ion mobility spectrometer/quadrupole time-of-flight mass spectrometer (IMS/QTOF MS). Specifically, signals of multiple interfering ions appeared in the MS spectra acquired at a relatively short ion filling time of the ion funnel trap for both selected protein samples, while they were absent at longer ion filling time. Further MS/MS studies confirmed that these interfering ions are derived from the fragmentation of the protein ions occurring before the IMS drift tube. The possible reasons for the ion fragmentation, including the ion fragmentation at the exit of the inlet capillary and the radio frequency (RF) heating in two ion funnels, were systematically studied. By carefully adjusting the voltages on each relevant component, the experimental results demonstrated that the ion fragmentation indeed occurs in the ion funnel trap due to excessive RF heating when the RF voltage was higher than 130 V. Therefore, it is extremely important to set the reasonable operation conditions for the ion funnel trap to avoid both ion fragmentation and ion storage bias in IMS/QTOF MS.
Anisi Stellati Fructus is the fruit of Illicium verum Hook. F. (Chinese star anise), which is a significant Chinese medicinal herb. It is reported to possess antimicrobial, antiviral, and antioxidant properties, and has also been documented to improve digestion and alleviate symptoms of dysentery, dyspepsia, asthma, flatulence, menstrual irregularities, colic, inflammation, bronchitis, and rheumatic diseases. At present, a variety of compounds have been isolated from Anisi Stellati Fructus. This study systematically investigated the chemical components of Anisi Stellati Fructus by a method of ultra-high-performance liquid chromatography-quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS) combined with comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS). For the non-volatile constituents, the methanol extract of Anisi Stellati Fructus was separated prior to MS analysis using a Waters BEH C18 column (100 mm×2.1 mm, 1.7 μm) with a gradient elution system consisting of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Mass spectrometric detection was performed under both positive and negative electrospray ionization (ESI) modes, and the major chromatographic peaks were identified based on accurate mass measurements, secondary mass spectral fragmentation patterns, consulting literature reports, mass spectrometry databases, fragmentation regularities of mass spectra, retention time, as well as reference compounds. For the volatile components, headspace solid-phase microextraction (HS-SPME) was employed for sample introduction. The separation was achieved using a combination of DB-Heavywax (30 m×250 μm, 0.25 μm) and DB-17 MS (1.1 m×180 μm, 0.18 μm) capillary columns under temperature programming. Electron ionization (EI) was used for ionization, with a mass scanning range of m/z 45-550. The identification of volatile compounds was accomplished by combining NIST standard spectral library searches with information from published literatures. The results showed that UPLC-Q-Orbitrap-HRMS identifies 45 compounds, including 13 phenylpropanoids, 8 phenolic acids, 18 flavonoids, and 6 other compounds. Meanwhile, GC×GC-MS identifies 108 volatile components, which are categorized into 66 terpenoids, 8 phenylpropanoids, 7 alkanes, 10 esters, 5 ketones, 3 alcohols, and 9 other compounds. This comprehensive and systematic qualitative analysis of the chemical constituents of Anisi Stellati Fructus provides a solid foundation for further research on its pharmacodynamic material basis, quality control, and pharmacological mechanisms.
Polysorbates are a class of hydrophilic non-ionic surfactants characterized by exceptional biocompatibility, rendering them the most frequently employed excipients in therapeutic monoclonal antibodies’ formulations. These compounds play a pivotal role in preventing the formation of antibody aggregates and their subsequent inactivation, while simultaneously mitigating non-specific adsorption onto surfaces of glass vials or intravenous infusion tubing. However, due to inherent variations in synthetic processes and diverse raw material sources, polysorbates exist as complex mixtures with significant batch-to-batch variability in their compositional profiles. Their inherent chemical instability predisposes them to degradation, with degradation products capable of interacting with protein particles present in therapeutic monoclonal antibodies to form insoluble particulates and visible foreign matters. These particulate contaminants pose substantial risks to both drug safety and therapeutic efficacy, thereby underscoring the critical importance of investigating the degradation mechanisms of polysorbates in antibody formulations. Chinese hamster ovary (CHO) cells represent the predominant expression system for antibody production, with trace amounts of host cell protein (HCP) residues persisting during the drug purification process. These trace amounts of HCP residues have been identified as a principal contributor to polysorbate degradation. In this study, a highly sensitive and specific mass spectrometry (MS) method was developed for quantifying trace amounts of polysorbate-degrading esterases in HCP residues. This study aims to elucidate the specific impact of HCP residues on polysorbate degradation, enable comprehensive quantitative analysis of polysorbate-degrading esterases, and thereby provide essential guidance for process optimization and quality control in antibody drug manufacturing. The methodology involved an initial non-targeted screening approach using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF MS) operating under data-dependent acquisition mode. This screening was performed on high-risk HCPs present in Protein A affinity eluates, which yielded comprehensive identification lists of polysorbate-degrading esterases at both the protein and peptide levels. Subsequently, a targeted quantitative approach was established using liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ MS), employing multiple reaction monitoring (MRM) based on characteristic peptide sequences to detect four common polysorbate-degrading esterases: lysosomal phospholipase A2, phospholipase B-like 2, lipase LIPH, and lysosomal acid lipase. The sample processing workflow underwent rigorous optimization, encompassing various parameters such as sample preparation procedures, extraction efficiency, and analytical conditions. The method underwent comprehensive validation, ultimately achieving a limit of quantification of 0.5 μg/g and a limit of detection of 0.1 μg/g. The developed method was successfully applied to quantify these four polysorbate-degrading esterases across various sample types, including purification process samples, bulk drug substances, and final drug products. The results demonstrated that the method exhibits exceptional rapidity, sensitivity, and specificity, making it well-suited for both identification and quantitative analysis of high-risk polysorbate-degrading esterases in therapeutic monoclonal antibodies. This capability provides a robust analytical tool for ensuring drug quality and safety throughout the manufacturing process, offering significant advantages for process development and quality control in antibody drug production.
In this study,a method of ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF MS)was used to analyze the in vitro components,blood-absorbed components,intestinal content components,as well as their metabolites of Codonopsis radix-Rehmanniae radix praeparata extract.The aqueous extract of Codonopsis radix-Rehmanniae radix praeparata was prepared by aqueous decoction and freeze-dried,and was administered to Wistar rats by gavage at a dose of 2.7 g/kg/d for 7 consecutive days;serum and colonic contents samples were collected at 0.25,0.5,1,and 2 h after the last administration.A Supelco Ascentis Express C18 column(50 mm×3.0 mm,2.7 μm)was used with 0.1％formic acid aqueous solution and acetonitrile as the mobile phase for gradient elution,and the mass spectrometry data were collected in both positive and negative ion modes of the electrospray ionisation source,respectively.The collected data and the self-composition database were imported into the UNIFI platform,and the manual identification and qualitative analysis of each component were performed by combining the relevant information of each compound in the database.A total of 103 chemical constituents including phenylethanol glycosides,phenylpropanoids,flavonoids,furans,alkaloids,terpenoids,amino acids,sugars,and organic acids,were identified in Codonopsis radix-Rehmanniae radix praeparata extract.The drug-containing serum samples and intestinal contents samples were further analyzed.A total of 28 prototypical blood-absorbed components and 45 intestinal content components were identified.The prototypical blood-absorbed components included 6 terpenoids,3 violet ketones,1 phenylethanol glycoside,1 phenylpropanoid,1 acetylide and polyacetylide,1 flavonoid,1 alkaloid,3 amino acids,1 saccharide,9 organic acids and 1 other compound.The prototypical composition of the intestinal content components consists of 4 cyclic enol ether terpene glycosides,3 sesquiterpene lactones,4 phenylethanol glycosides,2 violet ketones,1 furan,1 hexanol glycoside and hexenol glycoside,2 flavonoids,1 phenylpropanoid,1 amino acid,4 alkaloids,3 organic acids,1 saccharide,and 2 other compounds.The metabolites were mainly derived from cyclic enol ether terpene glycosides,sesquiterpene lactones,phenylethanol glycosides,alkaloids and furans.The main metabolic pathways included phase Ⅰ metabolic reactions such as deglycosylation,methylation,and hydroxylation,as well as phase Ⅱ metabolic reactions such as glucuronidation and sulfation.The UPLC-Q-TOF MS method enables rapid,efficient and accurate analysis of the chemical constituents,blood-absorbed components and intestinal content components of the Codonopsis radix-Rehmanniae radix praeparata extracts,thereby providing a methodological basis and reference for elucidating the pharmacological materials basis and mechanism of action of Codonopsis radix-Rehmanniae radixpraeparata.
Xanthii Fructus is the dried mature fruit with involucre of Xanthium sibiricum Patr. It has multiple pharmacological effects, including anti-inflammatory and analgesic, antibacterial, antiallergic, antitumor activities. Compared with the raw herb, stir-fried Xanthii Fructus has lower toxicity while retaining equivalent pharmacological efficacy. In traditional Chinese medicine, it is primarily used for treating symptoms such as allergic rhinitis. Our preliminary studies have indicated that the ethanol extract of stir-fried Xanthii Fructus demonstrates positive therapeutic effects on rats with rheumatoid arthritis, which can effectively reduce the levels of inflammatory mediators such as TNF-α and IL-1β. However, the precise chemical constituents responsible for its therapeutic effects remain to be elucidated. In this study, a method of ultra-performance liquid chromatography coupled with quadrupole-electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap MS) was established for the analysis of stir-fried Xanthii Fructus. Firstly, the components of stir-fried Xanthii Fructus were extracted by the heated reflux extraction method using 75% ethanol as the extraction solvent, with the extraction process repeated three times. Subsequently, the ethanol extract was separated on a Waters Acquity UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm). The mobile phase consisted 0.1% formic acid aqueous solution (A) and methanol (B) at a flow rate of 0.3 mL/min, with gradient elution applied. The electrospray ionization (ESI) source was utilized, which facilitated the execution of full scanning and secondary mass spectrometry scanning under both positive and negative ion modes, respectively. Finally, the acquired data were processed for peak extraction, peak alignment and deconvolution using Progenesis QI 3.0 software for comparison with reference substance databases and other theoretical databases, such as ChemSpider, PubChem, PubMed and Web of Science. A total of 26 chemical components were identified, including 15 phenylpropanoids, 4 organic acids, 2 flavones, 2 sesquiterpenes, 2 glycosides and 1 other component. Among them, phenylpropanoids and organic acids such as chlorogenic acid, caffeic acid, cynarin, ferulaldehyde, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C were more abundant and response in the mass spectra. The mass spectrometry fragmentation pathways of representative compounds in each class were also analyzed. This study provides a basis for the further exploration of the pharmacological effects and in vivo metabolic processes of stir-fried Xanthii Fructus.
