Journal of Chromatography B (v.1047, #C)
Editorial Board (i).
Mass spectrometry and the Mediterranean by Evangelos Gikas; Michaela Filiou (1).
Deciphering lymphoma pathogenesis via state-of-the-art mass spectrometry-based quantitative proteomics by Konstantina Psatha; Laxmikanth Kollipara; Chrysanthi Voutyraki; Peter Divanach; Albert Sickmann; George Z. Rassidakis; Elias Drakos; Michalis Aivaliotis (2-14).
Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future.
Keywords: Lymphoma; Quantitative proteomics; Mass spectrometry; Interactomics; Protein modifications; Targeted proteomics;
Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) based bioavailability determination of the major classes of phytochemicals by Evgenios Stylos; Maria V. Chatziathanasiadou; Aggeliki Syriopoulou; Andreas G. Tzakos (15-38).
Natural products derived from plants have served as an inexhausted source for drug discovery and drug development. They have been evolutionary amplified with drug-like properties and have already illustrated immense therapeutic potential over an array of different diseases. However, their incorporation in the drug discovery pipeline has been diminished the last two decades. This was probably due to barriers related to their inherent difficulties to be integrated in high-throughput screening assays as also their largely unexplored bioavailability. Analytical procedures have come into the spotlight, a result of the continuous development of the instrumentation’s capabilities as far as detection and separation is concerned. Integral part of this technological evolution is LC–MS instrumentation and its extended use for the determination of various compounds. The fact that it provides extra sensitivity, specificity and good separation in complex samples, makes LC–MS/MS the ultimate tool in the determination of many types of chemical compounds, such as phytochemicals. Herein, we focus on the achievements of the last five years in quantitative analysis of the major classes of phytochemicals (flavonoids, alkaloids, terpenes, glycosides and saponins) in plasma, through LC–MS/MS, as also their bioavailability.
Keywords: Liquid chromatography; Triple quadrupole; Phytochemicals; Plasma; Bioavailability;
The 2nd order focusing sector field type TOF mass analyzer with an orthogonal ion acceleration for LC-IMS-MS by S.S. Poteshin; A.I. Zarakovsky (39-44).
Original orthogonal acceleration (OA) electrostatic sector time of flight (TOF) mass analyzer is proposed those allows the second order focusing of time of flight by initial ions position. Resolving power aberration limit exceeding 80,000 FW (full width mass peak) was shown to be obtainable for mass analyzer with the total length of flight L = 133.2 cm, the average ion energy 3700 V and the ion energy spread of 2.5% on the entrance of sector field.
Keywords: Time of flight (TOF) mass analyzer; Time-of-flight aberration; Orthogonal accelerator; Mass resolution; Sector field TOF mass-analyzer;
Meaning and consequence of the coexistence of competitive hydrogen bond/salt forms on the dissociation orientation of non-covalent complexes by Ekaterina Darii; Sandra Alves; Yves Gimbert; Alain Perret; Jean-Claude Tabet (45-58).
Non-covalent complexes (NCC) between hexose monophosphates (HexP) and arginine (R) were analyzed using ESI MS and MS/MS in negative mode under different (hard, HC and soft, SC) desolvation conditions. High resolution mass spectrometry (HRMS) revealed the presence of different ionic species, namely, homo- and heteromultimers of R and HexP. Deprotonated heterodimers and corresponding sodiated species were enhanced under HC likely due to a decrease in available charge number associated with the reduction of H+/Na+ exchange. The quantum calculations showed that the formation of covalent systems is very little exothermic, therefore, such systems are disfavored. Desolvation dependent CID spectra of deprotonated [(HexP+R)‒H]− complexes demonstrated that they can exist within the hydrogen bond (HB) and salt bridge (SB) forms, yielding either NCC separation or covalent bond cleavages, respectively. Although HB forms are the main species, they cannot survive under HC; therefore, the minor SB forms became detectable. Energy-resolved mass spectrometry (ERMS) experiments revealed diagnostic fragment ions from both SB and HB forms, providing evidence that these isomeric forms are inconvertible. SB formation should result from the ionic interactions of highly acidic group of HexP with strongly basic guanidine group of arginine and thus requires an arginine zwitterion (ZW) form. This was confirmed by quantum calculations. Ion-ion interactions are significantly affected by the presence of sodium cation as demonstrated by the fragmentation patterns of sodiated complex species. Regarding CID data, only SB between protonated amino group of R and deprotonated phosphate group of HexP could be suggested, but the primary amine is not enough basic then, the SB must be fleeting. Nevertheless, the observation of the covalent bond cleavages suggests the presence of structures with a free negative charge able to induce fragmentations. Indeed, according to quantum calculations, solvated salt (SS) systems involving Na+/COO− salt solvated by neutral phosphate and negative charge on sugar ring are preferentially formed.
Some aspects of space-charge effect calculation in high-resolution mass spectrometry by D. Grinfeld; I. Kopaev; M. Skoblin; M. Monastyrskiy; A. Makarov; S. Alimpiev (59-63).
A variational 3D approach to the problem of simulating stationary distributions of ions in the radiofrequency low-vacuum ion traps with regard to Coulomb interaction and collisions of ions with buffer gas molecules is proposed. The software developed in the course of this work is employed to study the structure of stationary ion ensembles in the radiofrequency ion traps of various types. The effect of high-frequency and constant voltages, space-charge density, and buffer gas temperature on the formation of stationary distributions in the radiofrequency ion traps and their limiting capacitance is investigated. It is shown that the use of electrodes with a constant voltage in the presence of high enough ion density allows pre-filtering of ions directly in a high-frequency trap-accumulator.
Keywords: Ion trap; Space charge; Coulomb interaction; Buffer gas; Stationary distribution; Capacitance;
Mechanistic study of competitive releases of H2O, NH3 and CO2 from deprotonated aspartic and glutamic acids: Role of conformation by Pierre Barbier Saint Hilaire; Anna Warnet; Yves Gimbert; Ulli Martin Hohenester; Gianluca Giorgi; Marie-Françoise Olivier; François Fenaille; Benoît Colsch; Christophe Junot; Jean-Claude Tabet (64-74).
The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3 experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways.These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2 as observed in the MS3 experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D−H)−NH3]−during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4 kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.
Keywords: Electrospray high-resolution mass spectrometry; Regioselective dissociation; Unexpected cleavage of aspartate anion;
Observation of the side chain O-methylation of glutamic acid or aspartic acid containing model peptides by electrospray ionization-mass spectrometry by A. Emin Atik; Melda Z. Guray; Talat Yalcin (75-83).
O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dH2O) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC–MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions.
Keywords: O-methylation; Glutamic acid; Aspartic acid; ESI-MS;
Reliable tool for detection of novel Coxiella burnetii antigens, using immobilized human polyclonal antibodies by Gabriela Flores-Ramírez; Maksym Danchenko; Marco Quevedo-Díaz; Ludovit Skultety (84-91).
Coxiella burnetii (C. burnetii) is the etiological agent of a Q fever—the re-emerging disease with considerable economic impact. Due to many similar symptoms with commonly occurring infections, its clinical diagnosis is very difficult. Thus, a strong effort should be taken to raise the awareness and develop a robust strategy for an accurate diagnosis. The identification of specific C. burnetii biomarkers could be valuable for a sensitive and selective diagnosis of the disease. Herein, we described a workflow to identify immunoreactive proteins of C. burnetii with a high confidence. It is based on immunocapturing of bacterial antigens by biofunctionalized magnetic microspheres, followed by tandem mass spectrometry (MS/MS) identification. We detected dozens of previously reported antigens and proposed 15 novel biomarkers, which specificity was confirmed by in silico epitope analysis. Among them, the cardiolipin synthetase participating in the synthesis of cardiolipin was recognized. This biomarker could play a critical role in the early management of acute Q fever and prevention of Q fever endocarditis.
Keywords: Q fever; Biofunctionalized magnetic beads; LC–MS/MS; Immunoreactive proteins; Biomarkers; Serological proteome analysis (SERPA);
Yield of 6,000 proteins by 1D nLC–MS/MS without pre-fractionation by Athanasios K. Anagnostopoulos; Dimitrios J. Stravopodis; George Th. Tsangaris (92-96).
Mass spectrometry (MS) has dominated over other protein analysis methods that aspire to deliver rapid and sensitive protein annotation, due to its ability to acquire high-content biological information from samples of great complexity. Routinely, in-depth analysis of complex biological samples, such as total cell lysates, relies on the high separation power of two-dimensional liquid chromatography-tandem MS (2D LC–MS/MS), often combined with protein pre-fractionation. However, on the basis of recent advances in chromatographic and MS instrumentation, one-dimensional (1D) LC–MS/MS approaches have become the method-of-choice for high-volume/high-throughput protein experiments. Thousands of proteins can be identified in single-run LC–MS/MS experiments. In the present study a 1D LC–MS/MS approach was applied on whole-cell lysates of WM-266-4 human cells leading to identification of more than 5,300 protein groups, 6,000 proteins and 22,00 peptides, in a single run. Using no pre-fractionation steps, method optimization was achieved through experimentation on lysis and protein extraction solutions, as well as nLC gradient parameters.
Keywords: Method development; Liquid chromatography; Mass spectrometry; Single-run analysis; nLC–MS/MS; 1D–LC–MS/MS; Orbitrap; Protein identification;
Exhaled breath condensate analysis from intubated newborns by nano-HPLC coupled to high resolution MS by A.S. Kononikhin; N.L. Starodubtseva; V.V. Chagovets; A.Y. Ryndin; A.A. Burov; I.A. Popov; A.E. Bugrova; R.A. Dautov; A.O. Tokareva; Y.L. Podurovskaya; O.V. Ionov; V.E. Frankevich; E.N. Nikolaev; G.T. Sukhikh (97-105).
Display OmittedInvasiveness of examination and therapy methods is a serious problem for intensive care and nursing of premature infants. Exhaled breath condensate (EBC) is the most attractive biofluid for non-invasive methods development in neonatology for monitoring the status of intubated infants. The aim of the study was to propose an approach for EBC sampling and analysis from mechanically ventilated neonates. EBC collection system with good reproducibility of sampling was demonstrated. Discovery-based proteomic and metabolomic studies were performed using nano-HPLC coupled to high resolution MS. Label-free semi-quantitative data were compared for intubated neonates with congenital pneumonia (12 infants) and left-sided congenital diaphragmatic hernia (12 infants) in order to define disease-specific features. Totally 119 proteins and 164 metabolites were found. A number of proteins and metabolites that can act as potential biomarkers of respiratory diseases were proposed and require further validation.
Keywords: Exhaled breath condensate (EBC); HPLC; Proteomic; Metabolomics; Mass spectrometry; Newborns; Respiratory diseases; Pneumonia;
Post-acquisition spectral stitching. An alternative approach for data processing in untargeted metabolomics by UHPLC-ESI(−)-HRMS by Eirini Baira; Eleni Siapi; Panagiotis Zoumpoulakis; Deligeorgis Stelios G.; Alexios-Leandros Skaltsounis; Evagelos Gikas (106-114).
In the case of the MS-based metabolomics, the large number of false positives remains a fundamental issue.The aim of this study was to develop a new strategy, which highlights the number of the reliable features i.e. the detected features that correspond to a consistent peak according to chromatographic and mass spectrometric criteria.For the analysis blood samples from 20 chickens, which were administrated with naringin and 9 samples from control, were analyzed by UHPLC-HRMS (Orbitrap Velos). Two methodologies have been compared for data processing. In the first one (classical approach), all data in the 100–900 m/z mass-to charge range were included for the data processing procedure whereas for the newly developed methodology, the data were shred in 100 Da slices generating 8 datasets, which have been then subjected to the downstream MS data processing. Each dataset was treated separately and the m/z_tR features obtained by either VIP’s or t-test values were merged and used as the input for the construction of the general model.The new methodology resulted to a 4-fold increase of the peaks that could be considered chromatographically and mass spectrometrically valid.A new strategy was reported on the detection of chromatographically reliable features during a metabolomic approach. The shredding of the LC–MS chromatograms into multiple m/z ranges increased the number of the identified chromatographically reliable features.
Keywords: MZmine; PCA; PLS-DA; Permutation test; VIPs; t-test;
Sample preparation optimization in fecal metabolic profiling by Olga Deda; Anastasia Chrysovalantou Chatziioannou; Stella Fasoula; Dimitris Palachanis; Νicolaos Raikos; Georgios A. Theodoridis; Helen G. Gika (115-123).
Metabolomic analysis of feces can provide useful insight on the metabolic status, the health/disease state of the human/animal and the symbiosis with the gut microbiome. As a result, recently there is increased interest on the application of holistic analysis of feces for biomarker discovery. For metabolomics applications, the sample preparation process used prior to the analysis of fecal samples is of high importance, as it greatly affects the obtained metabolic profile, especially since feces, as matrix are diversifying in their physicochemical characteristics and molecular content. However there is still little information in the literature and lack of a universal approach on sample treatment for fecal metabolic profiling.The scope of the present work was to study the conditions for sample preparation of rat feces with the ultimate goal of the acquisition of comprehensive metabolic profiles either untargeted by NMR spectroscopy and GC–MS or targeted by HILIC-MS/MS. A fecal sample pooled from male and female Wistar rats was extracted under various conditions by modifying the pH value, the nature of the organic solvent and the sample weight to solvent volume ratio. It was found that the 1/2 (wf/vs) ratio provided the highest number of metabolites under neutral and basic conditions in both untargeted profiling techniques. Concerning LC–MS profiles, neutral acetonitrile and propanol provided higher signals and wide metabolite coverage, though extraction efficiency is metabolite dependent.
Keywords: Metabolomics; Metabolic profiling; Feces; Extraction optimization; Sample preparation;
Staphylococcus aureus methicillin resistance detected by HPLC-MS/MS targeted metabolic profiling by Katie Schelli; Joshua Rutowski; Julia Roubidoux; Jiangjiang Zhu (124-130).
Recently, novel bioanalytical methods, such as NMR and mass spectrometry based metabolomics approaches, have started to show promise in providing rapid, sensitive and reproducible detection of Staphylococcus aureus antibiotic resistance. Here we performed a proof-of-concept study focused on the application of HPLC-MS/MS based targeted metabolic profiling for detecting and monitoring the bacterial metabolic profile changes in response to sub-lethal levels of methicillin exposure. One hundred seventy-seven targeted metabolites from over 20 metabolic pathways were specifically screened and one hundred and thirty metabolites from in vitro bacterial tests were confidently detected from both methicillin susceptible and methicillin resistant Staphylococcus aureus (MSSA and MRSA, respectively). The metabolic profiles can be used to distinguish the isogenic pairs of MSSA strains from MRSA strains, without or with sub-lethal levels of methicillin exposure. In addition, better separation between MSSA and MRSA strains can be achieved in the latter case using principal component analysis (PCA). Metabolite data from isogenic pairs of MSSA and MRSA strains were further compared without and with sub-lethal levels of methicillin exposure, with metabolic pathway analyses additionally performed. Both analyses suggested that the metabolic activities of MSSA strains were more susceptible to the perturbation of the sub-lethal levels of methicillin exposure compared to the MRSA strains.
Keywords: Targeted metabolic profiling; Methicillin resistant Staphylococcus aureus; Antibiotic resistance test; Mass spectrometry based metabolomics; Metabolic pathway analysis;
MALDI imaging mass spectrometry analysis—A new approach for protein mapping in multiple sclerosis brain lesions by Giuseppina Maccarrone; Sandra Nischwitz; Sören-Oliver Deininger; Joachim Hornung; Fatima Barbara König; Christine Stadelmann; Christoph W. Turck; Frank Weber (131-140).
Multiple sclerosis is a disease of the central nervous system characterized by recurrent inflammatory demyelinating lesions in the early disease stage. Lesion formation and mechanisms leading to lesion remyelination are not fully understood. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry imaging (MALDI–IMS) is a technology which analyses proteins and peptides in tissue, preserves their spatial localization, and generates molecular maps within the tissue section. In a pilot study we employed MALDI imaging mass spectrometry to profile and identify peptides and proteins expressed in normal-appearing white matter, grey matter and multiple sclerosis brain lesions with different extents of remyelination. The unsupervised clustering analysis of the mass spectra generated images which reflected the tissue section morphology in luxol fast blue stain and in myelin basic protein immunohistochemistry. Lesions with low remyelination extent were defined by compounds with molecular weight smaller than 5300 Da, while more completely remyelinated lesions showed compounds with molecular weights greater than 15,200 Da. An in-depth analysis of the mass spectra enabled the detection of cortical lesions which were not seen by routine luxol fast blue histology. An ion mass, mainly distributed at the rim of multiple sclerosis lesions, was identified by liquid chromatography and tandem mass spectrometry as thymosin beta-4, a protein known to be involved in cell migration and in restorative processes. The ion mass of thymosin beta-4 was profiled by MALDI imaging mass spectrometry in brain slides of 12 multiple sclerosis patients and validated by immunohistochemical analysis. In summary, our results demonstrate the ability of the MALDI–IMS technology to map proteins within the brain parenchyma and multiple sclerosis lesions and to identify potential markers involved in multiple sclerosis pathogenesis and/or remyelination.
Keywords: MALDI imaging mass spectrometry; LC–ESI–MS/MS; Multiple sclerosis; Demyelination; Remyelination; Thymosin beta-4;
A GC–MS method for the detection and quantitation of ten major drugs of abuse in human hair samples by A. Orfanidis; O. Mastrogianni; A. Koukou; G. Psarros; H. Gika; G. Theodoridis; N. Raikos (141-150).
A sensitive analytical method has been developed in order to identify and quantify major drugs of abuse (DOA), namely morphine, codeine, 6-monoacetylmorphine, cocaine, ecgonine methyl ester, benzoylecgonine, amphetamine, methamphetamine, methylenedioxymethamphetamine and methylenedioxyamphetamine in human hair. Samples of hair were extracted with methanol under ultrasonication at 50 °C after a three step rinsing process to remove external contamination and dirt hair. Derivatization with BSTFA was selected in order to increase detection sensitivity of GC/MS analysis. Optimization of derivatization parameters was based on experiments for the selection of derivatization time, temperature and volume of derivatising agent. Validation of the method included evaluation of linearity which ranged from 2 to 350 ng/mg of hair mean concentration for all DOA, evaluation of sensitivity, accuracy, precision and repeatability. Limits of detection ranged from 0.05 to 0.46 ng/mg of hair. The developed method was applied for the analysis of hair samples obtained from three human subjects and were found positive in cocaine, and opiates.
Keywords: Drugs of abuse; Hair analysis; GC–MS; Derivatization;
Cross-validated stable-isotope dilution GC–MS and LC–MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol by Arslan Arinc Kayacelebi; Celina Schauerte; Katharina Kling; Jan Herbers; Bibiana Beckmann; Stefan Engeli; Jens Jordan; Alexander A. Zoerner; Dimitrios Tsikas (151-159).
2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC–MS and LC–MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10 μM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0–1 μM) as the MAGL product. Unlabelled AA (d0-AA, 1 μM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC–MS/MS analysis or in anhydrous acetonitrile for GC–MS analysis. LC–MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M–H]− → [M–H – ―CO2]−, i.e., m/z 311 → m/z 267 for d8-AA and m/z 303 → m/z 259 for d0-AA. Prior to GC–MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC–MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M–PFB]−, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC–MS and LC–MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0–1 μM) of d8-AA measured by LC–MS/MS (y) and that by GC–MS (x) revealed a straight line (r 2 = 0.9848) with the regression equation y = 0.003 + 0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC–MS/MS.
Keywords: Arachidonic acid; 2-Arachidonoyl glycerol; Cannabinoid receptors; Deuterium; Endocannabinoids; GC–MS; LC–MS/MS; MAGL; Prostaglandins;
Development of a multi-residue method for scrutinizing psychotropic compounds in natural waters by V. Brieudes; S. Lardy-Fontan; S. Vaslin-Reimann; H. Budzinski; B. Lalere (160-172).
The present work describes a multi-residue SPE-UPLC–MS/MS method aiming at the characterization of 68 compounds in natural waters, including parent compounds as well as their major metabolites and glucuronide conjugates. Development was conducted toward the quantitative determination of a broad range of analytes belonging to different class of psychotropic drugs such as benzodiazepines, antidepressants, stimulants, opiates and opioids, anticonvulsants, anti-dementia drugs, analgesic and anti-inflammatory drugs (as anthropic indicators) in the low ng L−1 range of concentration. Satisfactory extraction recoveries >70% were obtained for the majority of analytes (49 out of 68) allowing low limits of quantification. LOQ ranged between 0.1 and 17.8 ng L−1 and were lower than 5 ng L−1 for 94% of investigated analytes. Furthermore, addition of 25 isotopic labeled standards allowed to ensure reliability of the optimized method. Quantification errors were typically below 15% with relative standard variations <10% in intermediate precision conditions. Finally, the developed method was implemented in natural waters; sampling campaigns were conducted in the Seine River as a demonstration of the applicability and adequation of the method for its purpose. As a result, 48 out of 68 analytes were identified or quantified; some of them like memantine, rivastigmine, zolpidem 4-phenyl-carboxylic acid, zolpidem 6-carboxylic acid for one of the first time in surface waters. Among investigated psychotropic compounds and metabolites, tramadol, codeine, oxazepam, venlafaxine, O-desmethylvenlafaxine, gabapentin, carbamazepine and 10,11-dihydro-10,11-dihydroxycarbamazepine were found to be the most abundant.
Keywords: Pharmaceuticals; Illicit drugs; Metabolites; Multi-residue; Method development; Isotope dilution; SPE; UPLC–MS/MS; Surface water;
Study of the effect of CYP2C19 polymorphisms on omeprazole pharmacokinetics by utilizing validated LC–MS/MS and Real Time-PCR methods by Maria Koukoula; Yannis Dotsikas; Elina Molou; Kleopatra H. Schulpis; Georgia Thodi; Maria Chatzidaki; Olga Triantafylli; Yannis L. Loukas (173-179).
A fully automated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the determination of omeprazole in human plasma. Utilization of 96-well plates and robotic liquid handling workstations, rendered the whole procedure very fast, compared to the manual respective procedure of Liquid-Liquid Extraction (LLE). Sample analysis was performed by reversed phase LC–MS/MS, with positive electrospray ionization, using multiple reaction monitoring (MRM). The method required low plasma volumes and analysis of samples was completed in short run times. It was fully validated and applied to a pharmacokinetic study after per os administration of 20 mg tablet formulations of omeprazole. The obtained concentrations were used for the calculation of the basic omeprazole pharmacokinetic parameters. Some variations observed in pharmacokinetic parameters among subjects were attributed to differences of CYP2C19 genotype. Therefore, a novel molecular method was developed in which DNA analysis was conducted by using Real Time-Polymerase Chain Reaction (Real Time-PCR). As source of biological material, Dried Blood Spots (DBS) were utilized, offering an alternative and advantageous strategy for such kind of studies.
Keywords: Omeprazole; LC–MS/MS; Pharmacogenetic; PCR; Dried Blood Spots;
PCI-GC–MS–MS approach for identification of non-amino organic acid and amino acid profiles by Hemi Luan; Lin Yang; Fenfen Ji; Zongwei Cai (180-184).
Alkyl chloroformate have been wildly used for the fast derivatization of metabolites with amino and/or carboxyl groups, coupling of powerful separation and detection systems, such as GC–MS, which allows the comprehensive analysis of non-amino organic acids and amino acids. The reagents involving n-alkyl chloroformate and n-alcohol are generally employed for providing symmetric labeling terminal alkyl chain with the same length. Here, we developed an asymmetric labeling strategy and positive chemical ionization gas chromatography—tandem mass spectrometry (PCI-GC–MS–MS) approach for determination of non-amino organic acids and amino acids, as well as the short chain fatty acids. Carboxylic and amino groups could be selectively labelled by propyl and ethyl groups, respectively. The specific neutral loss of C3H8O (60 Da), C3H5O2 (74 Da) and C4H8O2 (88 Da) were useful in the selective identification for qualitative analysis of organic acids and amino acid derivatives. PCI-GC–MS–MS using multiple reaction monitoring (MRM) was applied for semi-quantification of typical non-amino organic acids and amino acids. This method exhibited a wide range of linear range, good regression coefficient (R2) and repeatability. The relative standard deviation (RSD) of targeted metabolites showed excellent intra- and inter-day precision (<5%). Our method provided a qualitative and semi-quantitative PCI-GC–MS–MS, coupled with alkyl chloroformate derivatization.
Keywords: Alkyl chloroformate; PCI-GC–MS–MS; Non-amino organic acids; Amino acids; Derivatization;
GC–ECNICI-MS/MS of eicosanoids as pentafluorobenzyl-trimethylsilyl (TMS) derivatives: Evidence of CAD-induced intramolecular TMS ether-to-ester rearrangement using carboxy-18O-labelled eicosanoids and possible implications in quantitative analysis by Dimitrios Tsikas (185-196).
GC–MS and GC–MS/MS of pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether (PFB-TMS) derivatives of hydroxylated long-chain fatty acids including arachidonic acid metabolites, the eicosanoids, in the electron-capture negative-ion chemical ionization (ECNICI) mode are the most sensitive and accurate approaches to quantify carboxyl groups-containing compounds in complex biological fluids such as plasma and urine. Under ECNICI conditions, PFB-TMS derivatives of eicosanoids ionize to form very few ions, with the carboxylates [M–PFB]− being typically the most intense. Less intense ions may be additionally formed by consecutive neutral loss (NL) of trimethylsilanol (TMSOH, 90 Da) groups ([M–PFB–(TMSOH)n]−). By using [1,1-18O2]- and [1,ω-18O2]-eicosanoids, we studied ion processes following collisionally activated dissociation (CAD) of the precursor ions [M–PFB]−. We found that CAD resulted in formation of product ions due to NL of a TMS18OH (92 Da) group in monocarboxylic and of a PFB18OH (200 Da) group in dicarboxylic eicosanoids. TMS18OH NL implies an intra-molecular transfer of the TMS group from hydroxyl groups to their carboxylate anions [M–PFB]−. From a mechanistic point of view, this rearrangement may explain formation of unique product ions in GC–MS/MS of eicosanoids under ECNICI conditions. From the quantitative point of view, quantification by GC–MS/MS of product ions due to [M–PFB–(TMSOH)n]− and [M–PFB–TMS18OH–(TMSOH)n-1]−would reveal incorrect data, if [1,1-18O2]-eicosanoids are used as internal standards and if no correction for the 18O-loss is performed. In 18O-labelled dicarboxylic eicosanoids, such as the major urinary metabolite (MUM) of E prostaglandins, i.e., [1,ω-18O2]-PGE-MUM), no TMS ester/TMS ether rearrangement was observed. Yet, 18O-loss occurred upon CAD of [M–PFB]− due to NL of PFB18OH (200 Da). In both cases the extent of 18O-loss needs to be determined and considered for accurate quantification of monocarboxylic acids such as 8-isoprostaglandin F2α (8-iso-PGF2α) and dicarboxylic eicosanoids such as PGE-MUM.
Keywords: Collisionally-activated dissociation; Derivatization; Electron-capture negative-ion chemical ionization; Oxygen-18; Pentafluorobenzyl bromide; Trimethylsilyl;
A hydrophilic interaction chromatography-tandem mass spectrometry method for amino acid profiling in mussels by Emmanouil D. Tsochatzis; Olga Begou; Helen G. Gika; Panayotis D. Karayannakidis; Stavros Kalogiannis (197-206).
A UHPLC-HILIC-tandem MS method has been developed and validated for the quantification of 21 amino acids (20 protein amino acids and cystine) in their free form (FAA) and as protein constituents (total amino acids, TAA) in a rich protein food matrix such as lyophilized mussels (Mytilus galloprovincialis) samples. FAA were analyzed after suspending the samples in the presence of trichloroacetic acid in order to prevent dissolving the proteins, while TAA were determined after acid hydrolysis with 6 M HCl in the presence of 4% v/v thioglycolic acid as a reducing agent. In hydrolysed samples 17 amino acids could be determined since tryptophan, cysteine, cystine and asparagine were degraded during acid hydrolysis. Linear regression coefficients (R2) were above 0.99 for all amino acids. Accuracy and precision, expressed as recovery (%) and relative standard deviation (RSD, %) were in acceptable levels, ranging from 78.2 to 123.3% and below 15%, respectively for both FAA and TAA. Uncertainty was also below 12% for FAA and below 22% for TAA. Sensitivity of the method was high with LOD values ranging from 0.003 to 0.034 g/100 g for FAA and 0.001 to 0.004 g/100 g for TAA, while LOQ ranged from 0.009 to 0.104 g/100 g for FAA and 0.002 to 0.011 g/100 g for TAA. The method proved to be a fast and reliable tool for acquiring information on free and total amino acids profile in high protein content foodstuffs such as mussels.
Keywords: HILIC; Tandem mass spectrometry; Free amino acids; Total amino acids; Mussels;
Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings by Erik Hanff; Moritz Lützow; Arslan Arinc Kayacelebi; Armin Finkel; Mirja Maassen; Georgi Radoslavov Yanchev; Arash Haghikia; Udo Bavendiek; Anna Buck; Thomas Lücke; Norbert Maassen; Dimitrios Tsikas (207-214).
Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO−) and nitrate (ONO2 −) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC–MS. Here, we report on the simultaneous PFB-Br derivatization (60 min, 50 °C) of ONO−, O15NO−, ONO2 −, O15NO2 −, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC–MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M−PFB]−, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO−), m/z 47 (O15NO−), m/z 62 (ONO2 −), m/z 63 (O15NO2 −), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97 min for PFB-ONO2/PFB-O15NO2, 3.1 min for PFB-NO2/PFB-15NO2, and 6.7 min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1 mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n = 8), NaNO3 (n = 9) supplementation increased UNOxR (1709 ± 355 vs. 369 ± 77, P < 0.05). Creatinine excretion did not differ between the groups (6.67 ± 1.34 mM vs. 5.72 ± 1.27 mM, P = 0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r = 0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P = 0.035 and P = 0.004, respectively) but not their concentrations (P = 0.68 and P = 0.40, respectively) differ between male (n = 54) and female (n = 24) CVD patients. No such differences were found between preterm newborn boys (n = 25) and girls (n = 22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.
Keywords: Renal carbonic anhydrase; Clinical studies; Creatinine; Nitric oxide; Quantification; Supplementation;
An ultra-high pressure liquid chromatography-tandem mass spectrometry method for the quantification of teicoplanin in plasma of neonates by O. Begou; A. Kontou; N. Raikos; K. Sarafidis; E. Roilides; I.N. Papadoyannis; H.G. Gika (215-222).
The development and validation of an ultra-high pressure liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was performed with the aim to be applied for the quantification of plasma teicoplanin concentrations in neonates. Pharmacokinetic data of teicoplanin in the neonatal population is very limited, therefore, a sensitive and reliable method for the determination of all isoforms of teicoplanin applied in a low volume of sample is of real importance. Teicoplanin main components were extracted by a simple acetonitrile precipitation step and analysed on a C18 chromatographic column by a triple quadrupole MS with electrospray ionization. The method provides quantitative data over a linear range of 25–6400 ng/mL with LOD 8.5 ng/mL and LOQ 25 ng/mL for total teicoplanin. The method was applied in plasma samples from neonates to support pharmacokinetic data and proved to be a reliable and fast method for the quantification of teicoplanin concentration levels in plasma of infants during therapy in Intensive Care Unit.
Keywords: Liquid chromatography–mass spectrometry; Teicoplanin; Antibiotic determination; Plasma;