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  • Kauppila, T. J.; Flink, A.; Pukkila, J.; Ketola, R. A. (2016)
    RATIONALE: Fast methods that allow the in situ analysis of explosives from a variety of surfaces are needed in crime scene investigations and home-land security. Here, the feasibility of the ambient mass spectrometry technique desorption atmospheric pressure photoionization (DAPPI) in the analysis of the most common nitrogen-based explosives is studied. METHODS: DAPPI and desorption electrospray ionization (DESI) were compared in the direct analysis of trinitrotoluene (TNT), trinitrophenol (picric acid), octogen (HMX), cyclonite (RDX), pentaerythritol tetranitrate (PETN), and nitroglycerin (NG). The effect of different additives in DAPPI dopant and in DESI spray solvent on the ionization efficiency was tested, as well as the suitability of DAPPI to detect explosives from a variety of surfaces. RESULTS: The analytes showed ions only in negative ion mode. With negative DAPPI, TNT and picric acid formed deprotonated molecules with all dopant systems, while RDX, HMX, PETN and NG were ionized by adduct formation. The formation of adducts was enhanced by addition of chloroform, formic acid, acetic acid or nitric acid to the DAPPI dopant. DAPPI was more sensitive than DESI for TNT, while DESI was more sensitive for HMX and picric acid. CONCLUSIONS: DAPPI could become an important method for the direct analysis of nitroaromatics from a variety of surfaces. For compounds that are thermally labile, or that have very low vapor pressure, however, DESI is better suited. Copyright (C) 2016 John Wiley & Sons, Ltd.
  • Pöhö, Paivi; Scholz, Karen; Kärkkäinen, Niina; Haapala, Markus; Räikkönen, Heikki; Kostiainen, Risto; Vaikkinen, Anu (2019)
    A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL(-1) and limits of quantitation (LOQ) were between 0.05 to 2 ng mL(-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples. (C) 2019 Elsevier B.V. All rights reserved.
  • Levanova, Alesia; Poranen, Minna Marjetta (2018)
    Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules. Elution is achieved by decreasing the PEG concentration. In this study, SXC applicability for the separation and purification of single-stranded (ss) and double-stranded (ds) RNA molecules was evaluated for the first time. The retention of ssRNA and dsRNA molecules of different lengths on convective interaction media (CIM) monolithic columns was systematically studied under variable PEG-6000 and NaCl concentrations. We determined that over 90% of long ssRNAs (700-6374 nucleotides) and long dsRNAs (500-6374 base pairs) are retained on the stationary phase in 15% PEG-6000 and >= 0.4 M NaCl. dsDNA and dsRNA molecules of the same length were partially separated by SXC. Separation of RNA molecules below 100 nucleotides from longer RNA species is easily achieved by SXC. Furthermore, SXC has the potential to separate dsRNAs from ssRNAs of the same length. We also demonstrated that SXC is suitable for the enrichment of ssRNA (PRR1 bacteriophage) and dsRNA (Phi6 bacteriophage) viral genomes from contaminating cellular RNA species. In summary, SXC on CIM monolithic columns is an appropriate tool for rapid RNA separation and concentration. (C) 2018 The Authors. Published by Elsevier B.V.
  • Knuuttila, Matias; Hämäläinen, Esa; Poutanen, Matti (2019)
    Recent development of gas chromatography and liquid chromatography-tandem mass spectrometry (GC-MS/MS, LC-MS/MS) has provided novel tools to define sex steroid concentrations. These new methods overcome several of the problems associated with immunoassays for sex steroids. With the novel MS-based applications we are now able to measure small concentrations of the steroid hormones reliably and with high accuracy in both body fluids and tissue homogenates. The sensitivity of the tandem mass spectrometry assays allows us also for the first time to reliably measure picomolar or even femtomolar concentrations of estrogens and androgens. Furthermore, due to a high sensitivity and specificity of MS technology, we are also able to measure low concentrations of steroid hormones of interest in the presence of pharmacological concentration of other steroids and structurally closely related compounds. Both of these features are essential for multiple preclinical models for prostate cancer. The MS assays are also valuable for the simultaneous measurement of multiple steroids and their metabolites in small sample volumes in serum and tissue biopsies of prostate cancer patients before and after drug interventions. As a result, novel information about steroid hormone synthesis and metabolic pathways in prostate cancer has been obtained. In our recent studies, we have extensively applied a GC-MS/MS method to study androgen biosynthesis and metabolism in VCaP prostate cancer xenografts in mice. In the present review, we shortly summarize some of the benefits of the GC-MS/MS and novel LC-MS/MS assays, and provide examples of their use in defining novel mechanisms of androgen action in prostate cancer.
  • Anton, Dea; Bender, Ingrid; Kaart, Tanel; Roasto, Mati; Heinonen, Marina; Luik, Anne; Puessa, Tonu (2017)
    Polyphenols of fruits and vegetables form an important part of human dietary compounds. Relatively little is known about accumulation of phenolics during fruits ripening process. The goal of this work was to study the changes in antioxidant activity and in content of 30 polyphenols during ripening of tomato fruits. Five organically and conventionally grown tomato cultivars were investigated at three different ripening stages. Phenolic compounds were extracted with methanol and extracts were analyzed by HPLC-DAD-MS/MS. During ripening, four different changing patterns were observed: (1) high level in green fruits with minimal changes; (2) continuous increase with maximum level in red-ripe fruits; (3) decrease; (4) increase and achieving maximum level at half-ripe stage. Similar change patterns were found for organic and conventional fruits. The accumulation patterns of phenolic compounds were similar in standard-type tomatoes but differed in several cases in cherry-type cultivar. Although contents of some polyphenols decreased during ripening, total phenolics and free radical scavenging activity increased in all studied cultivars and in case of both cultivationmodes. The changes in content of phenolic compounds during ripening were greatly influenced by cultivars, but cultivation mode had only minor impact on dynamics in polyphenols contents in tomato fruits.
  • Becker, Anna; Schalin-Jäntti, Camilla; Itkonen, Outi (2021)
    Context: Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. Objective: To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. Design: A retrospective study during 2016-2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. Methods: Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by 2 different liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays with samples prepared either by protein precipitation or solid phase extraction. Twenty-four-hour urine 5HIAA samples (n = 33) were analyzed by amperometric LC, and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. Results: We achieved 5 to 10 000 nmol/L linearity and Conclusion: Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling, we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.
  • Lindström, Mikael; Tohmola, Niina; Renkonen, Risto; Hämäläinen, Esa; Schalin-Jäntti, Camilla; Itkonen, Outi (2018)
    Background: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. Methods: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D-4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5hydroxyindoleacetic acid (5-HIAA) for detection of NENs. Results: Our assay is sensitive and has a wide linear range (10-10,000 nmo1/1). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 degrees C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmo1/1. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. Conclusions: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.
  • Daly, Paul; Lopez, Sara Casado; Peng, Mao; Lancefield, Christopher S.; Purvine, Samuel O.; Kim, Young-Mo; Zink, Erika M.; Dohnalkova, Alice; Singan, Vasanth R.; Lipzen, Anna; Dilworth, David; Wang, Mei; Ng, Vivian; Robinson, Errol; Orr, Galya; Baker, Scott E.; Bruijnincx, Pieter C. A.; Hilden, Kristiina S.; Grigoriev, Igor V.; Mäkelä, Miia R.; de Vries, Ronald P. (2018)
    White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
  • Semenova, Svetlana; Rozov, Stanislav; Panula, Pertti (2017)
    Catechol-O-methyltransferase (COMT; EC is an enzyme with multiple functions in vertebrates. COMT methylates and thus inactivates catecholamine neurotransmitters and metabolizes xenobiotic catechols. Gene polymorphism rs4680 that influences the enzymatic activity of COMT affects cognition and behavior in humans. The zebrafish is widely used as an experimental animal in many areas of biomedical research, but most aspects of COMT function in this species have remained uncharacterized. We hypothesized that both comt genes play essential roles in zebrafish. Both comt-a and comt-b were widely expressed in zebrafish tissues, but their relative abundance varied considerably. Homogenates of zebra fish organs, including the brain, showed enzymatic COMT activity that was the highest in the liver and kidney. Treatment of larval zebrafish with the COMT inhibitor Ro41-0960 shifted the balance of catecholamine metabolic pathways towards increased oxidative metabolism. Whole-body concentrations of dioxyphenylacetic acid (DOPAC), a product of dopamine oxidation, were increased in the inhibitor treated larvae, although the dopamine levels were unchanged. Thus, COMT is likely to participate in the processing of catecholamine neurotransmitters in the zebrafish, but the inhibition of COMT in larval fish is compensated efficiently and does not have pronounced effects on dopamine levels. (C) 2017 Elsevier Inc. All rights reserved.
  • Järvinen, Erkka; Deng, Feng; Kidron, Heidi; Finel, Moshe (2018)
    Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E-1-G), estradiol-3- and 17-glucuronides (E-2-3G and E-2-17G), as well as estriol-3- and 16-glucuronides (E-3-3G and E-3-16G) are found in human plasma and urine. Unlike E-2-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E-1-G, E-2-3G, E-3-3G, E-3-16G and estrone-3-sulfate (E-1-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E-1-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E-1-G and E-2-3G, were still transported by BCRP at 10-fold higher rates than E-1-S. BCRP also transported E-3-16G at higher rates than the studied MRPs, while it transported E-3-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E-1-G, E-2-3G, E-3-3G and E-3-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K-m values, between 180 and 790 mu M. MRP3 transported all the tested glucuronides at rather similar rates, at K-m values below 20 mu M, but lower V-max values than other transporters. In the case of E-3-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E-3-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.
  • Juvonen, Minna Katriina; Kotiranta, Markus; Jokela, Jouni Kalevi; Tuomainen, Päivi; Tenkanen, Tiina Maija (2019)
    Recent works provide evidence of the prebiotic potential of arabinoxylan-derived oligosaccharides (A)XOS. In this study, we developed a structural analysis for cereal-derived (A)XOS by negative ionization HILIC-MS/MS. Initially, we assessed twelve (A)XOS samples of known structures with different linkage positions and branching points by direct-infusion negative ESI-MSn. We subsequently developed the negative ion HILIC-MS/MS with a post-column addition of ammonium chloride. The selected (A)XOS represented both linear (arabinofuranosyl residue linked to the non-reducing end of xylooligosaccharide) and branched structures. Each (A)XOS sample produced a specific spectrum in negative ion ESI-MSn. By analyzing cross-ring fragment ions, we determined the linkage positions of linear (A)XOS. The presence or absence of diagnostic ions in the MS3 allowed us to detect different branches (O-2- or/and O-3-linked arabinofuranosyl with/or without O-4-linked xylopyranosyl at the non-reducing end). Furthermore, we could identify all analyzed samples by HILIC-MS/MS, based on the formed spectral library and chromatographic retention times.
  • Tienaho, Jenni; Karonen, Maarit; Muilu-Mäkelä, Riina; Wähälä, Kristiina; Leon Denegri, Eduardo; Franzén, Robert; Karp, Matti; Santala, Ville; Sarjala, Tytti (2019)
    Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some dierences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.
  • Chamlagain, Bhawani; Rautio, Saija; Edelmann, Minnamari; Ollilainen, Velimatti; Piironen, Vieno (2020)
    The niacin content of cereal raw materials reported in food-composition databases often differs considerably. One major reason for this discrepancy is the analytical method used for its measurement is that a significant part of the niacin in cereals exists in bound form. In this study, we compared the niacin content of some representative cereal raw materials analysed with a sensitive and validated ultra-high performance liquid chromatography-fluorescence method against the values found in five national food-composition databases. We used established extraction methods that are assumed to liberate niacin available for absorption (acid hydrolysis mimicking human digestion) or total niacin (strong acid-alkaline hydrolysis). The niacin content (mg/100 g dry weight) obtained with acid hydrolysis ranged from a low level in corn flour (0.26), white wheat flour (0.45) and oat flakes (0.48), to a higher level in wholegrain flours (rye: 0.79, barley: 0.99, wheat: 0.88), wheat bran (2.7) and wheat germ (2.7). The niacin content with the acid-alkaline hydrolysis, however, was 1.9-11-fold the value measured after extraction with acid hydrolysis. In general, the niacin content found in the databases is closer to the results obtained after the acid-alkaline extraction, suggesting that the niacin values reported in the databases may not reflect actual bioaccessible niacin but total niacin.
  • Kirwan, Jennifer A.; Brennan, Lorraine; Broadhurst, David; Fiehn, Oliver; Cascante, Marta; Dunn, Warwick B.; Schmidt, Michael A.; Velagapudi, Vidya (2018)
    BACKGROUND: The metabolome of any given biological system contains a diverse range of low molecular weight molecules (metabolites), whose abundances can be affected by the timing and method of sample collection, storage, and handling. Thus, it is necessary to consider the requirements for preanalytical processes and biobanking in metabolomics research. Poor practice can create bias and have deleterious effects on the robustness and reproducibility of acquired data. CONTENT: This review presents both current practice and latest evidence on preanalytical processes and biobanking of samples intended for metabolomics measurement of common biofluids and tissues. It highlights areas requiring more validation and research and provides some evidence-based guidelines on best practices. SUMMARY: Although many researchers and biobanking personnel are familiar with the necessity of standardizing sample collection procedures at the axiomatic level (e.g., fasting status, time of day, "time to freezer," sample volume), other less obvious factors can also negatively affect the validity of a study, such as vial size, material and batch, centrifuge speeds, storage temperature, time and conditions, and even environmental changes in the collection room. Any biobank or research study should establish and follow a well-defined and validated protocol for the collection of samples for metabolomics research. This protocol should be fully documented in any resulting study and should involve all stakeholders in its design. The use of samples that have been collected using standardized and validated protocols is a prerequisite to enable robust biological interpretation unhindered by unnecessary preanalytical factors that may complicate data analysis and interpretation. (C) 2018 American Association for Clinical Chemistry
  • Mesihää, Samuel; Rasanen, Ilpo; Ojanperä, Ilkka (2020)
    Purity assessment of seized material containing new psychoactive substances (NPS) is complicated without appropriate primary reference standards. Here we present a method for fast quantitative estimation of stimulant-type NPS with use of secondary reference standards, based on gas chromatography nitrogen chemiluminescence detection coupled with atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (GC-NCD-APCI-QTOFMS). Quantification was based on the detector’s N-equimolar response to nitrogen and using two external nitrogen-containing calibrators, MDMA for prim- and sec- amines and α-PVP for tert- amines. Sample preparation involved dissolving the seized powdery material in an organic solvent mixture followed by acylation with N-methyl-bis-trifluoroacetamide (MBTFA). The method’s between-day accuracy and precision over a five-day period was measured for twenty-eight stimulants: the grand mean equimolarity was 91.9% (CV 5.5%), as compared with primary reference standards. The GC-NCD-APCI-QTOFMS method was applied to the purity estimation of forty-two seized powder samples previously found to contain stimulant-type NPS by appropriate methods. The quantitative results were compared to those obtained by an established method relying on liquid chromatography chemiluminescence detection (LC-CLND), the latter using caffeine as an external calibrator. The mean difference of purity values between the methods was 8.1% (range 0.4 - 26.7%). The presented method might find use as a tool for instant purity assessment in forensic laboratories.
  • Kriikku, Pirkko; Pelander, Anna; Rasanen, Ilpo; Ojanperä, Ilkka (2019)
    U-47,700 is a synthetic opioid that emerged on the novel psychoactive substance market a few years ago. After incorporating the substance into the urine UPLC-TOF-MS screening used in post-mortem toxicology, the drug was detected in 10 autopsy cases within routine case work. In all cases, the cause of death was accidental poisoning by U-47,700 alone or in combination with other psychoactive substances. The concentration of U-47,700 in the blood samples ranged between 0.15-2.0 mg/L with a median of 0.30 mg/L. In one of the cases with a U-47,700 concentration of 0.27 mg/L, no other psychoactive substances were detected. The stored TOF-MS analytical data from the year preceding the incorporation of U-47,700 into the screening was reprocessed in order to search for more positive cases. The data-independent acquisition of the original screening allowed for retrospective re-analysis of the full-scan data without additional experiments on the actual sample. The retrospective data-analysis revealed two additional cases positive for U-47,700. The first mention of U-47,700 on a Finnish internet discussion forum was in March 2015. After having been detected in several death cases, the drug was put under national control in November 2016 and the last fatality occurred in 2017. The toxic lifespan of U-47,700 thus lasted for approximately 2 years in Finland. Forensic and clinical laboratories need to rapidly adjust their screening procedures in order to adapt to the continuously expanding field of novel psychoactive substances. Retrospective data-analysis is a practical tool for monitoring the emergence of new substances onto the market. (C) 2019 Elsevier B.V. All rights reserved.
  • Esterhuizen-Londt, Maranda; Pflugmacher, Stephan (2019)
    Bioaccumulation and biomagnification of β-N-methylamino-L-alanine (BMAA), a potent neurotoxin, has been demonstrated in various food webs. It is alarming as this intensification of BMAA will result in exposure to higher concentrations from a direct cyanobacterial source. As more food items are being identified as a source of BMAA and with the large variations in BMAA content, the aim of the present study was to evaluate BMAA uptake by, and accumulation in, two commonly consumed vegetables, Lactuca sativa and Allium fistulosum. Plants exposed to pure BMAA in controlled laboratory experiments, as well as vegetables naturally irrigated with water containing a BMAA producing cyanobacterial bloom were evaluated during growth and ripening. In the laboratory exposures, free BMAA was detected in both the edible ripe parts of L. sativa and A. fistulosum after 60 days of exposure to a total of 4.5 µg BMAA. However, in the bloom exposure samples no BMAA could be detected in the ripe vegetables of A. fistulosum, Cucurbita pepo, or Brassica rapa chinensis. The study emphasises the need to further screen items for BMAA to understand the human exposure risk as well as the difference between BMAA uptake patterns with free BMAA and that contained in cyanobacterial cells.