Comparative chemical fingerprinting of Oroxylum indicum and Scutellaria baicalensis using liquid chromatography and mass spectrometry

Introduction: Identification of Oroxylum indicum and Scutellaria baicalensis provides an interesting challenge in selection of biomarker compound to be used in routine analysis. Both plants have similar phytochemical profile and are rich sources of flavones and flavone glycosides. The objective of this study was to prepare the chemical fingerprinting of O. indicum bark and S. baicalensis roots using the liquid chromatography and mass spectroscopy in single chromatographic method. Materials and methods: Extracts prepared using various solvent systems (methanol, aqueous methanol, chloroform, hexane, and water) of both plants were analyzed using C18 reverse phase column with solvent system containing acetonitrile and 0.1% formic acid. Major flavonoids were identified based on mass spectra, fragmentation pattern, and UV spectra. Results: In this article, well-resolved high-performance liquid chromatographic (HPLC) separation in both plant extracts was obtained and chemical fingerprints for both plant extracts were established and flavonoids present (baicalin, oroxylin A-7-O-glucuronide, chrysin-7-O-glucuronide, baicalein, chrysin, oroxylin-A, wogonin, skullcap flavone II) were identified as possible biomarkers. Conclusion: Mass spectrometry coupled with HPLC can be a tool for fingerprinting of various natural products used in dietary supplement industry. The fingerprint developed in the article can be used for quality evaluation as well as identifying possible adulteration of extracts of both the plants.     

Elucidating the structure of carbon nanoparticles by ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry

A fast and accurate ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) method was developed for the separation and structural elucidation of fluorescent carbon nanoparticles (CNP). The CNP was synthesised from microwave-assisted pyrolysis of citric acid (CA) and 1,2-ethylenediamine (EDA). By using UPLC separation, the CNP product was well separated into ten fractions within 4.0 min. Based on high-accuracy MS and MS/MS analyses, the CNP species were revealed to display six kinds of chemical formulas, including (C₁₀H₂₀N₄O₅)_n, (C₈H₁₂N₂O₅)_n, (C₁₆H₂₂N₄O₉)_n, (C₆H₈O₇)_n, (C₁₄H₁₈N₂O₁₁)_n, and (C₁₄H₁₆N₂O₁₀)_n. In particular, our study revealed for the first time that the CNP species exist as supramolecular clusters with their individual monomers units linked together through non-covalent bonding forces. These findings clearly indicated the usefulness of UPLC-ESI-Q-TOF-MS/MS in identifying the chemical composition of CNP product. It is anticipated that our proposed methodology can be applied to study the structure-property relationships of CNP, facilitating in the production of CNP with desirable spectral features.     

Characterization of chemical components of Periplocae Cortex and their metabolites in rats using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Periplocae Cortex, named Xiang-Jia-Pi in China, has been widely used to treat autoimmune diseases, especially rheumatoid arthritis. However, the in vivo substances of Periplocae Cortex remain unknown yet. In this study, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used for profiling the chemical components and related metabolites of Periplocae Cortex. A total of 98 constituents were identified or tentatively characterized in Periplocae Cortex: 42 C21 steroidal glycosides, 10 cardiac glycosides, 23 organic acids, 4 aldehydes, 7 triterpenes, and 12 other types. Among them, 18 components were unambiguously identified by comparison with reference standards. In addition, 176 related xenobiotics (34 prototypes and 142 metabolites) were screened out and characterized in rats’ biosamples (plasma, urine, bile, and feces) after the oral administration of Periplocae Cortex. Moreover, the metabolic fate of periplocoside S-4a, a C21 steroidal glycoside, was proposed for the first time. In summary, phase II reactions (methylation, glucuronidation, and sulfation), phase I reactions (hydrolysis reactions, oxygenation, and reduction), and their combinations were the predominant metabolic reactions of Periplocae Cortex in rat. It is the first report to reveal the in vivo substances and metabolism feature of Periplocae Cortex. This study also provided meaningful information for further pharmacodynamics study of Periplocae Cortex, as well as its quality control research.     

Analysis of biologically active compounds in water by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry

A new method based on ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry ((Q-ToF)-MS) was developed for the analysis of 32 biologically active compounds including anti-inflammatories, analgesics, lipid regulators, psychiatric drugs, anti-ulcer agents, antibiotics, beta-blockers and phytoestrogens. This new method allows chromatographic analysis in 14 min, with instrumental detection limits from 2 to 84 pg, and limits of quantification ranging from 0.1 to 15 ng/L in tap water, and from 2 to 300 ng/L in wastewater. The potential of liquid chromatography with triple quadrupole mass spectrometry (LC/QqQ-MS) was compared with that of UPLC/(Q-ToF)-MS for the analysis of biologically active compounds in water samples. LC/Q-ToF provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. The available mass resolution of ToF instruments diminishes the problem of isobaric interferences and helps the analysis of trace compounds in complex samples. In this work UPLC/Q-ToF chromatograms were recorded containing full scan spectral data. The m/z values of analytes were extracted from the total ion chromatogram (TIC) and the accurate masses of the compounds were obtained. In addition, to increase the selectivity of ToF measurements a narrow accurate mass interval (20 m m/z units mass window) was used to reconstruct the chromatographic traces. However, regarding quantitative performance in terms of dynamic range and limits of detection (LODs), typical LODs achieved by QqQ instruments operating in multiple-reaction monitoring (MRM) mode ranged from 1 to 50 ng/L in wastewater, and the linear response for QqQ instruments generally covers three orders of magnitude. This is an important advantage over ToF instruments and one of the reasons why QqQ instruments are widely used in quantitative environmental analysis.     

Identifying chemical markers in wine-processed Salvia miltiorrhiza using ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry

To find the chemical markers of wine-processed Salvia miltiorrhiza (WSM), 76 constituents, including diterpenoid quinones and phenolic acids in Salvia miltiorrhiza (SM) and WSM, were profiled using ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS/MS) in positive- and the negative-ion modes. Thirty compounds were screened out as candidate differential components using chemometrics analysis, and the concentration of most compounds increased after processing with wine. Seven compounds, namely tanshinone IIA, magnesium lithospermate B, salvianolic acid G, cryptotanshinone, isocryptotanshinone, salvianolic acid B, and rosmarinic acid, were selected as chemical markers of WSM using variable importance of the project. This study revealed the chemical markers of WSM and confirmed that WSM can improve the extraction and solubility effect of chemical constituents.     

High-temperature ultra-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry applied to ibuprofen metabolites in human urine

The application of sub-2 microm porous particle liquid chromatography (LC) operated at elevated temperatures, coupled with time-of-flight mass spectrometry (MS), to the separation and identification of metabolites of ibuprofen present in human urine following oral administrations is illustrated. The LC/MS system generated a high-resolution analytical separation that, with an analysis time of 20 min, provided a peak capacity in the order of ca. 350. Using this system a total of nine glucuronides of the drug and its metabolites were detected, including a number of isomeric acyl glucuronides of ibuprofen itself, a side-chain-oxidized carboxylic acid acyl glucuronide and a number of acyl glucuronides of various hydroxylated metabolites. The identities of the metabolites were confirmed by their accurate mass values and the presence of the common fragment ions from ibuprofen.     

Characterization of glutathione conjugates of chloroacetanilide pesticides using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Glutathione S-transferases (GSTs) isolated from maize were used to catalyze the conjugation of glutathione (GSH) with chloroacetanilide herbicides, producing stable conjugates that were structurally characterized using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QqToF-MS) and liquid chromatography/ion trap mass spectrometry (LC/IT-MS). Enzyme-mediated dechlorination of alachlor, metolachlor, and propachlor resulted during GSH conjugation as revealed by the mass spectra of the conjugates, which was confirmed by the loss of the chlorine isotopic signature and from high accurate mass measurements. Several fragmentation patterns in the mass spectra of the chloroacetanilide-GSH conjugates can be used to verify the identities of the enzyme reaction products, such as characteristic ions corresponding to the neutral loss of glutamic acid residue (129 Da) and water (18 Da) observed in the product ion spectrum. For the first time, data are presented showing detection of chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates.     

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.     

Analyses of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry

Two liquid chromatography mass spectrometric techniques, i.e. ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-Tof MS) and high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS), were used for quantification, confirmation or identification of six macrolide antibiotic residues and/or their degradation products in eggs, raw milk, and/or honey. Macrolides were extracted from food samples by acetonitrile or phosphate buffer (0.1 M, pH 8.0), and sample extracts were further cleaned up using solid-phase extraction cartridges. UPLC/Q-Tof data were acquired in Tof MS full scan mode that allowed both quantification and confirmation of macrolides, and identification of their degradation products. LC/MS/MS data acquisition was achieved using multiple reaction monitoring (MRM), i.e. two transitions, to provide a high degree of sensitivity and repeatability. Matrix-matched standard calibration curves with the use of roxithromycin as an internal standard were utilized to achieve the best accuracy of the method. Both techniques demonstrated good quantitative performance in terms of accuracy and repeatability. LC/MS/MS had advantages over UPLC/Q-Tof MS in that its limits of detection were lower and repeatability was somewhat better. UPLC/Q-Tof provided ultimate and unequivocal confirmation of positive findings, and allowed degradation product identification based on accurate mass. The combination of the two techniques can be very beneficial or complementary in routine analysis of macrolide antibiotic residues and their degradation products in food matrices to ensure the safety of food supply.     

Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry

A lipidomics strategy, combining high resolution reversed-phase liquid chromatography (RPLC) with high resolution quadrupole time-of-flight mass spectrometry (QqTOF), is described. The method has carefully been assessed in both a qualitative and a quantitative fashion utilizing human blood plasma. The inherent low technical variability associated with the lipidomics method allows to measure 65% of the features with an intensity RSD value below 10%. Blood plasma lipid spike-in experiments demonstrate that relative concentration differences smaller than 25% can readily be revealed by means of a t-test. Utilizing an advanced identification strategy, it is shown that the detected features mainly originate from (lyso-)phospholipids, sphingolipids, mono-, di- and triacylglycerols and cholesterol esters. The high resolution offered by the up-front RPLC step further allows to discriminate various isomeric species associated with the different lipid classes. The added value of utilizing a Jetstream electrospray ionization (ESI) source over a regular ESI source in lipidomics is for the first time demonstrated. In addition, the application of ultra high performance LC (UHPLC) up to 1200 bar to extend the peak capacity or increase productivity is discussed.     

Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.     

Rapid isolation of new furostanol saponins from fenugreek seeds based on ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight tandem mass spectrometry

An ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight tandem mass spectrometry method was established to rapidly identify and guide the isolation of target saponins from fenugreek seeds. Based on the online screening performance, totally forty-six furostanol saponins were detected and elucidated. Among them, twenty compounds were predicted to be new. To rapidly obtain new furostanol saponins from these seeds, a further phytochemical study was carried out under the guidance of the ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight tandem mass spectrometry. Finally, six new furostanol saponins, named as trigoneosides XIV (1), XV (2), XVI (3), XVIIa (4), XVIIb (5), and XIV (6), together with one known furostanol saponin, parvifloside (7), were rapidly obtained, and their definitive structures were determined by NMR and chemical evidence.     

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.     

Rapid profiling of cantharidin analogs in Mylabris phalerata Pallas by ultra-performance liquid chromatography–quadrupole time-of-flight–tandem mass spectrometry

We evaluated the protective effect and toxicity of extracts from Mylabris phalerata Pallas by measuring the activated partial thromboplastin time, prothrombin time, venous thrombosis and acute toxicity in rats. Results showed the petroleum ether and water fractions of M. phalerata inhibited thrombosis but hardly prolonged the activated partial thromboplastin time and prothrombin time in rats. The trichloromethane fraction had obvious toxicity with an LD₅₀ of 0.2 g/kg in vivo, and contained many cantharidin analogs (CAs) by ultra-performance liquid chromatography–quadrupole ion trap–tandem mass spectrometry (UPLC–QTRAP–MS/MS). CAs are the major potential bioactivity constituent in M. phalerata. An effective and reliable UPLC–QTRAP–MS/MS method was successfully developed to separate and identify CAs. The fragmentation patterns of five purified compounds were applied to elucidate the structure of their analogs. Thirty-four CAs were characterized or tentatively identified, eight of which are proposed to be novel compounds ( 13 – 17 , 20 , 21 , 23 ), and their fragmentation patterns were investigated for the first time. Most importantly, a rapid and reliable UPLC–MS method was developed to identify the CAs of M. phalerata. This method has contributed to the discovery of most of these unknown analogs or their metabolites in M. phalerata effectively and quickly, and does not rely on limited chemical structural diversity libraries.     

High-resolution profiling of oxylipin-containing galactolipids in Arabidopsis extracts by ultra-performance liquid chromatography/time-of-flight mass spectrometry

A high-resolution ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) method using in-source collision-induced dissociation (CID) was developed for globally profiling oxylipin-containing galactolipids in Arabidopsis wounded leaves. MS and pseudo-MS/MS spectra were obtained during a single analytical run by switching a lens of the TOFMS transfer optics from low to high voltage. Numerous known galactolipids were observed, and four novel mono- or di-galactosyl monoacylglycerides (MGMGs or DGMGs) containing oxophytodienoic acid (OPDA) or dinor-oxophytodienoic acid (dn-OPDA), esterified respectively at the sn1 and the sn2 positions, were identified. Rapid microisolation of the galactolipids followed by alkaline and enzymatic hydrolyses enabled the release of the esterified oxylipins, which allowed for the unambiguous characterization of the oxylipin-containing monoacylglycerides. Their strong induction in response to wounding indicates that these compounds are probably lysogalactolipids formed from galactosyldiglycerides in the injured tissues.