Liquid Chromatography Orbitrap Mass Spectrometry Research Articles

Determination of toxic substances, pesticides and mycotoxins, in ginkgo biloba nutraceutical products by liquid chromatography Orbitrap-mass spectrometry

More than 250 toxic substances, including pesticides and mycotoxins, have been identified and quantified in ginkgo biloba nutraceutical products using liquid chromatography (LC) coupled to high resolution mass spectrometry (Exactive-Orbitrap). A “dilute and shoot” procedure, using acetonitrile with formic acid (1% v/v), was applied for the extraction of the target compounds. In this case, after this procedure, a clean-up step was necessary using a mixture of sorbents (PSA, GBC, C18 and Z-Sep+). Parameters such as trueness, repeatability, intermediate precision, limits of detection and quantification were evaluated. Recoveries between 70 and 120% with relative standard deviation (RSD) values lower than 20% were obtained for 260 of the compounds. Limits of detection and quantification below 5 and 10μgkg−1 were obtained, respectively. Nine samples of ginkgo biloba nutraceutical products were analyzed, finding pesticides in 8 out of 9 samples, such as hymexazol (10μgkg−1) and tebufenozide (55–459μgkg−1). In the case of mycotoxins, aflatoxin B1 (5–54μgkg−1), aflatoxin B2 (4–300μgkg−1) and T-2 toxin (18–20μgkg−1) were found in 6 samples.

Characterization of fumonisin A-series by high-resolution liquid chromatography-orbitrap mass spectrometry.

Fumonisin A-series (FAs) in a reference material of corn sample that was naturally contaminated with fumonisins was characterized using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitap MS). Peaks for fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3), in addition to three peaks corresponding to unknown compounds I, II, and III, were detected in the chromatogram for the corn sample. Fragment ion analysis for FB1, FB2, and FB3 showed that while the ions formed at m/z values of 200–800 were similar to those formed by the cleavage of the tricarballylic acids and the hydroxyl groups, the fragmentation patterns at m/z values of 50–200 varied depending on the hydroxyl group locations in the compounds. Fragment ion analysis of compounds I–III revealed structural similarities to FBs, only differing by an additional C2H2O in the unknown compounds. Using these results and by comparing the product ion mass spectra of compound I with fumonisin A1 (FA1) synthesized from FB1 standards, compounds I–III were hypothesized to be N-acetyl analogs of FBs: fumonisins A1 (FA1), A2 (FA2), and A3 (FA3). The method for determining concentrations was validated with FA1, FB1, FB2, and FB3 standards and applied to analyze the reference material. The FB1, FB2, and FB3 analytical levels were within acceptance limits and the amount of FA1 in the material was ~15% of FB1 amount at 4.2 mg/kg.

Large pesticide multiresidue screening method by liquid chromatography-Orbitrap mass spectrometry in full scan mode applied to fruit and vegetables

The present work is focused on evaluating the main operational parameters for multiresidue screening of an Orbitrap mass spectrometer for pesticide residue analysis in fruits and vegetables. Operational parameters such as resolution, software for the automatic detection, mass tolerance and retention time extraction window, along with the analytical performance, were evaluated in an updated UHPLC-Orbitrap-mass spectrometer working in full scan mode. The evaluation was performed using QuEChERS extracts of tomato, pepper, orange and green tea. The extracts were spiked with 170 selected pesticides at four concentration levels (10μg/kg, 50μg/kg, 100μg/kg and 500μg/kg). Extracts were diluted 5 fold before injection. Three different resolution settings (17,500, 35,000 and 70,000) were evaluated at various concentration levels. At 10μg/kg, using a resolution of 17,500 and 5ppm of mass tolerance, the detected pesticide rates were from 91% in tomato, to 83% in green tea. These percentages increased at higher resolution values. A resolution of 70,000 was adequate for such analysis even when a small percentage of false detect at low concentration was obtained. The rates of detected compounds increased and were from 98% in tomato to 88% in green tea. Mass tolerance of 5ppm was the most adequate for screening purposes. The observed false negative detects were mainly a consequence of a lack of compound sensitivity exacerbated by ion suppression effects in the experimental conditions applied. With reporting limits of 10μg/kg, reproducibility improved with resolution levels of 35,000 or higher. Linearity was investigated in the 2–100ng/mL (equivalent to 10–500μg/kg in the sample) range. Particularly good automatic screening effectiveness was obtained using the selected settings in the analysis of real samples where no false negatives detects and 5% of false positives detects were obtained.

Screening and confirmation of myo-inositol trispyrophosphate (ITPP) in human urine by hydrophilic interaction liquid chromatography high resolution / high accuracy mass spectrometry for doping control purposes.

Myo-inositol trispyrophosphate (ITPP) is a novel allosteric effector of haemoglobin with high permeation selectivity across the red blood cell plasma membrane. Due to its potential to reduce the oxygen affinity of haemoglobin, ITPP application results in an enhanced oxygen release in hypoxic tissues. Therefore, ITPP is being examined for the treatment of numerous illnesses that involve hypoxia, such as cardiovascular diseases, cancer or Alzheimer's disease. Similar to the prohibited substance Efaproxiral®, ITPP increases maximal exercise capacity in mice, providing high potential to be misused in sports. To keep up with cheating athletes, a fast and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for screening and confirmation of ITPP in human urine for doping control purposes was developed. According to the molecule's distinct hydrophilic properties, extraction from complex biological matrices is challenging and conventional reversed phase liquid chromatography (RPLC) separations are not suitable for its detection. Therefore an approach based on hydrophilic interaction liquid chromatography (HILIC) Orbitrap mass spectrometry was established. The methodology was fully validated for qualitative purposes. Screening and confirmation assay are characterized by satisfactory specificity and robustness, adequate intra-day (screening: 4.9-8.1%; confirmation: 2.0-6.7%) and inter-day precision (screening: 4.6-9.1%; confirmation: 1.8-6.6%), excellent linear correlations (>0.99) with sufficient LLOD in the sub ng/mL range (screening: 15 ng/mL; confirmation: 1 ng/mL). In addition it could be shown that ITPP is stable in human urine under the mandatory storage period and conditions for doping control laboratories. To our knowledge, this is the first validated 'dilute-and-inject' LC-MS/MS method for the reliable detection of ITPP in human urine.

TiO2 photocatalysis of 2-isopropyl-3-methoxy pyrazine taste and odor compound in aqueous phase: Kinetics, degradation pathways and toxicity evaluation

In recognition of the growing demand regarding the control of undesired taste and odor (T&O) problems in natural water resources, the photocatalytic degradation of 2-isopropyl-3 methoxy pyrazine (IPMP), a common metabolite of soil actinomycetes which contributes a rotten vegetable odor to water, was investigated under simulated solar irradiation. Under the studied conditions (C=10mgL−1, CTiO2=100 mg L−1 and I=600Wm−2), 95% of IPMP was removed within 20min of irradiation. The reaction intermediates were completely mineralized to CO2 and the nitrogen was predominantly released as NH4+ ions after 240min irradiation. The major transformation products of TiO2 photocatalysis of IPMP have been determined by the use of high resolution accurate liquid chromatography–orbitrap mass spectrometry as well as gas chromatography–mass spectrometry (GC–MS) techniques. Hydroxylation of the isopropyl and methoxy groups has been identified as the main reaction pathway. Scavenging experiments indicated the important role of HO•, h+ and O2•− in the photocatalytic process. Toxicity assessment revealed the efficiency of the photocatalytic treatment to achieve almost complete detoxification of the irradiated solution.

Liquid Chromatography-Orbitrap Mass Spectrometry Method for the Determination of Toxic Glycoalkaloids and their Aglycons in Potato Upper Soil

The fate of natural toxins in the environment has been in focus for the past years. Also, plant produced compounds that can be introduced to the environment by processes such as leaching, decomposition of plant residues or root exudation. The two toxic glycoalkaloids (α-solanine and α-chaconine) and theirs aglycons (demissidine and solasodine) produced by the potato plant (Solanum Tuberosum L.) have been found in upper soil collected each week in two months in varying levels. A new analytical method that uses liquid chromatography Orbitrap mass spectrometry has been developed for the analysis of two glycoalkaloids and two aglycons in potato upper soils, and method validation was performed including sensitivity, recovery, linearity, accuracy and precision and specificity. The application of this method for routine screening of these compounds has been achieved and this study represents a simple, fast and reliable method for the quantification of these compounds in potato upper soils.

Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.

Liquid Chromatography Orbitrap Mass Spectrometry Method for Analysis of Homocysteine and Related Aminothiols in Cereal Products

Liquid chromatography Orbitrap mass spectrometry method for quantification of biological aminothiols (cysteine, homocysteine, and glutathione) in cereal products has been developed. The assay is based on preliminary derivatization with N-(2-acridonyl)maleimide and high resolution accurate mass spectrometry with utilization of dl-Homocystine-3,3,3′,3′,4,4,4′,4′-d8 (homocystine-d8) as internal standard. The limits of quantification for homocysteine, cysteine, and glutathione are 19.44, 40.78, and 338.93 pg, respectively, per 10 μl injection. Intra- and inter-day precision expressed as relative standard deviations are in the range of 1.76 to 2.94 % and 1.06 to 4.13 %, respectively. The average recoveries were 98 % for Hcy, 87 % for Cys, and 92 % for GSH. Wheat, maize, and bakery products with different origin were analyzed. The content of Hcy in the investigated samples was found to be in range of 9–436 μg/100 g, Cys in range of 29–6,895 μg/100 g and GSH in range of 259–14,795 μg/100 g.

Analysis of (Functionalized) Fullerenes in Water Samples by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

One of the main challenges in environmental risk assessment of fullerenes is to develop analytical methods that detect and quantify fullerenes at low concentrations. In this paper we report on the development and optimization of a highly specific, robust, and relatively simple method for the quantitative determination of C60, C70, and six functionalized fullerenes, namely, [6,6]-phenyl-C61-butyric acid methyl ester, [6,6]-phenyl-C61-butyric acid butyl ester, [6,6]-phenyl-C61-butyric acid octyl ester, [6,6]-bis(phenyl)-C61-butyric acid methyl ester, [6,6]-thienyl-C61-butyric acid methyl ester, and [6,6]-phenyl-C71-butyric acid methyl ester ([70PCBM], in different aqueous matrixes. For this method fullerenes were extracted from the aqueous phase using solid-phase extraction (SPE), with subsequent analysis on a liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) system. SPE was optimized by varying different conditions to improve recovery of all fullerenes. Different SPE column materials (C18, C18e, C8, CN) were tested, and recoveries appeared to be the highest for the C18-material. Recoveries were improved by adding NaCl to the water during extraction. Very low limit of detection (LOD) values were obtained for all compounds with this method, ranging from 0.17 ng/L for [70]PCBM to 0.28 ng/L for C60, and subsequent limit of quantitation (LOQ) values of 0.57-0.91 ng/L. Recoveries for the fullerenes were on average 120% in ultrapure and drinking water. Recoveries appeared to be lower, but still acceptable (e.g., >78%), in surface water. The developed approach is promising and will be applied, for example, in (1) environmental monitoring, (2) a more in-depth study of environmental fate and transformation products, and (3) studying water treatment efficiency of C60, C70, and the various functionalized fullerenes.

Characterization of Oil Sands Process-Affected Waters by Liquid Chromatography Orbitrap Mass Spectrometry

Recovery of bitumen from oil sands in northern Alberta, Canada, occurs by surface mining or in situ thermal recovery, and both methods produce toxic oil sands process-affected water (OSPW). A new characterization strategy for surface mining OSPW (sm-OSPW) and in situ OSPW (is-OSPW) was achieved by combining liquid chromatography with orbitrap mass spectrometry (MS). In electrospray positive and negative ionization modes (ESI(+)/ESI(-)), mass spectral data were acquired with high resolving power (RP > 100,000-190,000) and mass accuracy (<2 ppm). The additional chromatographic resolution allowed for separation of various isomers and interference-free MS(n) experiments. Overall, ∼3000 elemental compositions were revealed in each OSPW sample, corresponding to a range of heteroatom-containing homologue classes: Ox (where x = 1-6), NOx (where x = 1-4), SOx (where x = 1-4), NO₂S, N, and S. Despite similarities between the OSPW samples at the level of heteroatom class, the two samples were very different when considering isomer patterns and double-bond equivalent profiles. The chromatographic separations also allowed for confirmation that, in both OSPW samples, the O₂ species detected in ESI(-) (i.e., naphthenic acids) were chemically distinct from the corresponding O₂ species detected in ESI(+). In comparison to model compounds, tandem MS spectra of these new O₂ species suggested a group of non-acidic compounds with dihydroxy, diketo, or ketohydroxy functionality. In light of the known endocrine-disrupting potential of sm-OSPW, the toxicity of these O₂ species deserves attention and the method should be further applied to environmental forensic analysis of water in the region.

Detection of Type A Trichothecene Di-Glucosides Produced in Corn by High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry

The existence of di-glucosylated derivative of T-2 toxin in plant (corn powder) was confirmed for the first time in addition to that of HT-2 toxin. These masked mycotoxins (mycotoxin glucosides) were identified as T-2 toxin-di-glucoside (T2GlcGlc) and HT-2 toxin-di-glucoside (HT2GlcGlc) based on accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometric (LC-Orbitrap MS) analysis. Although the absolute structure of T2GlcGlc was not clarified, two glucose molecules were suggested to be conjugated at 3-OH position in tandem when considering the structure of T-2 toxin. On the other hand, the specification of the structure seems to be more complicated in the case of HT2GlcGlc, since HT-2 toxin has two possible positions (at 3-OH and 4-OH) to be glusocylated. In addition, 15-monoacetoxyscirpenol-glucoside (MASGlc) was also detected in the identical sample.

Direct Peel Monitoring of Xenobiotics in Fruit by Direct Analysis in Real Time Coupled to a Linear Quadrupole Ion Trap–Orbitrap Mass Spectrometer

Study of xenobiotics present in fruit peel by exposing it (without any pretreatment) to direct analysis in real time coupled to a high-resolution orbitrap mass spectrometer (DART-HRMS) is reported for the first time. Variables such as DART gas heater temperature and pressure, source-to-MS distance, and sample velocity are investigated. The analysis of one sample by DART-MS lasts ca. 1 min, and the benefits of both high-resolution and tandem mass spectrometry to elucidate nontarget or unknown compounds are combined. Identification of postharvest fungicides, antioxidants, and sugars in fruit peel is performed in the positive ion mode. A possible elemental formula is suggested for marker components. The lowest imazalil concentration that could be detected by this system is 1 ng (equivalent to a concentration of ca. 300 μg kg(-1)), which is well below the maximum residue limit. For oranges and apples, direct peel exposition demonstrated good interday precision (within 20% for any concentration) and proper linearity (R(2) ≥ 0.99), with a dynamic range from 1 to 2500 ng for apple. A comparison of the results obtained using the direct peel screening DART-based method is made with those obtained by DART analysis of solvent extracts, as well as those obtained analyzing these extracts by ultrahigh-performance liquid chromatography orbitrap mass spectrometry (UHPLC-Orbitrap). The results are in good agreement. Thus, the proposed method proves to be quantitatively accurate with indisputable identification specificity. As an independent method, the approach of direct scanning of peel is of high interest and of potential future within food analysis to guarantee safety, quality, and authenticity.

Rapid and Semiautomated Method for the Analysis of Veterinary Drug Residues in Honey Based on Turbulent-Flow Liquid Chromatography Coupled to Ultrahigh-Performance Liquid Chromatography–Orbitrap Mass Spectrometry (TFC-UHPLC-Orbitrap-MS)

A simple and rapid method is described for the determination of veterinary drug (VD) residues in honey samples using turbulent flow chromatography coupled to ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (TFC-UHPLC-Orbitrap-MS). Honey samples were diluted with an aqueous solution of Na(2)EDTA (0.1 M). Then, they were injected into the chromatographic system including a TFC column. Afterward, the analytes were transferred to an UHPLC analytical column, where they were determined by UHPLC-Orbitrap-MS. Mean recoveries were obtained at three concentration levels (5, 10, and 50 μg/kg), ranging from 68 to 121% for most compounds. Repeatability (intraday precision) and interday precision (expressed as relative standard deviation, RSD) were <25% for most compounds. Limits of quantification (LOQs) ranged from 5 to 50 μg/kg and limits of identification (LOIs) from 0.1 to 50 μg/kg. The developed method was applied in honey samples, and it was fast and nonlaborious.

Comparison of the urinary protein patterns of athletes by 2D‐gel electrophoresis and mass spectrometry—a pilot study

Urinary proteins and exercise-induced proteinuria have been the subject of much research. Proteinuria has been studied in depth after different running and cycling intensities and durations and the different mechanisms of glomerular filtration and tubular dysfunction have been elucidated. The present study was carried out to compare urinary protein profiles of athletes in different sport categories (endurance sport, team sport, strength sport). Doping-control urine samples obtained from in-competition testing and specimens derived from a control group were analysed by means of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significantly deviating protein spots were enzymatically hydrolysed and identified by nanoflow liquid chromatography-orbitrap mass spectrometry. Endurance sport samples demonstrated a significant increase of mainly medium-sized urinary proteins such as transferrin, zinc alpha-2-glycoprotein and prostaglandin H2 D-isomerase (30-80 kDa) in 2D-PAGE experiments. Proteinuria was evident in all samples after protein concentration measurements (protein/creatinine > 15 mg/mmol). Alterations were also observed in strength sport samples, which showed an increase of low molecular weight proteins or protein fragments (<30 kDa, e.g., transthyretin, CD 59 antigen or an N-terminal transferrin fragment). In contrast, the concentration measurements did not imply proteinuria but total protein excretion was in a normal range. The study provides a first overview on 2D maps of the urinary proteome after different types of exercise. Future studies may lead to the establishment of urinary protein maps that are typical for a certain type of sport or even an individual athlete. These maps may complement the blood passport of athletes in doping control.