A review article on the multiple uses of LC-MS for new drug discovery applications from HTS to ADME optimization to development.
The use of high-performance liquid chromatography combined with mass ...spectrometry (HPLC-MS) or tandem mass spectrometry (HPLC-MS-MS) has proven to be the analytical technique of choice for most assays used in various stages of new drug discovery. A summary of the key components of HPLC-MS systems, as well as an overview of major application areas that use this technique as part of the drug discovery process, will be described here. This review will also provide an introduction into the various types of mass spectrometers that can be selected for the multiple tasks that can be performed using LC-MS as the analytical tool. The strategies for optimizing the use of this technique and also the potential problems and how to avoid them will be highlighted.
•A new method was developed to separate and determine oxypeucedanin enantiomers.•Stereoselective differences were found in Angelica dahuricae Radix and rat plasma.•(+)-oxypeucedanin was present at a ...relative high level in Angelica dahuricae Radix.•(−)-oxypeucedanin showed higher plasma concentration in stereoselectivity.
In this study, a new enantioseparation method was established for the quantitative analysis of the oxypeucedanin enantiomers by using cellulose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IC. For this method, enantiomeric separation of oxypeucedanin was achieved with the mobile phase consisting of acetonitrile-water (60:40, v/v) at a flow rate of 0.5 mL/min by changing the type and proportion of mobile phase. And the quantitative determination of racemic oxypeucedanin in Angelica Dahuricae Radix (in vitro) and rat plasma (in vivo) were performed on above-mentioned condition by High PerformanceLiquid Chromatography combined with diode arrangement detector (HPLC-DAD) and mass spectrometry (HPLC-MS/MS). The precision, repeatability, stability, recovery were within the acceptance criteria. And the method was validated in the concentration range of 1-400 μg/mL for the two enantiomers in vitro and 0.2–600 ng/mL in vivo. After validation, the established method was successfully applied to the stereoselective analysis of racemic oxypeucedanin in Angelica dahurica from different regions and the stereoselective pharmacokinetic investigation in rat. Results showed that the (+)-oxypeucedanin was at a relative high level in Angelica dahuricae Radix and (−)-oxypeucedanin performed a higher plasma concentration, which demonstrated the difference of oxypeucedanin enantiomers both in vitro and in vivo.
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•A multi-analytical approach based on four different analytical methods was used for the first time.•The approach is very effective in characterizing organic materials used in the ...past.•The ointment bases of historical ointment remains from the 18th century were identified.•Contemporary pharmacists often modified the formulations to suit their needs.
A multi-analytical approach based on four different analytical methods was used for the first time to study six historical ointments from the 18th century belonging to the baroque pharmacy of Capuchin Monastery in Hradčany (Prague, Czech Republic) in order to gain information on the ointment formulation, the presence of active substances, and also possible chemical modification deriving from the procedures used for their preparation. All ointments were initially characterized by gas chromatography/mass spectrometry following saponification, extraction and derivatization to identify the main classes of ingredients. The volatile organic compounds emitted by the ointments were then characterized by gas chromatographic analysis following solid-phase microextraction, providing complementary information on the possible more volatile and active ingredients present in the ointments. Finally, the botanical/animal origin of triacylglycerols was investigated by liquid chromatography-high resolution mass spectrometry, while the presence of beeswax was proved by flow injection analysis. The results obtained were used to hypothesize the probable original medical purpose of the ointments by comparing the results with historical recipes and the period literature. The use of this comprehensive multi-analytical approach allowed us to contribute to the knowledge of ancient ointments and to obtain information on the alteration of the chemical compounds in the ointment formulations.
The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-β-d-glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After ...enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of “isoGSLs”, i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.
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•List of glucosinolates characterized satisfactorily using NMR and MS data, in total 88.•Selected glucosinolate structures deduced indirectly, in total 47.•Review of glucosinolate nomenclature, chemistry, organic synthesis and plant biochemistry.•Review of glucosinolate identification and guidelines for scientific reporting.
•Fe3O4@SiO2@MOF core–shell magnetic microspheres were prepared as MSPE sorbent.•Fe3O4@SiO2@MOF core–shell magnetic microspheres were used for MSPE of polar analytes.•Fe3O4@SiO2@MOF core–shell ...magnetic microspheres achieved high extraction efficiency.
Fe3O4@SiO2@UiO-66 core–shell magnetic microspheres were synthesized and characterized by transmission electron microscopy, scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectrometry, vibrating sample magnetometry, nitrogen adsorption porosimetry and zeta potential analyzer. The synthesized Fe3O4@SiO2@UiO-66 microspheres were first used for magnetic solid-phase extraction (MSPE) of domoic acid (DA) in shellfish samples. Combined with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), a fast, simple and sensitive method for the determination of DA was established successfully. Under the optimized conditions, the developed method showed short analysis time, good linearity (r2=0.9990), low limit of detection (1.45pgmL−1; S/N=3:1), low limit of quantification (4.82pgmL−1; S/N=10:1), and good extraction repeatability (RSD≤5.0%; n=5). Real shellfish samples were processed using the developed method, and trace level of DA was detected. The results demonstrate that Fe3O4@SiO2@UiO-66 core–shell magnetic microspheres are the promising sorbents for rapid and efficient extraction of polar analytes from complex biological samples.
Non‐small‐cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has a poor prognosis, because overall survival after 5 years is 20–25% for all stages. Thus, it is extremely ...important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study, we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, α2 macroglobulin, and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, α2 macroglobulin, and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, αmicroglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.
It is extremely important to increase the survival rate in the early stages non‐small‐cell lung carcinomas by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging, and patient prognosis. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, α microglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.
Plants in natural environments are increasingly being subjected to a combination of abiotic stresses, such as drought and warming, in many regions. The effects of each stress and the combination of ...stresses on the functioning of shoots and roots have been studied extensively, but little is known about the simultaneous metabolome responses of the different organs of the plant to different stresses acting at once.
We studied the shift in metabolism and elemental composition of shoots and roots of two perennial grasses, Holcus lanatus and Alopecurus pratensis, in response to simultaneous drought and warming.
These species responded differently to individual and simultaneous stresses. These responses were even opposite in roots and shoots. In plants exposed to simultaneous drought and warming, terpenes, catechin and indole acetic acid accumulated in shoots, whereas amino acids, quinic acid, nitrogenous bases, the osmoprotectants choline and glycine betaine, and elements involved in growth (nitrogen, phosphorus and potassium) accumulated in roots. Under drought, warming further increased the allocation of primary metabolic activity to roots and changed the composition of secondary metabolites in shoots.
These results highlight the plasticity of plant metabolomes and stoichiometry, and the different complementary responses of shoots and roots to complex environmental conditions.
Nitrate commands genome‐wide gene expression changes that impact metabolism, physiology, plant growth, and development. In an effort to identify new components involved in nitrate responses in ...plants, we analyze the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. 176 phosphoproteins show significant changes at 5 or 20 min after nitrate treatments. Proteins identified by 5 min include signaling components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with transporter activity or hormone metabolism functions, among others. The phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants was significantly altered as compared to wild‐type plants, confirming its key role in nitrate signaling pathways that involves phosphorylation changes. Integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post‐translational level. We validated a new phosphorylation site in PIN2 and provide evidence that it functions in primary and lateral root growth responses to nitrate.
Synopsis
Phosphoproteomic analysis in response to nitrate treatment reveals early nitrate‐induced changes in Arabidopsis thaliana root phosphoproteins associated to signaling while late changes are associated to transport and hormone metabolism. Moreover, nitrate promotes the dephosphorylation of PIN2, impacting PIN2 subcellular localization and root development.
Several nitrate‐regulated phosphoproteins involved in signaling, metabolism and other functions were identified.
NRT1.1 is required for normal phosphoproteome responses to nitrate treatments.
Nitrate regulates root system architecture by modulating PIN2 phosphorylation status.
Phosphoproteomic analysis in response to nitrate treatment reveals early nitrate‐induced changes in Arabidopsis thaliana root phosphoproteins associated to signaling while late changes are associated to transport and hormone metabolism. Moreover, nitrate promotes the dephosphorylation of PIN2, impacting PIN2 subcellular localization and root development.
High molecular weight PAHs (HMW PAHs) are dangerous pollutants widely distributed in the environment. The use of microorganisms represents an important tool for HMW PAHs bioremediation, so, the ...understanding of their biochemical pathways facilitates the development of biodegradation strategies. For this reason, the potential role of species of microalgae, bacteria, and microalga-bacteria consortia in the degradation of HMW PAHs is discussed. The identification of their metabolites, mostly by GC–MS and LC-MS, allows a better approach to the enzymes involved in the key steps of the metabolic pathways of HMW PAHs biodegradation. So, this review intends to address the proteomic research on enzyme activities and their involvement in regulating essential biochemical functions that help bacteria and microalgae in the biodegradation processes of HMW PAHs. It is noteworthy that, given that to the best of our knowledge, this is the first review focused on the mass spectrometry identification of the HMW PAHs metabolites; whereby and due to the great concern of the presence of HMW PAHs in the environment, this material could help the urgency of developing new bioremediation methods. The elucidation of the metabolic pathways of persistent pollutant degrading microorganisms should lead to a better knowledge of the enzymes involved, which could contribute to a very ecological route to the control of environmental contamination in the future.
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•Compilation of HMW PAHs degradative microalgae, bacteria, and consortia species•Metabolite identification by GC/LC-MS is helpful to elucidate degradation pathways.•Enzymes related to the key metabolites during HMW PAHs degradation processes•Degradative enzymes as potential promising tools in bioremediation strategies
Aim of this study was to provide an up-to-date assessment of the antibiotics contaminating the aqueous environment in Italy, for a better understanding of risks for the ecosystem and human health. ...Antibiotics were first listed in order of their theoretical environmental loads, then were measured in wastewater of some sewage treatment plants (STPs) and in rivers in Italy. Macrolides, particularly clarithromycin and spiramycin, and quinolones, particularly ciprofloxacin and
l-floxacin/ofloxacin, were the most abundant antibiotics in untreated wastewater. Several of them were not removed in STPs and still remained in the treated wastewater, and a total estimate of 7–14
tons of active principles were discharged annually into the aqueous environment in Italy through this route. Results of the analysis of rivers in northern Italy agreed with these figures, with an average load of 5
kg/day, or about 1.8
tons/year, of antibiotics flowing in the River Po, at sampling sites covering a basin comprising about one-fifth of the Italian population. In conclusion, antibiotics, particularly macrolides and quinolones, are widespread environmental contaminants, and urban STPs are confirmed a major source of the contamination.
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