Radical Fragment Ions in Collision-Induced Dissociation Mass Spectrometry

Collision-induced dissociation (CID) is a common fragmentation strategy in mass spectrometry (MS) analysis. A conventional understanding is that fragment ions generated in low-energy CID should follow the even-electron rule. As such, (de)protonated precursor ions should predominately generate (de)protonated fragment ions with very few radical fragment ions (RFIs). However, the extent to which RFIs present in MS2 spectra has not been comprehensively investigated. This work uses the latest NIST 20 tandem mass spectral library to investigate of the occurrence of RFIs in CID MS2 experiments. In particular, RFIs were recognized using their integer double bond equivalent (DBE) values calculated from their annotated molecular formulas. Our study shows unexpected results as 65.4% and 68.8% of MS2 spectra contain at least 10% RFIs by ion-count (total number of ions) in positive and negative electrospray ionization (ESI) modes, respectively. Furthermore, we classified chemicals based on their compound classes and chemical substructures, and calculated the percentages of RFIs in each class. Results show that “Organic 1,3-dipolar compounds” and “Lignans, neolignans and related compounds” are the top 2 compound superclasses which tend to produce RFIs in their CID MS2 spectra. Moreover, aromatic, arylbromide, heteroaromatic, alkylarylether, phenol, and conjugated double bond-containing chemicals are more likely to produce RFIs. We also found four possible patterns of change in RFI percentages as a function of CID collision energy. Finally, we demonstrate that the inadequate consideration of RFIs in most conventional bioinformatic tools might cause problems during in silico fragmentation and de novo annotation of MS2 spectra. This work provides a further understanding of CID MS2 mechanism, and the unexpectedly large percentage of RFIs suggests a need for consideration in the development of bioinformatic software for MS2 interpretation.

Simultaneous Determination of Cis- and Trans-Palmitoleic acid in Rat Serum by LC-ESI-MS/MS

Background: Cis-palmitoleic acid (cPOA) and trans-palmitoleic acid (tPOA) are isomers of palmitoleic acid, which are monounsaturated fatty acids, affecting glucose and lipid metabolism, and reducing insulin resistance. tPOA was better than cPOA in regulating lipid metabolism in hyperlipidemia mice, but the metabolic transformation and structure-activity relationship haven’t been reported. Method: A precise and accurate liquid chromatography-tandem mass spectroscopy (LC–MS/MS) method was developed to determine cPOA and tPOA simultaneously. cPOA and tPOA were administered i.g. (intragastric gavage) to rats at 75 mg/kg respectively, serum samples were analyzed by LC-ESI-MS/MS on a reverse-phase BDS C18 column equilibrated and eluted with acetonitrile (A) and water (B) (A:B = 80:20, v/v) at a flow rate of 0.3 mL/min and the injection volume was 1 μL. Results: The calibration curves for cPOA and tPOA were linear over the range 0.1~12 μg/mL. Analytes were monitored by selected-reaction monitoring in negative electrospray ionization mode. The Tmax of cPOA was 0.94±0.44 h and the Cmax 8.17±1.97 μg/L, and the Tmax of tPOA was 1.50±0.98 h and the Cmax 14.77±11.91 μg/L. AUC0-24h of cPOA and tPOA were 59.45±29.83 and 113.88±72.25 mg/L*h.Conclusions: The method was applied in pharmacokinetic study of cPOA and tPOA successfully. Besides, it’s found the concentration of cPOA and tPOA fluctuated in serum of rats with the consistent trend, which may be reciprocal bio-convert in biological activity in the body.

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

A comprehensive non-targeted analysis study of the prenatal exposome

Recent technological advances in mass spectrometry have enabled us to screen biological samples for a very broad spectrum of chemical compounds allowing us to more comprehensively characterize the human exposome in critical periods of development. The goal of this study was three-fold: 1) to analyze 590 matched maternal and cord blood samples (total 295 pairs) using non-targeted analysis (NTA); 2) examine the differences in chemical abundance between maternal and cord blood samples; and 3) examine the associations between exogenous chemicals and endogenous metabolites. We analyzed all samples with high-resolution mass spectrometry (HRMS) using liquid chromatography – quadrupole time-of-flight mass spectrometry (LC-QTOF/MS), in both positive and negative electrospray ionization modes (ESI+ and ESI-) and in soft ionization (MS) and fragmentation (MS/MS) modes for prioritized features. We confirmed 19 unique compounds with analytical standards, we tentatively identified 73 compounds with MS/MS spectra matching, and we annotated 98 compounds using an annotation algorithm. We observed 103 significant associations in maternal and 128 in cord samples between compounds annotated as endogenous and compounds annotated as exogenous. An example of these relationships was an association between 3 poly and perfluoroalkyl substances (PFAS) and endogenous fatty acids in both the maternal and cord samples indicating potential interactions between PFAS and fatty acid regulating proteins.

Simultaneous determination of two galangin metabolites from Alpinia Officinarum Hance in rat plasma by UF LC-MS/MS and its application in pharmacokinetics study

Galangin has multiple pharmacological efficacies, such as anti-cancer, anti-inflammation and anti-oxidation. Galangin can be rapidly converted into glucuronidated metabolites in vivo. This study aimed to establish an UFLC-MS/MS analytical method to simultaneously determine the concentrations of two glucuronidated metabolites of galangin, galangin-3-O-β-D-glucuronic acid (GG-1) and galangin-7-O-β-D-glucuronic acid (GG-2) in rat plasma. After oral administration of galangal extract (0.3 g/kg), blood samples were collected from the orbital sinus, then treated by methanol precipitation and further gradient-eluted with Phenomenex Kinetex 2.6 µm XB-C18 column. The mass spectrometer was manipulated in the negative electrospray ionization (ESI) and selected multiple reaction monitoring (MRM) mode for the analytes. The precursor-to-product ion pairs applied for GG-1, GG-2 and chrysin (as the internal standard, IS) were m/z 445.2→269.0, 445.2→268.9 and 253.0→142.9, respectively. The results showed that the linear ranges for both GG-1 and GG-2 were 2.0–2000.0 ng/mL (r2 > 0.995). The inter- and intra-day precision were 89.3%–109.2%, RSD was less than 15%, and the repeatability was good. The recoveries of both metabolites and IS were over 89%, and matrix effect was within 15%. The validated analytical method was further applied to study the pharmacokinetic profiles of GG-1 and GG-2 in vivo. The pharmacokinetic parameters suggested that Tmax of GG-1 was equivalent to that of GG-2, and MRT0-t, t1/2 of GG-2 were a little higher than those of GG-1. Importantly, AUC0-t and Cmax of GG-2 were almost twice as those of GG-1. In short, the validated UFLCMS/MS analytical method was feasible to simultaneously determine two galangin metabolites GG-1 and GG-2 in rat plasma and further analyze in vivo metabolism of galangin.

Towards Extending the Detection Window of Gamma-Hydroxybutyric Acid—An Untargeted Metabolomics Study in Serum and Urine Following Controlled Administration in Healthy Men

In forensic toxicology, gamma-hydroxybutyrate (GHB) still represents one of the most challenging drugs of abuse in terms of analytical detection and interpretation. Given its rapid elimination, the detection window of GHB in common matrices is short (maximum 12 h in urine). Additionally, the differentiation from naturally occurring endogenous GHB, is challenging. Thus, novel biomarkers to extend the detection window of GHB are urgently needed. The present study aimed at searching new potential biomarkers of GHB use by means of mass spectrometry (MS) metabolomic profiling in serum (up to 16.5 h) and urine samples (up to 8 h after intake) collected during a placebo-controlled crossover study in healthy men. MS data acquired by different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization each) were filtered for significantly changed features applying univariate and mixed-effect model statistics. Complementary to a former study, conjugates of GHB with glycine, glutamate, taurine, carnitine and pentose (ribose) were identified in urine, with particularly GHB-pentose being promising for longer detection. None of the conjugates were detectable in serum. Therein, mainly energy metabolic substrates were identified, which may be useful for more detailed interpretation of underlying pathways but are too unspecific as biomarkers.

LC-MS/MS Based Metabolomics Reveal Candidate Biomarkers and Metabolic Changes in Different Buffalo Species

Consumers have shown more and more interest in high-quality and healthy dairy products and buffalo milk is commercially more viable than other milks in producing superior dairy products due to its higher contents of fat, crude protein, and total solids. Metabolomics is one of the most powerful strategies in molecular mechanism research however, little study has been focused on the milk metabolites in different buffalo species. Therefore, the aim of this study was to explore the underlying molecular mechanism of the fatty synthesis and candidate biomarkers by analyzing the metabolomic profiles. Milk of three groups of buffaloes, including 10 Mediterranean, 12 Murrah, and 10 crossbred buffaloes (Murrah × local swamp buffalo), were collected and UPLC-Q-Orbitrap HRMS was used to obtain the metabolomic profiles. Results showed that milk fatty acid in Mediterranean buffalo was significantly higher than Murrah buffalo and crossbred buffalo. A total of 1837/726 metabolites was identified in both positive and negative electrospray ionization (ESI±) mode, including 19 significantly different metabolites between Mediterranean and Murrah buffalo, and 18 different metabolites between Mediterranean and crossbred buffalo. We found 11 of the different metabolites were both significantly different between Mediterranean vs. Murrah group and Mediterranean vs crossbred group, indicating that they can be used as candidate biomarkers of Mediterranean buffalo milk. Further analysis found that the different metabolites were mainly enriched in fat synthesis related pathways such as fatty acid biosynthesis, unsaturated fatty acid biosynthesis, and linoleic acid metabolism, indicating that the priority of different pathways affected the milk fat content in different buffalo species. These specific metabolites may be used as biomarkers in the identification of milk quality and molecular breeding of high milk fat buffalo.

Simultaneous quantification method for 5-FU, uracil, and tegafur using UPLC-MS/MS and clinical application in monitoring UFT/LV combination therapy after hepatectomy

Combination therapy of tegafur/uracil (UFT) and leucovorin (LV) is widely used to treat colorectal cancers. Although this therapy has a significant therapeutic effect, severe adverse effects occur frequently. Therapeutic drug monitoring (TDM) may help to prevent adverse effects. A useful assay that can quantitate plasma levels of 5-FU, uracil, and tegafur simultaneously for TDM has been desired, but such a method is not currently available. In this study, we aimed to develop a sensitive method for simultaneous quantification of 5-FU, uracil, and tegafur in human plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). After preparing plasma samples by protein precipitation and liquid extraction, 5-FU, uracil, and tegafur were analyzed by UPLC-MS/MS in negative electrospray ionization mode. Validation was performed according to US Food and Drugs Administration guidance. The calibration curves were linear over concentration ranges of 2–500 ng/mL for 5-FU, 20–5000 ng/mL for uracil, and 200–50,000 ng/mL for tegafur. The corresponding average recovery rates were 79.9, 80.9, and 87.8%. The method provides accuracy within 11.6% and precision below 13.3% for all three analytes. Matrix effects of 5-FU, uracil, and tegafur were higher than 43.5, 84.9, and 100.2%, respectively. This assay was successfully applied to assess the time courses of plasma 5-FU, uracil, and tegafur concentrations in two patients with colorectal liver metastasis who received UFT/LV therapy after hepatectomy. In conclusion, we succeeded to develop a sensitive and robust UPLC-MS/MS method for simultaneous quantification of 5-FU, uracil, and tegafur in human plasma. This method is potentially useful for TDM in patients receiving UFT/LV combination therapy.

Chemical Components of Aqueous Extracts of Melia azedarach Fruits and Their Effects on The Transcriptome of Staphylococcus aureus

Staphylococcus aureus is the causative agent of numerous and varied clinical infections. Crude aqueous extracts of Melia azedarach fruits inhibit the planktonic growth and initial biofilm formation of S. aureus in a dose-dependent manner. Moreover, the biofilm topologies became sparse and decreased as the concentration of the aqueous extracts increased. RNA-Seq analyses revealed 532 differentially expressed genes (DEGs) after S. aureus exposure to 0.25 mg/l extracts; 319 of them were upregulated, and 213 were downregulated. The majority of DEGs were categorized into abundant sub-groups in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, untargeted UHPLC-MS/MS analyses of the aqueous extracts of M. azedarach fruits demonstrated a highly complex profile in positive and negative electrospray ionization modes. The extracts primarily consisted of lipids and lipid-like molecules, organic acids and their derivatives, phenylpropanoids, polyketides, organoheterocyclic compounds, and benzenoids, annotated by abundant lipid maps and KEGG pathways. Overall, this study provides evidence that the aqueous extracts of M. azedarach fruits can control S. aureus infections and sought to understand the mode of action of these extracts on S. aureus.