A defined amount, e.g. 30 µL of native whole blood is carefully pipetted and spotted onto a special marked filter paper/card without touching the card with the filter tip. After drying at room temperature for at least three hours, such a Dried Blood Spot (DBS) is punched, if necessary repeatedly. The target analyte(s) are extracted from the corresponding disk(s) by vortexing in the presence of an organic solvent for 60 min. Finally, only an aliquot of the supernatant is subjected to further clean-up. Thus the sample is diluted before the final analysis. Furthermore, the addition of the Internal Standard (IS) takes place late, at the extraction step (Figure 3).
Metabolomics is the scientific and systematic study of biochemical processes and pathways within cells, biofluids, tissues or organisms involving small substrates, intermediates and products of host and microbial metabolism, the metabolites, which can be influenced by genetic and environmental factors . It is one of the most complex methods of life science considering the high number and diversity of metabolites and their different physicochemical properties. Due to the divergent nature of small molecules no technique alone is able to measure the entire metabolome at once and many different complementary techniques evolved during the last decades to determine the small molecule content of bio specimens, most importantly nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The latter are often coupled with chromatographically methods (liquid chromatography- mass spectrometry (LC-MS), gas chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry) to increase resolution . The main focus lies on the comprehensive study of biochemical mechanism and interaction of the human body . Metabolomics is based on the idea that biological fluids like blood, plasma, urine, lymph or saliva reflect the health of an individual and can be used for medical analysis and diagnosis to identify disease and disease states . The metabolomics approach is an important tool in biomedical research, which contributes to the identification of new potential biomarkers . Biomarkers are accurately and reproducibly measurable and evaluable products of the organism which refer to a person’s individual health status . For medical purposes a high number of diagnostic and predictive biomarkers are already used for example specific metabolites linked to various diseases, such as cardiovascular disease [30, 46, 47], cancer [48- 50], diabetes  and Morbus Parkinson .
LC-MS/MSAnalysis of Modified Nucleosides LC-MS/MSanalysis of modified nucleosides using a triple quadrupole mass spectrometer was conducted under a variety of scientific tasks, requiring different kinds of experimental setups and subsequent data analysis. The rather standard DMRM mode, applicable for highly sensitive quantitative analysis of known RNA modifications, was mainly used for analysis of wobble U34 modifications in collaboration with the laboratories of Prof. Dr. R. Schaffrath and Prof. Dr. M. Stark in order to both prove the knockout effciency of mutants for the U34-modifying enzymes and to investigate their effects on the presence of the respective wobble U34 modifications [102, 246, 247]. In order to verify the successful isolation of several tRNAs from Saccharomyces cerevisiae, the second “standard” scan mode, namely the neutral loss scan, was used to evaluate the presence of both expected and unexpected modifications in the isolated tRNAs. The analyzed tRNAs contained, next to all of the expected modified nucleosides, only two more nucleosides, whose presence could be explained by either degradation or rearrangement of another modification. Hereby, LC-MS/MSanalysis confirmed the suitability of the isolation protocol for the isolation of pure tRNAs. A more sophisticated analysis was required for the nucleoside modification queuosine (Q), which displays an unusual fragmentation pattern . A product ion scan was performed to confirm the fragmentation at the aminomethyl-linker structure and to enable the programming of a suitable DMRM method, which was finally used to investigate the incorporation of Q into S. pombe tRNA under nutritional supply of queuine in collaboration with Dr. M. M ¨uller and Prof. Dr. A. Ehrenhofer-Murray . Remarkably, Q can occur in form of sugar-modified derivatives in mammals, manQ and galQ, which were readily detected in HeLa RNA but absent in S. pombe RNA. However, there might be additional, hitherto unknown
Data analysis with literature was very difficult because there were no data about LC- MS/MSanalysis for the six herbs. So identification of the main compounds was only a manual comparison with the reference data. However the references only should have one peak, but a lot of them had more than one. This was maybe caused by contamination by other substances. The references which had more than one main peak were atractylodine, oleanolic acid, tartaric acid and fumaric acid. So these references were useless, because it was not possible to identify the main peak. In some cases it was possible to differ a main peak from other smaller peaks. These substances are shown in the Tab. 14.
All LC-MS/MSanalysis were performed using an Agi- lent 1200 capillary-HPLC system (Germany) coupled to a HCT mass-spectrometer (Bruker, Bremen, Germany) with an electrospray ionisation (ESI) interface. A C18 Aq Zorbax column (150 × 0.5 mm; 5 μm) was used for sep- aration. HyStar-software (Bruker, Bremen, Germany) was used for data acquisition and processing. The mo- bile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The flow rate was set to 20 μl/min and the in- jection volume was 40 μl. The column temperature was maintained at 30°C. The plasma samples were separated using gradient given in the Table 1. The total time per run was 25 min. The mass-spectrometer was operated in the positive ion mode. The source temperature was set at 300°C. The nebuliser gas was set at 20 psi and the dry gas flow was 7 l/min. For the detection of angiotensin II, the MS/MS fragmentation mode was used. The accumula- tion time was 50 ms. The double charged parent ions of the native angiotensin II and the stable isotope ( 13 C- and 15
that catalyzes the conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP). Furthermore, an upregulation of fatty acid-binding protein (Fabp) and carbonic anhydrase III was detected, both pre- dicted to participate in aerobic metabolism of type I fibres, thus leading the authors to the conclusion of enhanced oxidative metabolism in dysferlinopathy. De Palma et al., applied a 2D-gel electrophoresis analysis, followed by measurement of altered protein contents by electrospray MS/MS (Q-TOF MS). Several proteins were detected in altered expressions, where NADH-ubiquinone oxidoreductase (O75489) and ubiquinol cytochrome c reductase (P22695) were up-regulated, whereas alpha-enolase (P06733) and phosphoglycerate mutase (P15259), both enzymes of the glycolytic pathway, were down- regulated. Taking into account that both studies analysed human muscle protein, it is surprising that there is a wide variability referring to the results reported, which might be attributed to the different MS meth- ods (matrix-assisted laser desorption/ionization, MALDI versus electrospray ionization, ESI) applied or to the lower sensitivity of 2D-electrophoresis. The SILAC-based LC-MS/MSanalysis applied by our group provides a global insight into the muscle protein turnover under various conditions. The usage of a SILAC reference as internal standard additionally enables a highly accurate comparison of wildtype and knockout mice (Kruger, Moser et al. 2008). Focusing on glycolytic and TCA-cycle enzymes, we found most of the protein expression levels unchanged between dysferlin-deficient and wildtype mice. Further- more, there were no alterations of enzymes connected to the mitochondrial respiratory chain, which clear- ly delineates the metabotype found in dysferlinopathy from other metabolic and hereditary muscular dis- eases, as outlined in chapter 5.4. Alterations between enzymes greatly varied between glycolytic
as illustrated by the method of peptaibiomics, target-oriented selection, and optimi- zation of separation techniques, as well as the use of state-of-the-art methods in mass spectrometry, enable the detection of homologues and positional isomers, even if present in trace amounts. In principle, only one fully-grown Petri dish or slope is required for a first routine analysis of the peptaibiome of a fungal culture. In this context, it should be clearly pointed out again that Iva is frequently found as d- or l- isomer in peptaibiotics . To continue, both enantiomers of Iva can be present in the same peptaibiotic, as was reported for neoefrapeptins and integramides (see above) as well as for the 15-residue peptaibol clonostachin: the latter contains d-Iva in positions 4 and 13, whereas l-Iva is found in positions 7 and 10 . On total hydrolysis, an apparently racemic mixture (RS)-dl-Iva is released. Separation of dl-Iva and dl-Pip, analyzed as N-trifluoroacyl amino acid propyl esters on Chirasil-l-Val TM (N-propionyl- l -valine-tert-butylamide polysiloxane), is not always satisfactory, as it depends both on the age and quality of the capillary columns provided by various manufacturers. However, Iva enantiomers can be resolved on Chirasil-l-Val TM after conversion into N- acetylisovaline propyl esters . Instead of Chirasil-l-Val TM , a Lipodex TM E column, representing a functionalized g-cyclodextrine, can be used providing excellent resolution of Iva and Pro enantiomers . Notably, all eight stereoisomers of 3- and 4-Hyp can be resolved on Chirasil-l-Val TM . Alternatively, pre-column derivatization with the chiral N a -(5-fluoro-2,4-dinitrophenyl)-l-alanine amide (FDAA; Marfey?s reagent), and subsequent HPLC or HPLC/ESI-MSanalysis of the resulting diastereoisomers is recommended : by this method, dl-Iva and dl-Pip are very well resolved. Last but not least, methanolysis of peptaibiotics, followed by trifluoroacetylation and analysis of the resulting dipeptides by GC/EI-MS, has been introduced as a method to solve the problem to assign the positions of isobaric amino acids Val/Iva and Leu/Ile, respectively . Recent advances in quantitation of dl- amino acids are summarized in .
To separate peptide mixtures prior to MSanalysis, nano reverse phase high-performance liquid chromatography (nanoRP-HPLC) was applied on the Ultimate 2 Dual Gra- dient HPLC system (Dionex, buffer A: 5% acetonitrile (ACN), 0.1% TFA, buffer B: 80% ACN, 0.1% TFA) on a PepMap separation column (Dionex, C18, 150 mm × 75 μm × 3 μm, 300 A). 500 fM of each mixture was separated three times using the same trapping and separation col- umn to reduce the quantification error which comes from HPLC and mass spectrometry. A gradient from 0% B to 50% B in 48 min was applied for the separation; peptides were detected at 214 and 280 nm in the UV detector. The exit of the HPLC was online coupled to the electrospray source of the LTQ mass spectrometer (Thermo Electron). Samples were analyzed in centroid mode first to test digest and labeling quality. For the quantitative analysis the LTQ was operated in enhanced profile mode for survey scans to gain higher mass accuracy. Samples were mass spectro- metrically analyzed using a top one method, in which the most abundant signal of the MS survey scan was frag- mented in the subsequent MS/MS event in the ion trap. Although with this method a lower number of MS/MS spectra were acquired, the increased number of MS scans leads to a better determination of the eluting peaks and therefore provides improved quantification of peptides. Data analysis was done with the Mascot Daemon  (Matrix Science), BioWorks 3.2  (Thermo Electron) software packages using an in house database. To demon- strate the merging of results from all of the mentioned search engines the ICPL labeled probes at an ratio of 1:1
Online LC-MSanalysis affords specific methods to inject and ionise the analyte. Apart from brute techniques, with fast atom bombardment (FAB) leading the way, the softer electrospray ionisation (ESI) has contributed to reach new levels of sensitivity. 1,17 The principle of ESI is based on the liquid sample flowing through a thin capillary whose end, the tip of the needle, is applied to voltage. 17 The electric potential, formed in relation to the counter electrode, reaches 3-4 kV. 15 When reaching the needle outlet, the sample solution becomes part of the electric field as well. In the case of positive ion-mode ESI, cations in the liquid appear at the surface while anions are removed by oxidation at the needle. Attracted by the anode, positive ions continuously move further, forming a liquid cone, the Taylor cone. When leaving the jet, droplets are formed. Due to a rising instability of equally charged units, droplets degrade at the characteristic Rayleigh limit. Having undergone Coulomb explosions, there are currently two schemes that describe the way the ultra-small drips are conveyed into the gas phase. The charged residue model (CRM) claims that desorption of single ions is obtained by multiple coulomb explosions and a subsequent desolvation. In turn, the ion evaporation model (IEM) suggests a direct emission of single ions from droplets, providing that the diameter is higher than required for the Rayleigh limit. Finally, when the gaseous cations reach the cathode, they enter the mass spectrometer. 16,17,18
Various types of mass spectrometers are currently used in metabolomics. Simply put, they differ on the one hand in the sample introduction system and the ion source, and on the other hand in the implemented mass analyzer. For metabolomics, the most relevant ion sources are ESI and electron impact ionization (EI). ESI, denoted as soft ionization source, is used in connection with LC or CE. Here the liquid sample is nebulised, evaporated and ionized under atmospheric pressure in a strong electric field. Small metabolites are usually ionized to single charged molecule ions, either positively charged or negatively charged depending on the applied polarity. A well known problem in ESI is ion suppression which is ideally alleviated by the use of isotope dilution mass spectrometry (IDMS), e.g. by use of fully 13 C labeled metabolites as internal standard  . EI is used in connection with GC, where the gaseous eluent and analytes are introduced into the ion source. Positive ionization of molecules is achieved under vacuum pressures by use of an electron beam at specific and reproducible energy. A typical fragmentation of the analytes occurs which is reproducible between instruments. A mass analyzer, as an integral part of a mass spectrometer, provides the separation in time or space, of ions according their mass-to-charge ratio (m/z). Criteria for mass analyzers are sensitivity, mass resolution, mass accuracy, scan speed or acquisition rate and MS/MS capability.
The omics revolution is closely related to recent technical revolution of modern and powerful mass spectrometric (MS) methods. Constantly evolving mass spectrometers enable researchers to characterise and quantify individual (bio)-molecules in living systems. MS is in the forefront of analytical techniques which measure the mass and count ionised molecule forms in a fast, selective, highly sensitive and reliable way. Other spectroscopy techniques such as ultraviolet, infrared and nuclear magnetic resonance spectroscopy are based on physical events resulting from the interaction of functional organic molecule groups with electromagnetic radiation. Important applications of MS in biological science include the structural characterisation of biomolecules such as carbohydrates, nucleic acids and steroids, sequencing of peptides and oligosaccharides, drug metabolism determination and their quantification. Selective portrayals of (bio)-molecules in tissues can be produced by monitoring characteristic patterns via a special MS technique called multiple reaction monitoring (MRM) .
Leslie Tais, Christoph Böttcher and Hartwig Schulz
Julius Kühn Institute, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Berlin
E-mail of corresponding author: firstname.lastname@example.org Wheat was one of the first domesticated
Due to the instability of Asn, Cys, Gln and Trp, only a few published methods allow the detailed analysis of all proteinogenic AA (24). Many procedures do not fully cover all analytes (28, 29). A major problem for detecting a huge spectrum of analytes is the insufficient peak sharpness of polar analytes like Asn, Cit, Gln (17, 18). Compared to our method, the commercial ITRAQ system for quantification of derivatives AA analyses 44 AA but is less sensitive and the required reagents are toxic and expensive. Using ITRAQ peak sharpness of Asn and Ser is not optimal either (19). In comparison, our method presents sharp peaks for all 22 measured analytes and efficient separation of the combined derivatization with ion-pair chromatography. The proposed method delivers high resolution chromatographic separation on standard C18 reversed-phase column which is extremely stable for thousands of samples. Because of a very favorable retention behavior, a separation within 16.5 min is possible using a modern HPLC technology. Improved chromatographic retention of polar AA allows for switching the beginning of the chromatogram to waste to reduce contamination of the mass spectrometers ion source. The flexible use of two ion sources (APCI, atmospheric pressure chemical ionization and ESI, electrospray ionization) provides a further advantage. Normally we used APCI because background signals and matrix effects of APCI have been reported to be often lower than with ESI (26). A minor drawback of derivatization with butanolic HCL is the formation of HCL gas during evaporation which may cause corrosion. Metallic parts of the evaporator device can be protected from rust by covering with adhesive film.
Folate deficiencies are often accompanied by the accumulation of tHcy, which causes HHCY (77). HHCY is classified into three types: moderate HHCY (tHcy = 12 – 30 µmol/L), intermediate HHCY (tHcy > 30 – 100 µmol/L), and severe HHCY (tHcy > 100 µmol/L) (111). Moderate HHCY is associated with several pathologic conditions, including cardiovascular and neurodegenerative diseases (91). Apart from oxidative stress, another main pathomechanism in HHCY is hypomethylation (30). Elevated tHcy concentrations can be lowered by supplementing low doses of FA in healthy subjects (27). In a meta-analysis, the Homocysteine Lowering Trialists’ Collaboration stated that daily doses of ≥ 800 µg FA are required to achieve the maximal reduction in plasma tHcy concentrations (100). Lower doses of 200 and 400 µg FA are associated with 60% and 90% tHcy lowering, respectively, of this maximal effect. However, the effect of tHcy lowering on pathologic conditions is ambiguous. On the one hand, tHcy lowering prevents stroke (189) and cardiovascular events in hemodialysis patients (184), on the other hand the risk of major cardiovascular events in patients with vascular disease could not been lowered (143), cognitive performance could not be improved in healthy subjects (150), and lower tHcy had no beneficial effect on inflammatory markers associated with atherosclerosis in patients with stable coronary artery diseases (22).
Evaluation of performance was mainly based on recovery values, but also on repeatability of the values and ease of handling during sample preparation. Experiments were conducted in duplicate as described in 2.2.5 and all four cartridges were treated the same way. The expected final concentration in the vials was 100 µg/l and was used for the calculation of the recovery shown in Figure 3-22, Figure 3-23 and Figure 3-24. These Figures indicate that the recovery is in the range of 75-98% for the first three cartridges, namely the Oasis HLB, Phenomenex Strata-X and Macherey-Nagel HR-X (all 3 ml and 60 mg). Only the Macherey-Nagel OH Diol (3 ml, 200 mg) shows lower recoveries for bacitracin (45-49%), while nearly no recovery for colistin and polymyxin (2-5%). The OH Diol column was used since it was mentioned in the application note 300750 from the Macherey-Nagel website when looking for SPE cartridges for the analysis of bacitracin. A possible explanation for the low recoveries using the OH Diol resin is the fact that not the recommended conditions and solutions were used. However since in literature mainly Oasis HLB and Phenomenex Strata-X cartridges are reported when analysing polypeptide antibiotics, the OH Diol cartridges were not further tested. The other three cartridges are all based on polystyrene divinyl benzene (PS-DVB) polymer resins.
Für die Untersuchung des Einflusses auf die Selektivität der MD-SPE Plattform wurden MS- Scans (m/z 100 – 1000) von Plasmaproben nach jeweiliger Analyse mit den untersuchten MMP-Materialien aufgezeichnet (Abbildung 53). Die Chromatogramme der vier MMP SPE- Säulen Oasis WCX 20 x 1 mm (1), 20 x 2,1 mm (2) sowie Strata X-CW 20 x 1 mm (3) und 20 x 2 mm (4) zeigen nur ein Systemsignal bei 3,4 min. In diesen Chromatogrammen wurden keine niedermolekularen Matrixbestandteile detektiert. In dem Chromatogramm des LiChrospher ® XDS Materials (5) wurden neben dem Systemsignal bei 3,7 min auch niedermolekulare Matrixbestandteile im Bereich von 3,9 – 4,5 min detektiert. Dies weist daraufhin, dass dieses Material eine Selektivität aufweist, die nicht für die MD-SPE Plattform geeignet ist.
Since the dyes are found in the wastewater as hydrolysates, procedures for hydrolysis were worked out first. It was possible to hydrolyse the reactive anchor groups gently, by pro- cesses similar to those used for dyeing, without changing or destroying the chromophore. The ozonisation of the hydrolysates was carried out in a bubble-column reactor. The nu- merous intermediate products were present only in low, stationary concentrations. For bet- ter study of these, they were first enriched by SPE, which separated polar from moderately polar degradation products. The main components of the moderately polar fraction could be characterised and partly quantified by RP chromatography with UV/VIS-diode-array or mass spectrometry (LC-QTOF with ESI/APCI) for detection. Plausible structures could be proposed from precision masses, which in most cases could be determined with a deviation of ≤ 5 ppm from the theoretical value. The structures were confirmed wherever possible by
Abb. 28 Die Abbildung stellt die Grundannahme der Aktivitäts-Korrelations-Analyse (AcorA) dar. Da ein Extrakt aus vielen Einzelsubstanzen besteht und jede einzelne durch mehrere MS- Peaks repräsentiert wird, ist es erforderlich, eine Korrelation zwischen der Gesamtbioaktivität und jedem einzelnen Peak bzw. dessen Peakintensität aufzustellen. Dabei wird angenommen, dass die aktivitätsrelevanten Peaks die beste Korrelation aufweisen. Voraussetzung für die Korrelationsanalyse ist jedoch ein gewisses Maß an Variation, sowohl bei der biologischen Aktivität, als auch bei den Peakintensitäten der Metabolitenprofile. Dies kann erreicht werden, indem zum Beispiel Extrakte eng verwandter Spezies, unter der Annahme, dass diese ähnliche Metabolitenprofile aufweisen, eingesetzt werden. Sofern nur ein biologisch aktiver Rohextrakt zur Verfügung steht, kann die notwendige Variation und Vervielfältigung, durch Anwendung von verschiedenen Modifikationsmethoden erzeugt werden. Dadurch wird ein großer Satz leicht veränderter Extraktvarianten erhalten. Durch die leichte Veränderung des Metabolitenprofils wird eine Erhöhung bzw. eine Verringerung der relativen Konzentration der enthaltenen Verbindungen erreicht, was anhand einer veränderten Gesamtbioaktivität und Peakintensität deutlich wird. Mittels AcorA erfolgt dann ein Vergleich des „Verhaltens“ bzw. des tendenziellen Verlaufs der Bioaktivität und der Peakintensität über alle Metabolitenprofile. Da kein linearer Zusammenhang zwischen Bioaktivität und Konzentration und somit Peakintensität besteht, bildet die Spearman Rangkorrelationsanalyse die Grundlage von AcorA. Als Ergebnis wird eine Hitliste von Peaks (m/z, t R , Korrelationskoeffizient, Peakintensität) erhalten, die eine statistisch signifikante
For oxidized lipids, biochemists often use elaborate terms describing a molecule at high structural resolution, e.g. 1-Palmitoyl-2-(9-keto-12-oxo-10-dodecenoic acid)-PC . It seems that using ox as a prefix has prevailed for shorthand notation of lipid classes, e.g. oxPC as used in the works of Reis et al. . Following our suggestions for shorthand notation in LDA, the aforementioned oxidized lipid would be reported as oxPC(28:1[2O])) at the MS 1 level, oxPC(16:0/12:1[2O]) at the MS 2 level with known sn-positions of the FA chains and oxPC(16:0/12:1[9-O,12-O]) when the structural resolution allows assignment of modification positions. Oxo, keto, epoxy and furan modifications will not be discernible, as they have the same masses, and oxo/keto/epoxy/furan and hydroxy modifications differ only in two hydrogen atoms, which could be interpreted as the mass difference of a single double bond. A similar issue emerges when the fatty acyl includes a hydroperoxy modification which has the same mass as a hydroxyl-hydroxy modification, and when the chain includes epidioxide, which has the same mass as an oxo-hydroxy modification. This ambiguity cannot be resolved by typical high-throughput MS techniques, as the complexity increases the more oxidation modifications a lipid carries. Taking an extreme case, oxTG(54:6[4OH]) has the exact same mass as oxTG(54:2[4O]). Due to a lack of adequate nomenclature for such cases, researchers currently do not have any means to report such ambiguities. Nevertheless, information to resolve said uncertainties can be obtained by studies of fragmentation patterns or elution profiles, but such studies are lacking for most of the “exotic” oxidation products.
Mit Hilfe der entwickelten Methode zur LC/MS-Trennung der Ceramide lassen sich auch Glucosylceramide chromatographieren, die als polare Lipidvorstufen in den unteren Schichten der Haut vorkommen. In Abbildung 18 ist ein Chromatogramm des Glucosylceramid-Mischstandards 1-O-[ E -D-Glucopyranosyl]-N-acyl-Sphingosin (GluCer(NS)) abgebildet. Die einzelnen enthaltenen Glucosylceramid-Spezies sind jeweils mit ihren Massenspuren [M-H] - dargestellt. Wie aus Abbildung 18 hervorgeht, werden die einzelnen Spezies analog den normalen Ceramiden nach ihrer Polarität und damit ebenfalls nach Kettenlänge bzw. Doppelbindung in der amidgebundenen Fettsäure aufgetrennt. Die Nachweisgrenze der Glucosylceramide wurde wegen fehlender einzelner Referenzsubstanzen nicht explizit bestimmt. Die Empfindlichkeit des Analysenverfahrens kann aber wegen der mit den nichtglucosylierten Ceramiden vergleichbaren Molekülstrukturen und Ionisierungsmechanismen mit dem unteren ppb-Bereich abgeschätzt werden (matrixfreie Meßlösungen).