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Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography-mass spectrometry (LC-MS) systems. In this study, to the best of our knowledge, we confirm for the first time the presence of six amino acids (GABA, Oxo-Pro, Asp, Gln, Glu, and Tyr) previously reported to be involved in response to and side effects from antiseizure medications in exhaled breath condensate and by extension in exhaled human breath. Raw data are publicly available at MetaboLights with the accession number MTBLS6760.

In this study, nine forced degradation products of maraviroc were found using chemometric analysis. This antiretroviral drug was subjected to photolytic, oxidative, as well as neutral, basic and acidic hydrolysis stress conditions. Additionally, its electrochemical transformation on platinum, gold and glassy carbon screen-printed electrodes was examined. This study showed that maraviroc is especially susceptible to UVA, H 2 O 2 and electrochemical degradation, while being resistant to neutral and acidic hydrolysis. A cluster analysis showed that the electrochemical transformation, with particular reference to the platinum electrode, is able to partially simulate the forced degradation processes, especially in the context of redox reactions. These findings indicate that the electrochemical methods can be considered as quick and relatively low-cost supplements to the commonly applied forced degradation procedures.

Inborn errors of monoamine neurotransmitter metabolism are rare genetic diseases classified as catecholamine and serotonin metabolism disorders or neurotransmitter transportopathies. To diagnose these orphan diseases, monoamine metabolites have been identified and validated as cerebrospinal fluid (CSF) biomarkers: 5-hydroxy-tryptophane, 5-hydroxy-indol-acetic acid, 3-ortho-methyl-DOPA, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol. The present work presents a UHPLC-MS/MS method developed for the quantification of these metabolites in CSF and compares it with a previously described UHPLC with fluorescence detection (UHPLC-FD) method. MS/MS detection was performed in positive electrospray ionization and multiple reaction monitoring mode. The UHPLC-MS/MS and UHPLC-FD methods were validated in terms of accuracy, linearity, precision and matrix effect. The lower limits of quantification (LLOQ) ranged between 0.5 and 10 nm and between 1 and 5 nm for the UHPLC-MS/MS method and the UHPLC-FD one, respectively. We verified the applicability of both methods by analyzing 30 CSF samples. The measured concentrations were comparable with the reference values described in the literature. The two methods allowed pathological samples to be distinguished from healthy ones for clinical diagnosis. UHPLC-MS/MS and UHPLC-FD methods exhibited very close LLOQs. As the UHPLC-MS/MS method is more selective, it allows faster analysis with a run time of 6 min per run vs. 10 min for the UHPLC-FD method., (© 2022 John Wiley & Sons Ltd.)

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U- 13 C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI + mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI - mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U- 13 C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U- 13 C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The alteration of lipid profile in biological specimens, such as plasma, mirrors abnormalities in their homeostasis and offers pivotal information for disease comprehension. Fast analytical methods are needed to highlight changes in plasma lipid profile and deliver rapid results. In this study we developed a fast reversed phase ultra high performance liquid chromatography-trapped ion mobility mass spectrometry (RP-UHPLC-TIMS-MS) method for untargeted lipidomics. A short, narrow-bore fully porous particle CSH column (50 mm × 2.1 mm, 1.7 µm) was used, and by selecting appropriate flow rate, temperature and gradient conditions, the total analysis time was reduced from 20 to 4 min. TIMS was operated in parallel accumulation serial fragmentation mode (PASEF) which allowed to select multiple precursors for MS/MS and separate co-eluting lipids based on their different mobility. Lipid annotation was performed by rule-based approach, comparison with LipidBlast spectral library and manual data curation, by taking into account class-specific fragmentation pattern, accurate mass, adduct form, retention behavior in RP and comparison of their collision cross-section (CCS) values for increased confidence. 306 unique lipids from 21 subclasses were annotated from 20 µL of plasma, while their concentration was estimated by class-specific deuterated internal standards. The analytical method was validated and finally applied to elucidate the alteration of plasma lipid profiles in a small cohort of amyotrophic lateral sclerosis (ALS) patients. Univariate and multivariate statistics evidenced significant differences with respect to control patients, particularly in the levels of ether linked lipids (PC-O, PE-O, PE-P and LPC-O), sphingolipids (Ceramides), and triacylglycerols, showing the usefulness of this fast approach in providing accurate and rapid results with respect to longer (≥15 min) untargeted UHPLC-HRMS methods., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Bacteria coordinate their behavior as a group via communication with their peers, known as 'quorum sensing'. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC-MS/MS) in multiple reaction monitoring (MRM) mode (in total <90 min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52 nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC-MS/MS method detected these RNPP peptides in wild-type murine feces samples.

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1 D RP-LC (stronger retention) and 2 D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1 D-RP run and the 68 s 2 D chiral separations of 30 heart-cuts (from the 1 D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient "smart peak parking" algorithm for the heart cuts and the resulting optimized analysis order thereof. 1 D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u- 13 C 15 N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cyclic peptides are considered collision-induced dissociation (CID)-resistant due to immobile protons, and the necessity of at least two consecutive dissociation reactions to produce fragments with deviating m/z values. Therefore, the bioanalysis of cyclic peptides by tandem mass spectrometry (MS/MS) poses a major challenge, especially on triple quadrupole (TQ) instruments. One of these peptides is the somatostatin analog pasireotide, a cyclic hexapeptide administered to treat Cushing's disease and acromegaly. To support oral formulation development, sub-therapeutic quantification of pasireotide is highly beneficial. Regardless of the considered CID-resistance, we investigated the CID-characteristics of pasireotide and subsequently developed an ultra-sensitive UHPLC-MS/MS assay with a lower limit of quantification (LLOQ) of 5 pg/mL (4.9 pM) when using 100 μL of plasma and validated it according to the guidelines of the FDA and EMA. The achieved sensitivity, which is the highest thus far reported, demonstrates that TQ-MS/MS is a feasible approach to sensitive quantification of cyclic peptides despite their CID-resistance. Pasireotide was fast and efficiently extracted by protein depletion via precipitation using acetonitrile. Correlation coefficients > 0.99 were achieved for all calibration curves with linear regression. Inter-run and intra-run accuracy ranged from 89.4 to 99.3 % with corresponding precision of ≤ 7.5 % in the calibrated range, and from 94.6 to 105.6 % with corresponding precision of ≤ 14.5 % at the LLOQ. Quantification of 10-fold diluted samples showed an accuracy of 90.8 % and corresponding precision of 4.0 %. The assay was applied to the quantification of pasireotide plasma concentrations after intravenous administration to beagle dogs., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An automated inline sample preparation (ILSP) method has been developed for pesticide residue analysis in spinach by LC-MS/MS. Chlorophyll pigments and other matrix constituents were removed from the sample extract using a UHPLC system equipped with an auxiliary pump, 6-port high pressure switching valve, and dual-directional ILSP cartridge containing bonded silica. The new procedure was evaluated as an entirely separate workflow using a simple solid-liquid extraction and as part of a cleanup strategy in conjunction with QuEChERS. Accuracy and precision experiments were conducted in spinach at two concentration levels (n = 6). Of the 63 pesticides tested, 86% (0.005 mg/kg) and 100% (0.05 mg/kg) displayed average recoveries within 70-120% and RSD values ≤20% for the ILSP method. In addition, low to moderate matrix effects (<50%) were calculated for 95% of the analytes. Overall performance of the proposed method was found to be better or comparable to a traditional QuEChERS procedure utilizing AOAC formulated salts and dSPE sorbents, while significantly reducing the amount of pigments reaching the MS source. The ILSP workflow is a simpler procedure with fewer steps that require less time than traditional extraction and cleanup techniques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Though marketed for over half a century, little is known about the pharmacokinetics of oral vancomycin except that its bioavailability is low, thus making accurate determination of plasma concentrations difficult. To quantify plasma concentrations of vancomycin after oral administration, we developed an ultra-sensitive UHPLC-MS/MS assay and validated it according to FDA´s and EMA´s pertinent guidelines. A fast and simple protein precipitation method followed by short UHPLC chromatography was developed for extraction and separation of vancomycin from plasma. Quantification was performed via heated positive electrospray tandem mass spectrometry with multiple reaction monitoring using deuterated internal standard. The assay was linear in the calibrated concentration range of 0.05-100 ng/mL showing correlation coefficients >0.997. Intraday and interday accuracy showed coefficients of variation <12% at the lower limit of quantification (LLOQ) of 0.05 ng/mL and <6% in the calibrated range while corresponding values for precision were <13% and <8%, respectively. With its high sensitivity, the assay allows for the accurate quantification of therapeutic plasma concentrations in the therapeutic range (up to 100 μg/mL) in 1000-fold diluted samples with a sample volume decreased down to 1 μL. The UHPLC-MS/MS assay was successfully used for the determination of trough plasma concentrations of two patients with Clostridium difficile infection receiving oral vancomycin therapy and its performance was compared to a commercial immunoassay for concentrations close to its LLOQ., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new method using ultra high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) methodology was developed for the determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) contents in liquid and solid vegetable samples. The advantages of this method are speed, high sensitivity and practical application. In accordance with these advantages, the present method allows the simultaneous determination of AA and DHAA without previous reduction/derivatization of DHAA and without the use of internal standards in the samples. This is of high interest in routine analysis, providing a simpler sample preparation, as well as enhanced accuracy and robustness. Its validation included selectivity, sensitivity and linearity, precision and accuracy, matrix effect, and recovery. The results showed high selectivity and sensitivity, with calibration curves ranging from 10 to 500 ng mL -1 and from 50 to 500 ng mL -1 for AA and DHAA, respectively. Appropriate dilutions for each sample are necessary to avoid the matrix effect with accepted recoveries.

Alternanthera sessilis (red) (ASR) is an edible herbal plant with many beneficial health effects. This study aimed to investigate the antioxidant components and antioxidant activities of the edible leaves and stems of ASR extracted using solvent of varying polarities namely water, ethanol, ethyl acetate and hexane. ASR leaf extracts showed higher in both antioxidant components and activities than the stem extracts. Among the antioxidant components, the ethanol leaf extract showed higher phenolic (77.29 ± 1.02 mg GAE/g extract) content while the ethyl acetate leaf extract was rich in flavonoids (157.44 ± 10.19 mg RE/g extract), carotenoids (782.97 ± 10.78 mg BE/g extract) and betalains (betanin: 67.08 ± 0.49 mg/g extract; amaranthin: 93.94 ± 0.68 mg/g extract and betaxanthin: 53.92 ± 0.88 mg/g extract). Nevertheless, the ethanol leaf extract showed the highest DPPH radical scavenging activity and ABTS radical cation scavenging activity. It also exhibited highest ferric reducing activity among all the extracts. Four polyphenolic compounds from ASR leaf, namely ferulic acid, rutin, quercetin and apigenin, were identified and quantified using ultra high performance liquid chromatography. The existence of these compounds was further verified using tandem mass spectrometry. These current results indicate that ASR leaf particularly the ethanol extract has the potential to be exploited as a source of natural antioxidants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study the comparison of human liver microsomes in in vitro incubation as well as ZnO- and TiO 2 -assisted photocatalytic degradation of clozapine as a mimicking method of phase I metabolism transformation was performed. Based on reversed-phase UHPLC separation and high-resolution MS/MS data, eight transformation products were identified and seven of them were found to be hepatic metabolites of the parent compound. The multivariate chemometric comparison of the obtained results shows ZnO-assisted photocatalysis to be a more suitable approach to phase I metabolism simulation. The photocatalytic experiments demonstrated that the disappearance of clozapine followed pseudo-zero order kinetics., (© 2018 John Wiley & Sons, Ltd.)

A fast analytical method for the simultaneous detection of 24 β 2 -agonists in human urine was developed and validated. The method covers the therapeutic drugs most commonly administered, but also potentially abused β 2 -agonists. The procedure is based on enzymatic deconjugation with β-glucuronidase followed by SPE clean up using mixed-phase cartridges with both ion-exchange and lipophilic properties. Instrumental analysis conducted by UHPLC-MS/MS allowed high peak resolution and rapid chromatographic separation, with reduced time and costs. The method was fully validated according ISO 17025:2005 principles. The following parameters were determined for each analyte: specificity, selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and carry-over. The method was tested on real samples obtained from patients subjected to clinical treatment under chronic or acute therapy with either formoterol, indacaterol, salbutamol, or salmeterol. The drugs were administered using pressurized metered dose inhalers. All β 2 -agonists administered to the patients were detected in the real samples. The method proved adequate to accurately measure the concentration of these analytes in the real samples. The observed analytical data are discussed with reference to the administered dose and the duration of the therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present work a Hydrophilic Interaction Liquid Chromatography-tandem Mass Spectrometry (HILIC-MS/MS) method was developed for the efficient separation and quantification of a large number of small polar bioactive molecules in Royal Jelly. The method was validated and provided satisfactory detection sensitivity for 88 components. Quantification was proven to be precise for 64 components exhibiting good linearity, recoveries R% >90% for the majority of analytes and intra- and inter-day precision from 0.14 to 20% RSD. Analysis of 125 fresh royal jelly samples of Greek origin provided useful information on royal jelly's hydrophilic bioactive components revealing lysine, ribose, proline, melezitose and glutamic acid to be in high abundance. In addition the occurrence of 18 hydrophilic nutrients which have not been reported previously as royal jelly constituents is shown., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (t G ), temperature (T) and ternary eluent composition (t C ) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography-Mass Spectrometry (LC-MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured R s,crit -values., (Copyright © 2016. Published by Elsevier B.V.)

Plant growth regulators (PGRs) are very different chemical compounds that play essential roles in plant development and the regulation of physiological processes. They exert their functions by a mechanism called cross-talk (involving either synergistic or antagonistic actions) thus; it is for great interest to study as many PGRs as possible to obtain accurate information about plant status. Much effort has been applied to develop methods capable of analyze large numbers of these compounds but frequently excluding some chemical families or important PGRs within each family. In addition, most of the methods are specially designed for matrices easy to work with. Therefore, we wanted to develop a method which achieved the requirements lacking in the literature and also being fast and reliable. Here we present a simple, fast and robust method for the extraction and quantification of 20 different PGRs using UHPLC-MS/MS optimized in complex matrices., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lipidomics refers to the large-scale study of lipids in biological systems (Wenk, Nat Rev Drug Discov 4(7):594-610, 2005; Rolim et al., Gene 554(2):131-139, 2015). From a mass spectrometric point of view, by lipidomics we understand targeted or untargeted mass spectrometric analysis of lipids using either liquid chromatography (LC) (Castro-Perez et al., J Proteome Res 9(5):2377-2389, 2010) or shotgun (Han and Gross, Mass Spectrom Rev 24(3):367-412, 2005) approaches coupled with tandem mass spectrometry. This chapter describes the former methodology, which is becoming rapidly the preferred method for lipid identification owing to similarities with established omics workflows, such as proteomics (Washburn et al., Nat Biotechnol 19(3):242-247, 2001) or genomics (Yadav, J Biomol Tech: JBT 18(5):277, 2007). The workflow described consists in lipid extraction using a modified Bligh and Dyer method (Bligh and Dyer, Can J Biochem Physiol 37(8):911-917, 1959), ultra high pressure liquid chromatography fractionation of lipid samples on a reverse phase C18 column, followed by tandem mass spectrometric analysis and in silico database search for lipid identification based on MSMS spectrum matching (Kind et al., Nat Methods 10(8):755-758, 2013; Yamada et al., J Chromatogr A 1292:211-218, 2013; Taguchi and Ishikawa, J Chromatogr A 1217(25):4229-4239, 2010; Peake et al., Thermoscientifices 1-3, 2015) and accurate mass of parent ion (Sud et al., Nucleic Acids Res 35(database issue):D527-D532, 2007; Wishart et al., Nucleic Acids Res 35(database):D521-D526, 2007).

The impact of viscous friction on eluent temperature and column efficiency in liquid chromatography is of renewed interest as the need for pressures exceeding 1000bar to use with columns packed with sub-2μm particles has grown. One way the development of axial and radial temperature gradients that arise due to viscous friction can be affected is by the thermal environment the column is placed in. In this study, a new column oven integrated into an ultrahigh pressure liquid chromatograph that enables both still-air and forced-air operating modes is investigated to find the magnitude of the effect of the axial thermal gradient that forms in 2.1×100mm columns packed with sub-2μm particles in these modes. Temperature increases of nearly 30K were observed when the generated power of the column exceeded 25W/m. The impact of the heating due to viscous friction on the repeatability of peak capacity, elution time, and peak area ratio to an internal standard for a gradient UHPLC-MS/MS method to analyze neurotransmitters was found to be limited. This result indicates that high speed UHPLC-MS/MS gradient methods under conditions of high viscous friction may be possible without the negative effects typically observed with isocratic separations under similar conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hydrolyzable tannins are measured routinely during the characterization of food and beverage samples. Most methods for the determination of hydrolyzable tannins use hydrolysis or methanolysis to convert complex tannins to small molecules (gallic acid, methyl gallate, and ellagic acid) for quantification by HPLC-UV. Often unrecognized, analytical limitations and variability inherent in these approaches for the measurement of hydrolyzable tannins include the variable mass fraction (0-0.90) that is released as analyte, contributions of sources other than tannins to hydrolyzable gallate (can exceed >10 wt %/wt), the measurement of both free and total analyte, and lack of controls to account for degradation. An accurate, specific, sensitive, and higher-throughput approach for the determination of hydrolyzable gallate based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that overcomes these limitations was developed.

To date five nucleoside analogs are used in the treatment of chronic hepatitis B: among these, entecavir is the most used. Nevertheless a few information about its distribution in tissues is currently known. Since the determination of entecavir disposition in the hepatocytes is impracticable because of its invasiveness, the quantification in an "easier-to-obtain" cellular model could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry assay based on an automated on-line SPE, to quantify entecavir concentrations in peripheral blood mononucleated cells (PBMCs), in both its phosphorylated and un-phosphorylated forms. To achieve this, each PBMC isolate was divided in two aliquots, one was treated with acid phosphatase to convert entecavir phosphorylated metabolites into free form, the other one was not-treated. Standards and quality controls were prepared in PBMCs, isolated from healthy donors, and underwent the same process. 20 μL of the resulting solutions were injected in the on-line SPE system. Thymidine was used as internal standard. Calibration curves fitted a linear model for entecavir levels in a range from 0.039 ng to 5 ng (mean r(2)=0.998). Accuracy, intra-day and inter-day precision of the method fitted FDA guidelines recommendations. Moreover, recovery was consistent and matrix effect resulted low and reproducible. We tested this method by monitoring entecavir concentrations in PBMCs from 28HBV mono-infected patients, confirming its reliability and suitability for the evaluation of intracellular entecavir penetration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, we propose a new quick and accurate analytical method by UHPLC-QqQ-MS/MS which is able to identify free phytoprostanes in olive and refined sunflower oils. The recovery provided high extraction efficiencies ranging from 102.90% to 140.64% using Strata-XAW cartridge. The intra-day and inter-day variations for all target compounds ranged from 2.24% to 13.64% and 0.01% to 13.69%, respectively, and the accuracies for these parameters varied from 80.33% to 119.64% and from 80.34% to 119.90%, respectively. Results obtained reflect that refined sunflower presented more series of phytoprostanes and a 20 and 8-fold higher quantity than two types of olive oil: Extra virgin olive oil and olive oil, (containing half virgin extra olive oil and half refined olive oil). The manufacture process could be the key for the different phytoprostane production since most of the plant oils are subjected to a refining treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In 2004, a new multi-residue pesticides method had been published using methanol as extraction solvent. Our goal for this study was to optimize the analytical scheme while extending the compound scope from 19 to 200 pesticides. The main changes from the original method take place at the sample extraction and processing with a special attention to make the overall method fit for routine analysis with minimal cost. Hence, after a quick Ultra-Turrax homogenization with a methanolic solution, the sample is simply diluted before the separation and detection by ultra-high-performance liquid chromatography and MS/MS detection for quantitative and confirmatory purposes. The performance of the method including limits of quantification (LOQs), linearity, matrix effect, precision was evaluated during validation in accordance with the European Union SANCO/12571/2013 regulatory guidelines. Two representative matrices, lettuce and orange, were selected and fortified at two concentration levels for these experiments. At the LOQ and ten times the LOQ, recoveries of the analytes were mostly within 70-120%, with coefficients of variation lower than 25% in intra-day repeatability conditions. In addition to being simple and fast, these results demonstrate the suitability of the optimized method for the analysis of large scope pesticides in routine laboratories., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC-MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD<5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24h at 28 °C., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Physalins, uniquely discovered from genus physalis, showed significant bioactivities in many aspects. It is therefore very important for the exploration of natural resources rich of physalins. However, there is no efficient approach for rapid discovery and identification of this class of compounds due to their structural complexity. To address the issue, the fragmentation pathways and correspondingly fragmentation rules of physalins in negative MS/MS mode were thoroughly investigated in this study using seven physalin standards. As a result, diagnostic ions for the rapid screening of physalins and classification of different types of physalins were determined based on their MS/MS fragmentation patterns. On top of that, an integrated approach using UHPLC-QTOF-MS/MS together with a novel three-step data mining strategy was developed for the systematic analysis of physalins in complex samples. Consequently, 46 physalins including 20 novel ones were efficiently discovered and identified from the crude extracts of Ph. alkekengi calyx. The present study laid a foundation for future study of different parts of Ph. alkekengi and other physalis species with regard to rapid discovery of novel physalins. In addition, this study provided a base for establishing a quality control method of the raw materials of Ph. alkekengi according to the profile of physalins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Today, there is an increasing number of liquid chromatography tandem-mass spectrometric (LC-MS/MS) methods for the analysis of eicosanoids and related lipids in biological matrices. An overview of currently applied LC-MS/MS methods is given with attention to sample preparation strategies, chromatographic separation including ultra high performance liquid chromatography (UHPLC) and chiral separation, as well as to mass spectrometric detection using multiple reacting monitoring (MRM). Further, the application in recent clinical research is reviewed with focus on preanalytical aspects prior to LC-MS/MS analysis as well as applications in major diseases of Western civilization including respiratory diseases, diabetes, cancer, liver diseases, atherosclerosis, and neurovascular diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel reversed-phase ultra high performance liquid chromatography coupled with high resolution quantitation mass spectrometry (UHPLC/HRQMS) method was developed to quantify 2,5-dichlorobenzoyl chloride (DCBC), a genotoxic impurity, in MLN9708 drug substance. A surrogate strategy was utilized whereby DCBC was intentionally hydrolyzed to 2,5-dichlorobenzoic acid (DCBA) to provide a stable and reliable detection target. The hydrolysis approach was conservative since the measured signal represented the sum of DCBC and DCBA in MLN9708 drug substance, and such approach was acknowledged and accepted by food and drug administration (FDA). HRQMS was used as the detection method since conventional MS/MS methodology gave poor sensitivity and selectivity due to non-specific fragmentation of carbon dioxide loss upon collision activation dissociation. Profile algorithm mass spectrometry data were acquired with mass resolving power (MRP) of 60,000. Quantitation was based on the extracted ion chromatography (EIC) peak area signal, which was extracted at m/z 188.9515 with a mass extraction window (MEW) of 5ppm. The UHPLC/HRQMS method was validated based on International Conference on Harmonization (ICH) guidelines, which included selectivity, limit of detection (LOD), limit of quantitation (LOQ), repeatability, linearity, accuracy, and stability., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Carbohydrates have a wide variety of structures whose complexity and heterogeneity challenge the field of analytical chemistry. Tandem mass spectrometry, with its remarkable sensitivity and high information content, provides key advantages to addressing the structural elucidation of polysaccharides. Yet, classical fragmentation by collision-activated dissociation (CAD) in many cases fails to reach a comprehensive structural determination, especially when isomers have to be differentiated. In this work, for the first time, vacuum ultra-violet (VUV) synchrotron radiation is used as the activation process in tandem mass spectrometry of large oligosaccharides. Compared to low energy CAD (LE-CAD), photon activated dissociation brought more straightforward and valuable structural information. The outstanding feature was that complete series of informative ions were produced, with only minor neutral losses. Moreover, systematic fragmentation rules could be drawn thus facilitating the definitive assignments of fragment identities. As a result, most of the structures present in a complex mixture of oligogalacturonans could be comprehensively resolved, including many isomers differing in the position of methyl groups along the galacturonic acid backbone., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Sensitive and reliable analytical methodology has been developed for the measurement of patulin in regulated foodstuffs by using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with triple quadrupole analyser. Solid samples were extracted with ethyl acetate, while liquid samples were directly injected into the chromatographic system after dilution and filtration without any clean-up step. Chromatographic separation was achieved in less than 4min. Electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) sources were evaluated, in order to assess matrix effects. The use of ESI source caused strong signal suppression in samples; however, matrix effect was negligible using APCI, allowing quantification with calibration standards prepared in solvent. The method was validated in four different apple matrices (juice, fruit, puree and compote) at two concentrations at the low μgkg(-1) level. Average recoveries (n=5) ranged from 71% to 108%, with RSDs lower than 14%., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report an accurate quantification of free sialic acid (SA) in human plasma using LC-MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC-MS/MS analysis of free SA. In addition, the use of (13)C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20ng/mL and a total running time of 4.2min, which is the most sensitive and quick measurement for free SA in biomatrices., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tropomyosin-related kinases (Trk) are tyrosine kinase receptors implicated in tumor proliferation, invasion, and survival signaling across a number of tumors, making them potentially attractive targets for the treatment of cancer. AZD7451 is a potent and selective inhibitor of Trk kinases currently undergoing a Phase I dose escalation in glioblastoma multiforme at the National Cancer Institute. A key part of early clinical testing for AZD7451 involves demonstrating that pharmacokinetic half-life and clinical exposures of AZD7451 are sufficient to inhibit Trk receptors in preclinical models. To address this need, an ultra sensitive analytical method was developed to measure the AZD7451 profile in human plasma. A liquid-liquid extraction recovered >80% of AZD7451 before quantitative analysis by ultra HPLC-MS/MS. A Varian Polaris(®) C18-A column and a mass transition of m/z 383.5→340.5 (m/z 389.6→342.0 for the internal standard [(2)H6]-AZD7451) was used, and a dynamic calibration range of 0.5-1000ng/mL was established, which provided a sensitive (<8.5% deviation), and precise (<6%) quantitative assay for AZD7451. AZD7451 demonstrated stability in human plasma at room temperature for 24h (<7% change) and after extraction at 4°C for 24h (<8% change), and was stable through 4 freeze/thaw cycles (<8% change). This method was used to measure AZD7451 plasma levels in clinical samples to confirm the sensitivity at several time points following AZD7451 treatment in subjects with glioblastoma., (Published by Elsevier B.V.)

A sensitive and specific ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the measurement of climbazole deposition from hair care products onto artificial skin and human scalp. Deuterated climbazole was used as the internal standard. Atmospheric pressure chemical ionization (APCI) in positive mode was applied for the detection of climbazole. For quantification, multiple reaction monitoring (MRM) transition 293.0>69.0 was monitored for climbazole, and MRM transition 296.0>225.1 for the deuterated climbazole. The linear range ran from 4 to 2000 ng mL(-1). The limit of detection (LOD) and the limit of quantification (LOQ) were 1 ng mL(-1) and 4 ng mL(-1), respectively, which enabled quantification of climbazole on artificial skin and human scalp at ppb level (corresponding to 16 ng cm(-2)). For the sampling of climbazole from human scalp the buffer scrub method using a surfactant-modified phosphate buffered saline (PBS) solution was selected based on a performance comparison of tape stripping, the buffer scrub method and solvent extraction in in vitro studies. Using this method, climbazole deposition in in vitro and in vivo studies was successfully quantified., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Buds of poplar trees (Populus species) are often covered with sticky, usually polyphenol-rich, exudates. Moreover, accessible data showed that some Populus bud extracts may be excellent antibacterial agents, especially against Gram-positive bacteria. Due to the fragmentary nature of the data found, we conducted a systematic screening study. The antimicrobial activity of two extract types (semi-polar—ethanolic and polar—ethanolic-water (50/50; V/V)) from 27 bud samples of different poplar taxons were compared. Antimicrobial assays were performed against Gram-positive (five strains) and Gram-negative (six strains) bacteria as well as fungi (three strains) and covered the determination of minimal inhibitory, bactericidal, and fungicidal concentrations. The composition of extracts was later investigated by ultra-high-performance liquid chromatography coupled with ultraviolet detection (UHPLC-DAD) and with electrospray-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-ESI-qTOF-MS). As a result, most of the extracts exhibited good (MIC ≤ 62.5 µg/mL) or moderate (62.5 < MIC ≤ 500 µg/mL) activity against Gram-positives and Helicobacter pylori, as well as fungi. The most active were ethanolic extracts from P. trichocarpa, P. trichocarpa clone 'Robusta', and P. tacamahaca × P. trichocarpa. The strongest activity was observed for P. tacamahaca × P. trichocarpa. Antibacterial activity was supposedly connected with the abundant presence of flavonoids (pinobanksin, pinobanksin 3-acetate, chrysin, pinocembrin, galangin, isosakuranetin dihydrochalcone, pinocembrin dihydrochalcone, and 2′,6′-dihydroxy-4′-methoxydihydrochalcone), hydroxycinnamic acids monoesters (p-methoxycinnamic acid cinnamyl ester, caffeic acid phenethylate and different isomers of prenyl esters), and some minor components (balsacones). [ABSTRACT FROM AUTHOR]

Background: Fosfomycin acts against aerobic Gram−/+ bacteria by blocking the synthesis of peptidoglycan. Its use has been currently re-evaluated for intravenous administration for the treatment of systemic infections by multidrug-resistant bacteria. Concentration-/time-dependent activity has been suggested, with potential clinical advantages from prolonged or continuous infusion. Nevertheless, little is known about Fosfomycin stability in elastomeric pumps. The aim of the present work was stability investigation before administration at 4 °C and during administration at 34 °C. Methods: InfectoFos® (InfectoPharm s.r.l., Milan, Italy) preparation for intravenous use in elastomeric pumps at 4 °C and 34 °C was analyzed following EMA guidelines for drug stability. Samples were analyzed with an ultra-high performance liquid chromatography coupled with tandem mass spectrometry method on a LX50® UHPLC system equipped with a QSight 220® (Perkin Elmer, Milan, Italy) tandem mass spectrometer. Results: Fosfomycin in elastomeric preparation is stable for at least 5 days at a storage temperature of 4 °C and 34 °C. Conclusions: The results suggest Fosfomycin eligibility for continuous infusion even in the context of outpatient parenteral antibiotic therapy. Therefore, this approach should be tested in clinical and pharmacokinetic studies, in order to evaluate the possible gains in the pharmacokinetic profile and the clinical effectiveness. [ABSTRACT FROM AUTHOR]

A simple, rapid, sensitive, and reliable ultra‐high‐performance liquid chromatography‐hybrid linear ion trap triple quadrupole mass spectrometry method was developed and validated for simultaneous determination of 10 bioactive compounds in stem bark of Betula utilis grown in high altitude of Himalaya, India. The objective of the study is to develop and validate ultra‐high‐performance liquid chromatography‐hybrid linear ion trap triple quadrupole mass spectrometry for investigation of geographical variations of triterpenoids, phenolics, and flavonoids contents in stem bark of B. utilis. The validated method was successfully applied to investigate geographical variations of triterpenoids, phenolics, and flavonoids in stem bark of B. utilis. The contents of betulinic acid and oleanolic acid were detected higher among selected analytes. The present variation study reveals great importance for the application and overall assessment of B. utilis. [ABSTRACT FROM AUTHOR]

In order to further gain a comprehensive chemical understanding of erythritol tetranitrate (ETN), the synthesis impurities of ETN were investigated using ultrahigh performance liquid chromatography with tandem high resolution mass spectrometry (UHPLC‐MS/MS). A total of 12 impurities were identified relating to the synthesis of ETN. We have synthesized pure analytical standards of erythritol‐1,4‐dinitrate (1,4‐EDiN) and erythritol‐1,2,4‐trinitrate (1,2,4‐ETriN). These isomerically pure standards have allowed for confirmation of the identity of some of the discovered impurities. Additionally, partially nitrated with sulfate substituted erythritol was identified which is theorized to have formed from the incomplete displacement of the formed sulfate group on the alcohol position during nitration. [ABSTRACT FROM AUTHOR]

Glycosidic aroma compounds are the important precursors of volatile aroma in grapes, and they can be added with odorous aglycones via enzyme- or acid-catalyzed hydrolysis during wine fermentation and storage. Developing an analytical method for intact glycosides can provide the possibility to study the accumulation of these aroma precursors in grape berries. For this purpose, a Tandem Mass Spectrometry (MS/MS). database based on ultra-high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry was built, covering multiple aglycone classes. Subsequently, the profiles of glycosidic aroma compounds in Vitis vinifera L. cv. Muscat Blanc, Riesling, and Chardonnay berries during maturation were investigated. Pentosyl-hexosides were the most abundant glycosides in all three varieties. Both composition and concentration of glycosidic aroma compounds varied obviously among grape varieties. Except for monoterpenol pentosyl-hexosides, most glycosides were kept almost stable in their concentrations during berry maturation. This research provides an approach to understand the variation of glycosidic aroma components from the perspective of aglycones and glycosides. [ABSTRACT FROM AUTHOR]

Sudan I and metanil yellow are azo dyes, illegally used to impart bright red and yellow color to foods respectively. It is assumed that these appearances are due to adulteration. Regulations must be able to detect the substances in a cost-effective, efficient, and sensitive manner. A simple and precise ultra-high-performance liquid chromatography with photodiode array detector based analytical method is developed for detection and quantification of metanil yellow and Sudan 1 from turmeric and chili powders. The method necessitated no sample clean up from matrix components and the separation of the analytes was performed on a reversed-phase C18 column with an optimized solvent system consisting of 0.6305 g of ammonium formate + 100 µL of formic acid in HPLC grade water-methanol: acetonitrile (70:30 V/V) for metanil yellow at 413 nm and 0.2% formic acid in water-acetonitrile (30:70, V/V) for curcumin at 425 nm. Depending on the metanil yellow and Sudan I dye involved, LOD and LOQ were in the range of 0.05–0.12 μg/Kg and 0.3–0.5 μg/Kg in the turmeric and red chili powders respectively. The recovery, repeatability and reproducibility varied from 80.6 to 112.60% from 0.3 to 0.83%, and less than 20% RSD. Linearity obtained for metanil yellow and Sudan I was all r2 > 0.9999 and in the range of 0.5–1.0 mg/L and 0.05–5 mg/L respectively. The proposed method was applied to determine the concentration of curcumin and metanil yellow in 11 market samples, 24 rhizome powder samples and Sudan1 from 9 chili powder samples. This method has the potential to be used for illegal Sudan dyes and metanil yellow in turmeric and red chili powders due to its reliable, rapid and excellent precision. [ABSTRACT FROM AUTHOR]

Highlights • A rapid method was developed for the analysis of vitamin K compounds. • Method was used to screen and quantitate vitamin K from 17 fermented food products. • Chinese fermented soybean pastes contained significant amount of MK-6. • Highest MK-7 content was detected in natto, and MK-9 was present in kefir. Abstract A rapid ultra-high performance liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method was developed for the analysis of vitamin K compounds: phylloquinone (PK) and menaquinones (MK-n). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 min. Sample treatment with liquid and solid phase extractions and by the use of MK-4 as an internal standard enabled the quantitation of microgram level of vitamin K compounds in food. The method was used to screen and quantitate vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42 µg/100 g), whereas the highest MK-7 content was detected in natto (902 µg/100 g). Some MK-9 was present in kefir (5 µg/100 g). Two Chinese fermented soybean pastes contained significant amount of MK-6 (5–36 µg/100 g), MK-7 (12–86 µg/100 g), and MK-8 (22–44 µg/100 g). [ABSTRACT FROM AUTHOR]

Abstract: A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed‐phase liquid chromatography–tandem mass spectrometry (LC–MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra‐batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high‐throughput sample analysis studies. [ABSTRACT FROM AUTHOR]

To study effects of high power ultrasound treatment (20 kHz) on β-carotene degradation, a second-order central composite design (CCD) was performed to investigate maximum β-carotene loss with three independent factors (ultrasonic intensity, sonication time, and temperature). Results based on variance analysis and Pareto chart have shown that sonication time is the most important factor, followed by ultrasonic intensity level. The evolved degradation products have been tentatively identified using ultra high performance liquid chromatography coupled to both diode array detector and a mass spectrometer (UHPLC–DAD–MS). The main degradation products, tentatively identified, are three Z -isomers of β-carotene and seven β-apo-carotenals/ones. Hypothesis on the degradation mechanism of carotenoids are presented. [ABSTRACT FROM AUTHOR]

A method for the determination and quantification of ketosteroid hormones in meat by mass spectrometry, based on the derivatization of the carbonyl moiety of steroids by O-methylhydroxylamine, is presented. The quantitative assay is performed by means of multiple-reaction-monitoring (MRM) scan mode and using the corresponding labelled species, obtained by reaction with d-methoxylamine, as internal standard. The accuracy of the method was established by evaluating artificially spiked samples, obtaining values in the range 90-110 %. Recovery tests were performed on blank matrix samples spiked with non-natural steroids including trenbolone and melengestrol acetate. The latter experiment revealed that the yield of the extraction processes was approximately 60 %. Good values of LOQ and LOD were achieved, making this method competitive with current hormone assay methods. [ABSTRACT FROM AUTHOR]

Abstract: A simple and effective method was developed for characterization of phenolic constituents in the roots of Polygonum multiflorum by combination of ultra-high-pressure liquid chromatography with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC–LTQ-Orbitrap). Stilbenes, anthraquinones, tannins and naphthalenes were differentiated by diagnostic fragment ions with accurate mass measurements and characteristic fragmentation pathways. Based on the proposed strategy, fifty-nine constituents were characterized or tentatively identified, of which twenty-two constituents were the first to be reported in P. multiflorum and twelve compounds were characterized as potential new compounds. The identification and structure elucidation of these chemicals provided essential data for further phytochemical studies and quality control of P. multiflorum. The results also demonstrated that our novel method can be extended to screen and characterize other phenolic constituents and their metabolites in botanical extracts. [Copyright &y& Elsevier]

Abstract: In this paper two fast and highly sensitive ultra-high performance liquid chromatography (UHPLC) methods for the determination of tetracycline antibiotics (oxytetracycline, tetracycline, doxycycline, demeclocycline, chlortetracycline, minocycline and degradation product epitetracycline) in surface waters have been developed using fluorescence (FL) and mass spectrometry (MS) detection. ACQUITY UPLC BEH C8 and ACQUITY CSH C18 columns were employed for FL and MS detection, respectively, both packed with 1.7μm particles. Mixed-mode separation mechanism of CSH (charged surface technology) sorbent was found particularly useful in analysis of TCs, which possess problematic amphoteric structures. The FL methodology was based on chelation of tetracyclines with calcium ions to perform on-column derivatisation. The developed methods were compared in the terms of validation parameters including linearity, sensitivity, precision and accuracy. The linearity range for FL detection was within 7ngmL−1 to 50μgmL−1 with method limit of detection (MLOD) as low as 0.2ngmL−1 for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003ngmL−1, which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01–50ngmL−1 were used for quantification to compensate for matrix effects with the correlation coefficients demonstrating good linearity (0.9940–0.9999). The extraction of the antibiotics from surface waters was performed using solid phase extraction with Oasis HLB cartridges. Accuracy was expressed as recovery with values ranging from 96.52% to 127.30% and from 91.66% to 123.70% for FL and MS detection, respectively. [Copyright &y& Elsevier]

Abstract: The development and use of UHPLC-based methods for the identification, validation and analysis of biomarkers for diseases is reviewed. The currents trends in types of stationary phases and modes of detection are discussed. Afterwards, examples are provided on the use of UHPLC-MS for finding novel biomarkers in samples from in vitro or in vivo animal models of human diseases, as well as in biofluid samples (mainly urine and plasma) obtained from patients. Molecular profiling and targeted analysis are considered, providing an overview of recent experimental or clinical works carried out using UHPLC analysis of compounds from various chemical classes, such as low molecular weight metabolites, hormones, lipids, peptides and proteins. [Copyright &y& Elsevier]

Abstract: The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called “classical doping agents” (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography–mass spectrometry (GC–MS) techniques allowed for the development of comprehensive screening methods. GC–MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography–mass spectrometry (LC–MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2μm) column packing, which provides high peak capacity within analysis times even 5–10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices. [Copyright &y& Elsevier]

Abstract: A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78–103.78μg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20μL) and a run time of 1.5min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution. [Copyright &y& Elsevier]

ABSTRACT Because liver cancer is rarely suitable for surgery, transcatheter arterial chemoembolization (TACE) is used for palliative therapy. In this procedure, an emulsion of doxorubicin in iodized oil is injected directly into liver tumors through a catheter positioned within the artery supplying blood flow to the tumor. At present, there is limited understanding of factors affecting the delivery and dispersion of doxorubicin within treated tumors during TACE. This study addresses the development and application of an ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) method for rapid confirmation of drug delivery after TACE in a rabbit VX2 liver cancer model. Doxorubicin levels in liver tumors were measured using UHPLC-MS-MS and compared with computed tomography measured levels of iodized oil, a metric used clinically to indicate drug delivery. We found that tissue drug levels determined using UHPLC-MS-MS did not correlate with the regional iodized oil concentration (vehicle) within tumors following TACE, suggesting that chemotherapeutic drugs like doxorubicin spread throughout tumors, and that lack of iodized oil staining in portions of a tumor does not necessarily indicate inadequate therapy during TACE. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The quantification of acetaminophen (APAP) and two of its metabolites, i.e. acetaminophen-glucuronide (APAP-GLUC) and acetaminophen-cysteine (APAP-CYS), is described in human plasma using ultra-high performance liquid chromatography coupled to a triple quadrupole linear ion trap mass spectrometer operating in the selected reaction monitoring (SRM/MS) mode and to a high resolution quadrupole time-of -flight mass spectrometer operating in the MS/MS (HR-SRM/MS) mode. Starting with a 50μL plasma aliquot, a generic sample preparation was performed using protein precipitation with methanol/ethanol. Both methods were found to be linear over 2.5 orders of magnitude. Similar performances to the SRM/MS assay were obtained for APAP, APAP-CYS and APAP-GLUC using high resolution-selected reaction monitoring mode with LLOQ of 20, 50 and 50ng/mL, respectively. For all analytes, precision was found to be better than 12% and accuracy in the range 90.3–109%. The present study demonstrates the ability of QqTOF platforms for accurate and precise quantification in MS/MS mode using short duty cycle with similar sensitivity to LC–SRM/MS. Additionally, as full scan data MSALL are available qualitative and quantitative information on metabolites can also be obtained in a single LC–MS run. [Copyright &y& Elsevier]

Abstract: The aim of the study was the use of a fast and simple method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) for the simultaneous determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), fumonisins (FB1 and FB2), deoxynivalenol (DON), T2 and HT2 toxins in 58 samples of raw wheat (n =34), barley (n =5), sorghum (n =3), processed wheat (n =13) and breakfast cereals (n =3) from Tunisian markets. The frequency of contamination of total samples with the analyzed mycotoxins was 50%. AFG2 was the most frequently detected in 11 samples (4 wheat, 4 barley and 3 sorghum) and it was detected at 5.2–52.4μg/kg. HT2 toxin contaminated seven samples (4 wheat and 3 barley) and it was detected at 5.0–11.1μg/kg. FB2 was detected in one wheat, sorghum, semolina and breakfast cereal samples at 5.0–61.5μg/kg. FB1 was detected in three samples (2 sorghum and one barley) at 6.4–120μg/kg. AFB1 was only found in two sorghum samples at 14 and 79.9μg/kg. OTA was detected in one sorghum sample at concentrations below limit of quantification (5μg/kg). The analytical results also showed that all the analyzed samples were not contaminated with DON, AFG1, AFB2 and T2 toxin. [Copyright &y& Elsevier]