Performance Liquid Chromatography-ion Trap Mass Research Articles

Development of an in-house mixed-mode solid-phase extraction for the determination of 16 basic drugs in urine by High Performance Liquid Chromatography-Ion Trap Mass Spectrometry

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine.By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA).The sorbent characterization analyses brought to the selection of the AMPSA based phase.Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate.To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine.

Water-Soluble Organic Composition of the Arctic Sea Surface Microlayer and Association with Ice Nucleation Ability.

Organic matter in the sea surface microlayer (SML) may be transferred to the atmosphere as sea spray and hence influence the composition and properties of marine aerosol. Recent work has demonstrated that the SML contains material capable of heterogeneously nucleating ice, but the nature of this material remains largely unknown. Water-soluble organic matter was extracted from SML and underlying seawater from the Arctic and analyzed using a combination of mass spectrometric approaches. High performance liquid chromatography-ion trap mass spectrometry (LC-IT-MS), and Fourier transform ion cyclotron resonance MS (FT-ICR-MS), showed seawater extracts to be compositionally similar across all stations, whereas microlayer extracts had a different and more variable composition. LC-IT-MS demonstrated the enrichment of particular ions in the microlayer. Ice nucleation ability (defined as the median droplet freezing temperature) appeared to be related to the relative abundances of some ions, although the extracts themselves did not retain this property. Molecular formulas were assigned using LC-quadrupole time-of-flight MS (LC-TOF-MS2) and FT-ICR-MS. The ice nucleation tracer ions were associated with elevated biogenic trace gases, and were also observed in atmospheric aerosol collected during the summer, but not early spring suggesting a biogenic source of ice nuclei in the Arctic microlayer.

Yew (Taxus) intoxication in free-ranging cervids.

Wild ruminants, including deer species (cervids) have incorrectly been regarded as refractory to yew (Taxus) intoxication. This assumption has been based upon anecdotal observations of individual deer browsing on yew over time without apparent adverse effect. A single case of yew intoxication was reported in a free-ranging Norwegian moose (Alces alces) in 2008. The current report describes five additional cases of yew toxicosis in moose, seven in roe deer (Capreolus capreolus) and two in reindeer (Rangifer tarandus tarandus), all in Norway. The animals were found dead during the winter, close to or within gardens containing yew plants showing signs of browsing. Gross findings included lung congestion and edema, thoracic and pericardial effusion, bilateral heart dilatation, epi- and endocardial hemorrhage, and enlarged (congested) spleen. Yew plant remnants were detected in the rumen of all animals with the exception of a single moose. Histology revealed multifocal acute myocardial degeneration and necrosis with hemorrhage in roe deer, but not in the two other species. A qualitative high performance liquid chromatography–ion trap mass spectrometry analysis was used to tentatively identify five major Taxus alkaloids (taxines) in crude yew extracts and in heart and liver samples from the moose cases. All five major taxines were detected with good signal/noise ratio in tissue samples from the four moose with visible ruminal yew content, whereas lower levels of taxines were detected in the moose without visible ruminal yew content. Possible differences in interspecies tolerance to taxines and role of individual protective adaptation are discussed.

Ultrahigh-performance liquid chromatography-ion trap mass spectrometry characterization of the steroidal saponins of Dioscorea panthaica Prain et Burkill and its application for accelerating the isolation and structural elucidation of steroidal saponins

Dioscorea panthaica is a traditional Chinese medicinal herb used in the treatment of various physiological conditions, including cardiovascular disease, gastropathy and hypertension. Steroidal saponins (SS) are the main active ingredients of this herb and have effects on myocardial ischemia and cancer. The phytochemical evaluation of SS is both time-consuming and laborious, and the isolation and structural determination steps can be especially demanding. For this reason, the development of new methods to accelerate the processes involved in the identification, isolation and structural elucidation of SS is highly desirable. In this study, a new ultrahigh performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT/MSn) method has been developed for the identification of the SS in D. panthaica Prain et Burkill. Notably, the current method can distinguish between spirostanol and furostanol-type compounds based on the fragmentation patterns observed by electrospray ionization-ion trap mass spectrometry (ESI-IT/MSn) analysis. UHPLC-IT/MSn was used to conduct a detailed investigation of the number, structural class and order of the sugar moieties in the sugar chains of the SS present in D. panthaica. The established fragmentation features were used to analyze the compounds found in the 65% ethanol fraction of the water extracts of D. panthaica. Twenty-three SS were identified, including 11 potential new compounds and six groups of isomers. Two of these newly identified SS were selected as representative examples, and their chemical structures were confirmed by 1H and 13C NMR analyses. This newly developed UHPLC-IT/MSn method therefore allowed for the efficient identification, isolation and structural determination of the SS in D. panthaica.

Fast screening of 24 sedative hypnotics illegally added in improving sleep health foods by high performance liquid chromatography-ion trap mass spectrometry

A fast screening method was established for the simultaneous determination of 24 sedative hypnotics illegally added in improving sleep health foods by high performance liquid chromatography-ion trap mass spectrometry (HPLC-IT MS). The method was based on the sonication assisted extraction of the improving sleep health food samples using methanol. The extract was then filtrated with 0.45 µm filter membrane and the filtrate was separated on a Phenomenex Luna C18 column with isocratic elution at a flow rate of 0.3 mL/min. A binary mobile phase was 0.05% (v/v) formic acid (solvent A)-methanol/acetonitrile (15:25, v/v, solvent B). The electrospray ionization (ESI) source in positive ion mode or negative ion mode was used to scan MS1-MS3 spectra for the 24 sedative hypnotics. The MS2 and MS3 spectra were used for qualitative analysis of samples. The calibration graphs were linear in their concentration ranges with the correlation coefficients (r2) more than 0.999. The limits of detection (LODs) were 4.0-446.6 µg/L. The recoveries for all the drugs in the improving sleep health foods were 88.6%-110.3% with the relative standard deviations no more than 9.8% at three spiked levels. Twenty-seven batches of the improving sleep health foods were tested. Melatonin was found in eighteen batches. The method is fast, specific, sensitive, easy and suitable for fast screening of 24 sedative hypnotics illegally added in improving sleep health foods.

The simultaneous determination of vitamins A, E and β-carotene in bovine milk by high performance liquid chromatography–ion trap mass spectrometry (HPLC–MSn)

Bovine milk is considered to be a good dietary source of vitamins A, E and β-carotene. This paper describes a robust method for the simultaneous determination of these compounds using high performance liquid chromatography–ion trap mass spectrometry (HPLC–MSn) as the determinative step. The compounds were separated with a 5 μm Polaris 2.1 × 150 mm C18-A column and an aqueous-methanol mobile phase and the levels determined using the MS2 fragments (vitamin A, m/z 213 and m/z 199, vitamin E m/z 165 and β-carotene m/z 413). The HPLC–MSn was operated in the positive ion APCI mode. The method was validated using repeatability studies, duplicate analyses, recovery experiments, proficiency study data and comparison with previously validated high performance liquid chromatographic ultraviolet/visible (HPLC–UV/Vis) and fluorescence (HPLC–Fl) procedures used in our laboratory. Expanded measurement uncertainties were similar for HPLC–MSn, HPLC–UV/Vis and HPLC–Fl, e.g. vitamin A 45 μg/100 ml, HPLC–MSn ±8%, HPLC–UV ±9%; vitamin E 150 μg/100 ml, HPLC–MSn ±11%, HPLC–Fl ±9%; β-carotene 12 μg/100 ml, HPLC–MSn ±18%, HPLC–Vis ±21%.

Determination of Fluoroquinolones in Grass Carp by High Performance Liquid Chromatography-Ion Trap Mass Spectrometry Using Mixed-templates Imprinted Polymer Extraction

The molecularly imprinted polymer(MIP) has been prepared using levofloxacin and ciprofloxacin as the mixed-templates,α-methacrylic acid(MAA) as functional monomers and trimethylolpropane trimethylacrylate(TRIM) as crosslinker.A method has been established for simultaneous determination of residues of ten fluoroquinolones in grass carp by high performance liquid chromatography-ion trap mass spectrometry(HPLC-ITMS) combining with molecularly imprinted polymer solid phase extraction(MISPE).The samples were extracted with 2% acetic acid in acetonitrile and defatted with n-hexane,then purified by the self-made molecularly imprinted cartridge.The preparation of samples and the separation conditions had been optimized,HPLC was performed with a gradient elution program while acetonitrile and water(containing 0.05% formic acid) were used as mobile phase.The identification and quantification were done with ion trap mass spectrometry.The recove-ries ranged from 80.6% to 104.6% with a relative standard deviations of 8.6%.The limits of detection(LOD) were 0.11-0.25 μg/kg and the limits of quantification(LOQ) were 0.35-0.84 μg/kg.This method is suitable for validation and multi-residue analysis of fluoroquolones in aquatic products.

Determination of <I>β</I><SUB>2</SUB>-agonists and <I>β</I>-blockers in urine using high performance liquid chromatography-ion trap mass spectrometry

A method has been developed for the determination of 23 beta2-agonists and 5 beta-blockers in urine samples using high performance liquid chromatography-ion trap mass spectrometry (HPLC-IT-MS). Urine samples were first deproteinized by high-speed frozen centrifugation, and the supernatants were loaded on an Extrelut diatomite column for clean-up. The analytes were eluted by ethyl acetate and concentrated for further analysis. The analytical separation was performed on an AtlantisT3-150 mm chromatographic column with the gradient elution using methanol and water (containing 0.1% formic acid). The detection was carried on a linear ion trap mass spectrometer under multiple reaction monitoring (MRM) mode with the source operated in positive mode of electrospray ionization (ESI+). Nine deuterium labeled beta2-agonists were used as internal standards for quantitative analysis. The results showed that the linear ranges for 23 beta2-agonists and 5 beta-blockers were 0.005-0.16 mg/L, and the limits of detection were all around 0.2 microg/L. The mixed standard solution was added into the blank urine samples, and the recoveries of 23 beta2-agonists and 5 beta-blockers were ranged from 57.1% to 127.7% with the relative standard deviations of 1.1%-31.1%. The results demonstrate that the method is easy, fast, sensitive, and suitable for the confirmation and quantification of 23 beta2-agonists and 5 beta-blockers in urine samples.

LC–MS characterization of metoclopramide photolysis products

The aim of this study was to investigate the photodegradation of the antiemetic drug metoclopramide. Metoclopramide aqueous solutions were photoirradiated at 254 nm under argon atmosphere. Irradiated metoclopramide solutions were analyzed by high performance liquid chromatography–ion trap mass spectrometry in order to characterize photolysis products. Rapid decrease in metoclopramide purity, following first-order kinetic, was observed following irradiation. The structures of 18 photolysis products were tentatively identified based on their mass spectra and fragmentation. The main degradation mechanism was scission of the chlorine which could be followed by polymerization of the resulting products since dimeric and trimeric products were observed.

Determination of the main bioactive metabolites of Radix Salvia miltiorrhizae in Compound Danshen Dripping Pills and the tissue distribution of Danshensu in rabbit by SPE‐HPLC‐MSn

A fast solid phase extraction-high performance liquid chromatography-ion trap mass spectrometry (SPE-HPLC-MSn) method is presented for quantitative determination of 3-(3,4-dihydroxyphenyl)-2-hydroxylpropionic acid (Danshensu, DSS) from Compound Danshen Dripping Pills (CDDP) in rabbits' heart, liver, kidney, and lung and for qualitative analysis of the bioactive metabolites of Radix Salvia miltiorrhizae (DS) from CDDP in these tissues. The proposed method was validated with a detection limit of 0.06 ng/mL and a linear range of 5.0-200 ng/mL for DSS. The main metabolites of DS were pyrocatechol, vanillic acid, isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, and 2-hydroxyl-3-(3,4-dihydroxyphenyl)propinonic sulfuric anhydride. In vitro pharmacological experiments demonstrated that isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate had a distinct vasodilatating action.

Pharmacokinetics and metabolism of 3,4-dichlorophenyl-propenoyl-sec.-butylamine in rats by high performance liquid chromatography–ion trap mass spectrometry

The pharmacokinetics (PK) and metabolism of 3,4-dichlorophenyl-propenoyl-sec.-butylamine (3,4-DCPB), a novel antiepileptic drug, were investigated after its oral administration to rats (100 mg/kg) by HPLC. The absorption and elimination of 3,4-DCPB were rapid. 3,4-DCPB was found to undergo extensive metabolism as the major route of elimination. Structures of the metabolites present in rat plasma were identified with liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS/MS). It was concluded that 3,4-DCPB was involved in the multiple metabolic pathways (hydrolysis, dealkylation and oxidation) and the hydrolysis product, 3,4-dichloro-cinnamic acid (M1) appeared to be the major metabolite.

A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation

Callus cells of Arabidopsis thaliana (cv. Landsberg erecta) were exposed for 8 h to a horizontal clinostat rotation (H, simulated weightlessness), a vertical clinostat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and fructose apparently decreased, while starch content increased in the H compared with the V- and S-treated cells. In order to investigate the influences of clinostat rotation on the cellular proteome further, the proteome alterations induced by horizontal and vertical clinostat rotation have been comparatively analysed by high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 80 protein spots showed quantitative and qualitative variations that were significantly (P <0.01) and reproducibly different between the clinorotated (H or V) and the stationary control samples. Protein spots excised from 2-D gels were analysed by microbe high performance liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. 18 protein spots, which showed significant expression alteration only under the H condition compared with those under V and S conditions, were identified. Of these proteins, seven were involved in stress responses, and four protein spots were identified as key enzymes in carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated in the H samples. Other proteins such as protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins also exhibited altered expression under the H condition. The data presented in this study illustrate that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins involved in general stress responses, metabolic pathways, gene activation/transcription, protein synthesis, and cell wall biosynthesis.

Association of Acyl-CoA Synthetase-1 with GLUT4-containing Vesicles

GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.