Analytical and Bioanalytical Chemistry (v.405, #6)
Metallomics: an emerging interdisciplinary science by Michael Sperling; Uwe Karst (1789-1790).
is Chief Executive Officer of the European Virtual Institute of Speciation Analysis (EVISA), an association formed to promote speciation analysis, especially by fostering interdisciplinary cooperation, transfer of know-how, education, and exchange of information. He gained his broad experience in atomic spectrometry and trace element and speciation analysis as the head of the laboratory for applied research of a renowned instrument manufacturer. More recently, Michael Sperling joined the Institute of Inorganic and Analytical Chemistry of the University of Münster, where he is investigating techniques and methods for speciation analysis. His main research interest is the development of hyphenated analytical techniques for study of metal species in biological systems. received his PhD at the University of Münster, Germany, in Karl Cammann’s group and moved to the University of Colorado in Boulder for a postdoctoral fellowship with Robert E. Sievers. After finishing his Habilitation in Münster, he was appointed Full Professor of Chemical Analysis at the University of Twente, The Netherlands. In 2005, he took his current position as Chair of Analytical Chemistry in Münster. His main research interests include hyphenated techniques, with particular focus on pharmaceutical analysis, elemental speciation, and metallomics.
Anticancer metallodrug research analytically painting the “omics” picture—current developments and future trends by Michael Groessl; Christian G. Hartinger (1791-1808).
Anticancer metallodrug development has for a long time been characterised by the similarity of new drug candidates to cisplatin and DNA as the primary target. Recent advances in bioanalytical techniques with high sensitivity and selectivity have revealed that metal-based drugs can undergo a wide range of biomolecular interactions beyond DNA and have generated interest in proteins as possible targets for metallodrugs. In fact, implementation of metallomics approaches that are able to reveal the fate of the compounds in biological systems can help to move drug development towards more targeted and rational design of novel metallodrugs. Additionally, proteomic screening and gene expression analysis can provide insight into physiological response to drug treatment and identify the reasons for drug resistance. Herein, we review selected applications which led to a better understanding of the mode of action of clinically established metal-based anticancer agents and novel metallodrug candidates.
Keywords: Anticancer chemotherapeutics; Bioanalytical chemistry; Medicinal chemistry; Metal-based drugs; Metallomics; Proteomics
Opportunities in multidimensional trace metal imaging: taking copper-associated disease research to the next level by Stefan Vogt; Martina Ralle (1809-1820).
Copper plays an important role in numerous biological processes across all living systems predominantly because of its versatile redox behavior. Cellular copper homeostasis is tightly regulated and disturbances lead to severe disorders such as Wilson disease and Menkes disease. Age-related changes of copper metabolism have been implicated in other neurodegenerative disorders such as Alzheimer disease. The role of copper in these diseases has been a topic of mostly bioinorganic research efforts for more than a decade, metal–protein interactions have been characterized, and cellular copper pathways have been described. Despite these efforts, crucial aspects of how copper is associated with Alzheimer disease, for example, are still only poorly understood. To take metal-related disease research to the next level, emerging multidimensional imaging techniques are now revealing the copper metallome as the basis to better understand disease mechanisms. This review describes how recent advances in X-ray fluorescence microscopy and fluorescent copper probes have started to contribute to this field, specifically in Wilson disease and Alzheimer disease. It furthermore provides an overview of current developments and future applications in X-ray microscopic methods. Figure 3 mm × 3 mm P, Fe, and Cu elemental maps of a lateral ventricle from a mouse brain. An H & E image is shown for comparison. The images are displayed as red temperature maps where lighter color indicates higher elemental concentration. The image emphasizes the power of XFM: the copper distribution around the lateral ventricle is extremely heterogenous with local copper concentrations exceeding 25 mM while the average is approximately 100 μM.
Keywords: Imaging; X-ray; Fluorescence; Copper; Neurological disease
The unravelling of metabolic dysfunctions linked to metal-associated diseases by blue native polyacrylamide gel electrophoresis by Sungwon Han; Christopher Auger; Zachary Castonguay; Varun P. Appanna; Sean C. Thomas; Vasu D. Appanna (1821-1831).
Gel electrophoresis is routinely used to separate and analyse macromolecules in biological systems. Although many of these electrophoretic techniques necessitate the denaturing of the analytes prior to their analysis, blue native polyacrylamide gel electrophoresis (BN-PAGE) permits the investigation of proteins/enzymes and their supramolecular structures such as the metabolon in native form. This attribute renders this analytical tool conducive to deciphering the metabolic perturbations invoked by metal toxicity. In this review, we elaborate on how BN-PAGE has led to the discovery of the dysfunctional metabolic pathways associated with disorders such as Alzheimer’s disease, Parkinson’s disease, and obesity that have been observed as a consequence of exposure to various metal toxicants.
Keywords: Blue native polyacrylamide gel electrophoresis; Metabolic dysfunction; Metal toxicity; Enzymes; Functional proteomics; Diseases
Plasmonics for the study of metal ion–protein interactions by Giuseppe Grasso; Giuseppe Spoto (1833-1843).
The study of metal–protein interactions is an expanding field of research investigated by bioinorganic chemists as it has wide applications in biological systems. Very recently, it has been reported that it is possible to study metal–protein interactions by immobilizing biomolecules on metal surfaces and applying experimental approaches based on plasmonics which have usually been used to investigate protein–protein interactions. This is possible because the electronic structure of metals generates plasmons whose properties can be exploited to obtain information from biomolecules that interact not only with other molecules but also with ions in solution. One major challenge of such approaches is to immobilize the protein to be studied on a metal surface with preserved native structure. This review reports and discusses all the works that deal with such an expanding new field of application of plasmonics with specific attention to surface plasmon resonance, highlighting the advantages and drawbacks of such approaches in comparison with other experimental techniques traditionally used to study metal–protein interactions. Figure Plasmonics is a powerful tool for the study of metal ion-protein interactions
Keywords: Bioanalytical methods; Biosensors; Metals/heavy metals; Enzymes; Clinical/biomedical analysis; Surface plasmon resonance
Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE–ICP–MS by Tam T. T. N. Nguyen; Jesper Østergaard; Stefan Stürup; Bente Gammelgaard (1845-1854).
A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 μg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics. Figure A method for distinguishing free cisplatin from liposome-encapsulated and protein-bound platinum in human plasma allows for studies of stability and kinetics of new drug formulations during drug development
Keywords: CE–ICP–MS; Cisplatin; PEGylated liposomes; Human plasma; Drug development
The interaction of platinum-based drugs with native biologically relevant proteins by Christine Brauckmann; Christoph A. Wehe; Michael Kieshauer; Claudia Lanvers-Kaminsky; Michael Sperling; Uwe Karst (1855-1864).
This study focuses on the identification of the products that are formed upon binding of therapeutically relevant platinum complexes to proteins like β-lactoglobulin A (LGA), human serum albumin (HSA), or human hemoglobin (HB). The respective proteins were incubated with the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin. LGA was selected as the model protein in addition to the two most abundant blood proteins HSA and HB. In case of the model protein, the effect of free thiol groups on the affinity of cisplatin, carboplatin, and oxaliplatin was investigated by means of liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS). The reduced form of LGA, which contains four free thiol groups more than the native LGA, shows a much higher affinity to the platinum-based drugs. By means of liquid chromatography coupled to inductively coupled plasma mass spectrometry, the reaction behavior of the platinum-based drugs towards HSA and HB was investigated under different conditions considering the chloride concentration (4 or 100 mM) and the incubation time (24 and 48 h). In case of carboplatin, less than 6 % protein-bound platinum was detected. However, both cisplatin and oxaliplatin display a high affinity to the proteins investigated. Further information was obtained by means of LC/ESI-ToF-MS. In case of oxaliplatin, the complex [Pt(DACH)]2+ (DACH = C6N2H14) was identified interacting with HSA and HB. For cisplatin, different results were observed for the two proteins. The complex [Pt(NH3)2Cl]+ interacted predominantly with HSA and [Pt(NH3)2]2+ with HB. Figure
Keywords: Cisplatin; Carboplatin; Oxaliplatin; Human albumin; Human hemoglobin; ESI-MS
Speciation of gadolinium in surface water samples and plants by hydrophilic interaction chromatography hyphenated with inductively coupled plasma mass spectrometry by Uwe Lindner; Jana Lingott; Silke Richter; Norbert Jakubowski; Ulrich Panne (1865-1873).
Hydrophilic interaction chromatography (HILIC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was optimized for speciation analysis of gadolinium-based contrast agents in environmental samples, in particular surface river waters and plants. Surface water samples from the Teltow channel, near Berlin, were investigated over a distance of 5 km downstream from the influx of a wastewater treatment plant. The total concentration of gadolinium increased significantly from 50 to 990 ng L−1 due to the influx of the contrast agents. After complete mixing with the river water, the concentration remained constant over a distance of at least 4 km. Two main substances [Dotarem® (Gd-DOTA) and Gadovist® (Gd-BT-DO3A)] have been identified in the river water using standards. A gadolinium-based contrast agent, possibly Gd-DOTA (Dotarem®), was also detected in water plant samples taken from the Teltow channel. Therefore, uptake of contrast agents [Gadovist® (Gd-BTDO3A), Magnevist® (Gd-DTPA), Omniscan® (Gd-DTPA-BMA), Dotarem® (Gd-DOTA), and Multihance® (Gd-BOPTA)] by plants was investigated in a model experiment using Lepidium sativum (cress plants). HILIC–ICP-MS was used for identification of different contrast agents, and a first approach for quantification using aqueous standard solutions was tested. For speciation analysis, all investigated contrast agents could be extracted from the plant tissues with a recovery of about 54 % for Multihance® (Gd-BOPTA) up to 106 % for Gadovist® (Gd-BT-DO3A). These experiments demonstrate that all contrast agents investigated are transported from the roots to the leaves where the highest content was measured.
Keywords: Gadolinium-based contrast agents; Hydrophilic interaction chromatography (HILIC); Speciation; Inductively coupled plasma mass spectrometry (ICP-MS); Plants; Surface water
Selenium speciation in paired serum and cerebrospinal fluid samples by Nikolay Solovyev; Achim Berthele; Bernhard Michalke (1875-1884).
Se speciation was performed in 24 individual paired serum and cerebrospinal fluid (CSF) samples from neurologically healthy persons. Strong anion exchange (SAX) separation, coupled to inductively coupled plasma–dynamic reaction cell–mass spectrometry (ICP-DRC-MS), was employed. Species identification was done by standard matched retention time, standard addition and by size exclusion chromatography followed from SAX (2-D SEC-SAX-ICP-DRC-MS) and by SAX followed from CE-ICP-DRC-MS (2-D SAX-CE-ICP-DRC-MS). Limit of detection (LoD, 3 × standard deviation (SD) of noise) was in the range of 0.026–0.031 μg/L for all investigated species and thus was set uniformly to 0.032 μg/L. Quality control for total Se determination was performed by analysing control materials “human serum” and “urine”, where determined values met target values. Several Se species were found in both sample types having following median values (sequence: serum/CSF, each in μg Se/L): total Se, 58.39/0.86; selenoprotein P (SePP), 5.19/0.47; Se-methionine (SeM), 0.23/-serum is significantly correlated to total Se-serum when the latter was > 65 μg/L; however, SePP-CSF appeared independent of SePP-serum. For Se-HSA-serum versus (vs.) Se-HSA-CSF, a weak linear relationship was found (r 2 = 0.1722). On the contrary, for anti-oxidative Se-enzymes, higher r 2 values were calculated: GPx-serum vs. GPx-CSF, r 2 = 0.3837; TrxR-serum vs. TrxR-CSF, r 2 = 0.6293. Q -Se-species values (= ratios of CSF-Se-species/serum-Se-species) were compared with the Q -Alb value (HSA-CSF/HSA-serum = clinical index of NB integrity) for deeper information about NB passage of Se species. The Q -Se-HSA value (3.8 × 10−3) was in accordance to the molecular mass dependent restriction at NB (Q -Alb at 5.25 × 10−3). Increased Q values were seen for TrxR (21.3 × 10−3) and GPx (8.3 × 10−3) which are not (completely) explained by molecular size. For these two anti-oxidative Se-enzymes (GPx, TrxR), we hypothesize that there might be either a facilitated diffusion across NB or they might be additionally synthesized in the brain.
Keywords: Selenium speciation; Cerebrospinal fluid; Serum; Thioredoxin reductase; Glutathione peroxidase
Formation of methylated oxyarsenicals and thioarsenicals in wild-type and arsenic (+3 oxidation state) methyltransferase knockout mice exposed to arsenate by Hua Naranmandura; Kanwal Rehman; X. Chris Le; David J. Thomas (1885-1891).
Arsenic (+3 oxidation state) methyltransferase (As3mt) plays a central role in the enzymatically catalyzed conversion of inorganic arsenic into methylated metabolites. Most studies of the metabolism and disposition of arsenicals following exposure to inorganic arsenic focus on the formation and fate of methylated oxyarsenicals. However, recent research has shown methylated thioarsenicals to be another important class of metabolites of inorganic arsenic. Here, we report on the presence of methylated oxy- and thioarsenicals in urine and liver from wild-type mice that efficiently methylate inorganic arsenic and from As3mt knockout mice that lack arsenic methyltransferase activity. Following a single oral dose of 0.5 mg of arsenic as arsenate/kg body weight, urine from wild-type mice contained methylated oxyarsenicals and unknown arsenicals. Further analysis identified one unknown arsenical in urine of wild-type mice as dimethylmonothioarsinic acid. In addition, another unknown arsenical in urine of wild-type mice that occurred in the urine of about 20 % of arsenate-treated mice. The presence of low levels of methylated arsenicals in liver digests of As3mt knockout mice may reflect the activity of other methyltransferases or the absorption of methylated arsenicals formed by the microbiota of the gastrointestinal tract. The lack of methylated thioarsenicals in urine of As3mt knockout mice suggests a close link between the processes that form methylated oxy- and thioarsenicals.
Keywords: Arsenic; Arsenite; Cytotoxicity; Dimethylmonothioarsinic acid; Methylated oxyarsenical; Thioarsenicals
Effect of transfection with PLP2 antisense oligonucleotides on gene expression of cadmium-treated MDA-MB231 breast cancer cells by Alessandra Longo; Mariangela Librizzi; Claudio Luparello (1893-1901).
Emerging evidence indicates that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in human normal and pathological cells. We have already shown that exposure of MDA-MB231 breast cancer cells to 5 μM CdCl2 for 96 h, apart from significantly affecting mitochondrial metabolism, induces modifications of the expression level of genes coding for members of stress response-, mitochondrial respiration-, MAP kinase-, NF–κB-, and apoptosis-related pathways. In the present study, we have expanded the knowledge on the biological effects of Cd–breast cancer cell interactions, indicating PLP2 (proteolipid protein-2) as a novel member of the list of Cd-upregulated genes by MDA-MB231 cancer cells and, through the application of transfection techniques with specific antisense oligonucleotides, we have demonstrated that such over-expression may be an upstream event to some of the changes of gene expression levels already observed in Cd-treated cells, thus unveiling new possible molecular relationship between PLP2 and genes linked to the stress and apoptotic responses.
Keywords: Cadmium; PLP2; Breast cancer; Differential display-PCR; Caspase; Gene expression
Arsenic speciation in saliva of acute promyelocytic leukemia patients undergoing arsenic trioxide treatment by Baowei Chen; Fenglin Cao; Chungang Yuan; Xiufen Lu; Shengwen Shen; Jin Zhou; X. Chris Le (1903-1911).
Arsenic trioxide has been successfully used as a therapeutic in the treatment of acute promyelocytic leukemia (APL). Detailed monitoring of the therapeutic arsenic and its metabolites in various accessible specimens of APL patients can contribute to improving treatment efficacy and minimizing arsenic-induced side effects. This article focuses on the determination of arsenic species in saliva samples from APL patients undergoing arsenic treatment. Saliva samples were collected from nine APL patients over three consecutive days. The patients received 10 mg arsenic trioxide each day via intravenous infusion. The saliva samples were analyzed using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Monomethylarsonous acid and monomethylmonothioarsonic acid were identified along with arsenite, dimethylarsinic acid, monomethylarsonic acid, and arsenate. Arsenite was the predominant arsenic species, accounting for 71.8 % of total arsenic in the saliva. Following the arsenic infusion each day, the percentage of methylated arsenicals significantly decreased, possibly suggesting that the arsenic methylation process was saturated by the high doses immediately after the arsenic infusion. The temporal profiles of arsenic species in saliva following each arsenic infusion over 3 days have provided information on arsenic exposure, metabolism, and excretion. These results suggest that saliva can be used as an appropriate clinical biomarker for monitoring arsenic species in APL patients. Figure Arsenic species and temporal profiles over three days from nine patients
Keywords: Acute promyelocytic leukemia; Arsenic speciation; Saliva; Metabolism; Arsenic trioxide treatment
Improving species-specific IDMS: the advantages of triple IDMS by Claudia Frank; Olaf Rienitz; Claudia Swart; Detlef Schiel (1913-1919).
Triple isotope dilution mass spectrometry (triple IDMS) has been applied for the first time on protein quantification, especially on transferrin. Transferrin as an acute phase protein is a marker for several inflammation processes in the human body. Therefore, in Germany, the accurate and precise measurement of this important analyte is required. In this work, a new approach to triple IDMS is described and compared to double IDMS. Also, complete uncertainty budgets for both methods were set up to demonstrate the ability of this method to be used as a reference procedure. The relative expanded uncertainty (k = 2) for triple IDMS (3.6 %) is smaller than the one for double IDMS (4.0 %). The content of transferrin found in the human serum reference material ERM-DA470k/IFCC ((2.41 ± 0.08) g/kg) with both methods was in good agreement with each other and with the certificate. For triple IDMS ((2.426 ± 0.086) g/kg) and for double IDMS ((2.317 ± 0.092) g/kg), transferrin was determined. Although triple IDMS is a little more time consuming compared to double IDMS, there is the advantage that the isotopic composition of the spike material does not have to be determined. This is very useful especially in case of a marginal isotopic enrichment in the spike or problems with the accurate measurement of the spike isotope ratio. Figure Using triple instead of double species-specific IDMS helps to reduce the uncertainty and improves the reliability of the results, especially in cases where an accurate determination of the spike isotope ratio is difficult or impossible, because the spike ratio cancels from the equation
Keywords: Transferrin; Species-specific IDMS; Triple IDMS; Human serum
Emission (57Co) Mössbauer spectroscopy as a tool for probing speciation and metabolic transformations of cobalt(II) in bacterial cells by Alexander A. Kamnev; Anna V. Tugarova; Krisztina Kovács; Ernő Kuzmann; Borbála Biró; Petros A. Tarantilis; Zoltán Homonnay (1921-1927).
The emission (57Co) variant of Mössbauer spectroscopy, rarely used in biology-related studies, was applied to study binding and possible transformations of 57CoII traces in live and dead (hydrothermally treated) cells of the rhizobacterium Azospirillum brasilense (strain Sp7) at T = 80 K in frozen aqueous suspensions and as their dried residues. The Mössbauer parameters calculated from the spectra were compared with the similarly obtained data reported earlier for another A. brasilense strain, Sp245 (which differs from strain Sp7 by the ecological niche occupied in the rhizosphere and was found earlier to exhibit different metabolic responses under similar environmental conditions). Similarly to strain Sp245, live cells of strain Sp7, rapidly frozen 2 min and 1 h after their contact with 57Co2+ (measured in frozen suspensions), showed marked differences in their Mössbauer parameters, reflecting metabolic transformations of 57Co2+ occurring within an hour. However, the parameters for strains Sp7 (this work) and Sp245 (reported earlier), obtained under similar conditions, were found to significantly differ, implying dissimilarity in their metabolic response to Co2+. This is in line with their different metabolic responses to several heavy metals, including Co2+, detected earlier using Fourier transform infrared spectroscopy.
Keywords: Cobalt(II) metabolism; Azospirillum brasilense ; 57Co emission Mössbauer spectroscopy; Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy
Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides by Sarojini J. K. A. Ubhayasekera; Johan Staaf; Anders Forslund; Peter Bergsten; Jonas Bergquist (1929-1935).
Circulating free fatty acids (FFAs) play important physiological roles as contributing components in cellular structure as well as energy utilization. Elevated levels of circulating FFAs are associated with metabolic aberrations in humans. FFAs differ in chain length and saturation and may be altered in metabolically dysregulated conditions, such as type 2 diabetes mellitus. Potentially, alterations in circulating levels of specific FFAs could also be important in terms of prognostic value. Various methods have been used to analyze FFAs. In this study, a straightforward and accurate method for the determination of FFAs in plasma has been established and evaluated, through conversion of plasma FFAs into acid fluorides followed by conversion to Weinreb amides (dimethylamide). The method is mild, efficient, selective, and quantitative for FFAs, when analyzed with capillary gas chromatography tandem mass spectrometry. Standard curves were linear over the range of 1,000–20,000 ng/mL with a correlation coefficient (r 2) of 0.998, and coefficient of variation of triplicate analysis was <10 %. The gas chromatography–mass spectrometry (GC-MS) technique was reproducible and repeatable, and recoveries were above 90 %. From the generated MS spectra, five specific FFAs were identified. An explicit interest was the quantification of palmitate (C16:0) and palmitoleate (C16:1), which have been connected with detrimental and positive effects on the insulin-producing beta cells, respectively. The results demonstrate the suitability of Weinreb amides for efficient and rapid isolation of FFAs in plasma, prior to quantitative GC-MS analysis. We suggest that the method can be used as a routine standardized way of quantifying FFAs.
Keywords: Free fatty acids; Obesity; Weinreb amides
A novel UPLC–MS–MS method for simultaneous determination of seven uremic retention toxins with cardiovascular relevance in chronic kidney disease patients by Jente Boelaert; Frédéric Lynen; Griet Glorieux; Sunny Eloot; Maria Van Landschoot; Marie-Anne Waterloos; Pat Sandra; Raymond Vanholder (1937-1947).
Chronic kidney disease (CKD) is a devastating illness characterized by accumulation of uremic retention solutes in the body. The objective of this study was to develop and validate a simple, rapid, and robust UPLC–MS–MS method for simultaneous determination, in serum, of seven organic acid uremic retention toxins, namely uric acid (UA), hippuric acid (HA), indoxylsulfate (IS), p-cresylglucuronide (pCG), p-cresylsulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF). Isotopically labeled internal standards (d5-HA; 1,3-15N2-UA, and d5-IAA) were used to correct for variations in sample preparation and system performance. Separation on a C18 column was followed by negative electrospray ionization and tandem mass spectrometric detection. Accuracy was below the 15 % threshold. Within-day precision varied from 0.60 to 4.54 % and between-day precision was below 13.33 % for all compounds. The applicability of the method was evaluated by analyzing 78 serum samples originating both from healthy controls and from patients at different stages of CKD. These results were compared with those obtained by use of conventional HPLC–PDA–FLD methods. A good correlation was obtained between both methods for all compounds.
Keywords: Chronic kidney disease; Uremic retention solutes; UPLC; Tandem mass spectrometry
Quantification of insulin-like growth factor-1 in dried blood spots for detection of growth hormone abuse in sport by Holly D. Cox; Jessica Rampton; Daniel Eichner (1949-1958).
There is significant evidence that athletes are using recombinant human growth hormone (rhGH) to enhance performance, and its use is banned by the World Anti-Doping Agency and professional sports leagues. Insulin-like growth factor-1 (IGF-1) is the primary mediator of growth hormone action and is used as a biomarker for the detection of rhGH abuse. The current biomarker-based method requires collection and expedited shipment of venous blood which is costly and may decrease the number of tests performed. Measurement of GH biomarkers in dried blood spots (DBS) would considerably simplify sample collection and shipping methods to allow testing of a greater number of samples regardless of location. A method was developed to quantify intact IGF-1 protein in DBS by liquid chromatography–tandem mass spectrometry. A step-wise acid–acetonitrile extraction was optimized to achieve a sensitive assay with a lower limit of quantification of 50 ng/mL. IGF-1 remained stable at room temperature for up to 8 days, which would allow shipment of DBS cards at ambient temperature. In a comparison between plasma concentrations of IGF-1 and concentrations measured from venous and finger prick DBS, there was good correlation and agreement, r 2 of 0.8551 and accuracy of 86–113 % for venous DBS and r 2 of 0.9586 and accuracy of 89–122 % for finger prick DBS. The method is intended for use as a rapid screening method for IGF-1 to be used in the biomarker method of rhGH abuse detection.
Keywords: Amino acids; Peptides; Bioanalytical methods; Drug monitoring; Drug screening; Mass spectrometry; ICP-M
Neuronal metabolomics by ion mobility mass spectrometry: cocaine effects on glucose and selected biogenic amine metabolites in the frontal cortex, striatum, and thalamus of the rat by Kimberly A. Kaplan; Veronica M. Chiu; Peter A. Lukus; Xing Zhang; William F. Siems; James O. Schenk; Herbert H. Hill Jr (1959-1968).
We report results of studies of global and targeted neuronal metabolomes by ambient pressure ion mobility mass spectrometry. The rat frontal cortex, striatum, and thalamus were sampled from control nontreated rats and those treated with acute cocaine or pargyline. Quantitative evaluations were made by standard additions or isotopic dilution. The mass detection limit was ∼100 pmol varying with the analyte. Targeted metabolites of dopamine, serotonin, and glucose followed the rank order of distribution expected between the anatomical areas. Data was evaluated by principal component analysis on 764 common metabolites (identified by m/z and reduced mobility). Differences between anatomical areas and treatment groups were observed for 53 % of these metabolites using principal component analysis. Global and targeted metabolic differences were observed between the three anatomical areas with contralateral differences between some areas. Following drug treatments, global and targeted metabolomes were found to shift relative to controls and still maintained anatomical differences. Pargyline reduced 3,4-dihydroxyphenylacetic acid below detection limits, and 5-HIAA varied between anatomical regions. Notable findings were: (1) global metabolomes were different between anatomical areas and were altered by acute cocaine providing a broad but targeted window of discovery for metabolic changes produced by drugs of abuse; (2) quantitative analysis was demonstrated using isotope dilution and standard addition; (3) cocaine changed glucose and biogenic amine metabolism in the anatomical areas tested; and (4) the largest effect of cocaine was on the glycolysis metabolome in the thalamus confirming inferences from previous positron emission tomography studies using 2-deoxyglucose. Figure Instrumental schematic of an ion mobility mass spectrometer used for measuring changes in neuronal metabolomes of varying anatomical regions. Two-dimensional data is generated for each anatomical area of interest
Keywords: Ion mobility mass spectrometry; Cocaine; Isotope dilution; Glucose; Standard addition
Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method by Chun-Chi Wang; Shu-Fang Cheng; Hui-Ling Cheng; Yen-Ling Chen (1969-1976).
This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid–liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at −20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r ≥ 0.990) over a range of 50–1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n = 3) and inter-day (n = 5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N = 5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.
Keywords: Full-capillary sample injection combined with sweeping; Capillary electrophoresis; Anabolic androgenic steroids; Urine
Rapid extraction and preservation of genomic DNA from human samples by D. Kalyanasundaram; J.-H. Kim; W.-H. Yeo; K. Oh; K.-H. Lee; M.-H. Kim; S.-M. Ryew; S.-G. Ahn; D. Gao; G. A. Cangelosi; J.-H. Chung (1977-1983).
Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol. Figure Portable microtip device for human genomic DNA extraction
Keywords: DNA extraction; Microtip; Electric field; Human genomic DNA; Human samples
Immobilization of proteins on carboxylic acid functionalized nanopatterns by Johnpeter N. Ngunjiri; Daniel J. Stark; Tian Tian; Kimberly A. Briggman; Jayne C. Garno (1985-1993).
The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.
Keywords: Atomic force microscopy; Nanografting; Scanning probe lithography; Self-assembled monolayers; Infrared reflection–absorption spectroscopy; Protein immobilization
Oral cancer diagnostics based on infrared spectral markers and wax physisorption kinetics by Li-Fang Chiu; Pei-Yu Huang; Wei-Fan Chiang; Tung-Yiu Wong; Sheng-Hsiang Lin; Yao-Chang Lee; Dar-Bin Shieh (1995-2007).
Infrared microspectroscopy is an emerging approach for disease analysis owing to its capability for in situ chemical characterization of pathological processes. Synchrotron-based infrared microspectroscopy (SR-IMS) provides ultra-high spatial resolution for profiling biochemical events associated with disease progression. Spectral alterations were observed in cultured oral cells derived from healthy, precancerous, primary, and metastatic cancers. An innovative wax-physisorption-based kinetic FTIR imaging method for the detection of oral precancer and cancer was demonstrated successfully. The approach is based on determining the residual amount of paraffin wax (C25H52) or beeswax (C46H92O2) on a sample surface after xylene washing. This amount is used as a signpost of the degree of physisorption that altered during malignant transformation. The results of linear discriminant analysis (LDA) of oral cell lines indicated that the methylene (CH2) and methyl group (CH3) stretching vibrations in the range of 3,000–2,800 cm−1 have the highest accuracy rate (89.6 %) to discriminate the healthy keratinocytes (NHOK) from cancer cells. The results of wax-physisorption-based FTIR imaging showed a stronger physisorption with beeswax in oral precancerous and cancer cells as compared with that of NHOK, which showed a strong capability with paraffin wax. The infrared kinetic study of oral cavity tissue showed a consistency in the wax physisorption of the cell lines. On the basis of our findings, these results show the potential use of wax-physisorption-based kinetic FTIR imaging for the early screening of oral cancer lesions and the chemical changes during oral carcinogenesis. Figure Synchrotron-based infrared microspectroscopy (SR-IMS) provides ultra-high spatial resolution for profiling biochemical events associated with disease progression. FTIR spectra collected by SR-IMS were classified by linear discriminant analysis (LDA). The results of LDA of oral cell lines indicate the optical absorption in the range of 3,000–2,800 cm−1 have the highest accuracy to discriminate normal healthy oral keratinocytes (NHOK) from cancer cells. Two types of organic waxes with different polarity were used as adsorbents for cancer screening. The results of wax-physisorption-based FTIR imaging showed a stronger physisorption of beeswax in tumor tissues as compared with that of normal oral mucosa, which showed a stronger capability of physisorption to paraffin wax.
Keywords: Infrared spectrophotometry; FTIR; Carcinogenesis test; Oral cancer
High-throughput, low-volume, multianalyte quantification of plasma metabolites related to one-carbon metabolism using HPLC-MS/MS by Øivind Midttun; Gry Kvalheim; Per Magne Ueland (2009-2017).
Risk of chronic diseases, like cardiovascular disease and cancer, has been associated with biomarkers related to one-carbon metabolism, which comprises a metabolic network of cross-talking pathways. To address this complexity in epidemiological studies, we have established an isotope dilution HPLC-MS/MS method for quantification of 12 biomarkers and metabolites. All sample handling is performed by a robotic workstation. The assay uses 45 μL of plasma, and sample treatment consists of protein precipitation by trichloroacetic acid. The analytes were separated on a Fortis Phenyl column using an isocratic mobile phase that contained water, methanol and acetic acid. Methionine, methionine sulfoxide, choline, betaine, dimethylglycine, arginine, asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine, creatinine, cystathionine and trimethyllysine all showed limits of detection well below the 5th percentile of plasma distributions in healthy humans, coefficients of variation were in the range 2.2–12.3 %, and recoveries were 80–131 %. Simple sample processing, low-volume consumption, multiplexing and high capacity/short run time of this method make it suitable for large-scale metabolic profiling of precious biobank samples.
Keywords: One-carbon metabolism; HPLC-MS/MS; High throughput; Epidemiology
Simultaneous monitoring of seven phenolic metabolites of endocrine disrupting compounds (EDC) in human urine using gas chromatography with tandem mass spectrometry by Lukas Schmidt; Johannes Müller; Thomas Göen (2019-2029).
A gas chromatographic-tandem mass spectrometric (GC-MS/MS) method for the simultaneous determination of the three well-known endocrine disruptors, bisphenol A, daidzein and genistein, as well as of four human pesticide metabolites which are supposed to have proper endocrine activity or which are metabolites of endocrine-disrupting compounds, viz., 1- and 2-naphthol, 2-isopropoxyphenol and 3,5,6-trichloropyridinol, has been developed and validated. The method involves enzymatic cleavage of the conjugates using β-glucuronidase/arylsulfatase followed by solid-phase extraction and derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Isotopically labelled internal standards were used for all analytes, to achieve best analytical error correction. The method proved to be both sensitive and reliable in human urine with detection limits ranging from 0.1 to 0.6 μg/L for all analytes. Precision and repeatability was determined to range from 1 to 15 %. Compared with other published analytical procedures, the present method enables the simultaneous determination of a couple of phenolic agents with competitive or improved analytical reliability. Thus, the present method is suitable for a combined monitoring of the exposure to prominent xenobiotics with effects on the human endocrine system (bisphenol A, carbaryl, chlorpyrifos, chlorpyrifos-methyl, naphthalene, propoxur, triclopyr) and phytoestrogens (daidzein, genistein) in population studies.
Keywords: Biomonitoring; Endocrine disruptors; Bisphenol A; Naphthol; Isoflavones; Pesticide metabolites
Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC–MS/MS by Christina Haberhauer-Troyer; Marizela Delic; Brigitte Gasser; Diethard Mattanovich; Stephan Hann; Gunda Koellensperger (2031-2039).
A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography–MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-13C2,15N) was established using H2O2 as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25–30 % (coverage factor = 2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.
Keywords: Glutathione; Glutathione disulfide; LC-MS; Extraction; Sample preparation
Ni speciation in tea infusions by monolithic chromatography—ICP-MS and Q-TOF-MS by Janez Ščančar; Tea Zuliani; Dušan Žigon; Radmila Milačič (2041-2051).
For humans, Ni is not considered to be an essential trace element. Its compounds, at levels present in foodstuffs and drinks, are generally considered to be safe for consumption, but for individuals who already suffer from contact allergy to Ni and may be subject to develop systemic reactions from its dietary ingestion, dietary exposure to Ni must be kept under control. Being the second most popular beverage, tea is a potential source of dietary Ni. Present knowledge on its speciation in tea infusions is poor. Therefore, complete speciation analysis, consisting of separation by liquid chromatography using a weak CIM DEAE-1 monolithic column, “on-line” detection by inductively coupled plasma mass spectrometry (ICP-MS) and “off-line” identification of ligands by hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MS), was implemented for the first time to study Ni speciation in tea infusions. Total concentrations of Ni in dry leaves of white, green, oolong and black tea (Camellia sinensis) and flowers of herbal chamomile (Matricaria chamomilla) and hibiscus (Hibiscus sabdariffa) tea were determined after microwave digestion by ICP-MS. They lay between 1.21 and 14.4 mg kg−1. Good agreement between the determined and the certified values of the Ni content in the standard reference material SRM 1573a tomato leaves confirmed the accuracy of the total Ni determination. During the infusion process, up to 85 % of Ni was extracted from tea leaves or flowers. Separation of Ni species was completed in 10 min by applying aqueous linear gradient elution with 0.6 mol L−1 NH4NO3. Ni was found to be present in the chromatographic fraction in which quinic acid was identified by Q-TOF in all the tea infusions analysed, which had pH values between 5.6 and 6.0. The only exception was the infusion of hibiscus tea with a pH of 2.7, where results of speciation analysis showed that Ni is present in its divalent ionic form.
Keywords: Nickel speciation; Monolithic chromatography; Inductively coupled plasma mass spectrometry; Time-of-flight mass spectrometry; Tea infusions; Quinic acid
ToF-S-SIMS molecular 3D analysis of micro-objects as an alternative to ion beam erosion at large depth: application to single inkjet dots by Yannick Vercammen; Jaymes Van Luppen; Christiaan Van Roost; Roel De Mondt; Frank Vangaever; Luc Van Vaeck (2053-2064).
Molecular depth profiling is needed to develop high-tech materials optimised to the μm or even up to the nm scale. Recent progress in time-of-flight static secondary ion mass spectrometry (ToF-S-SIMS) offers perspectives to molecular depth profiling. However, at this moment, the methodology is not yet capable to deal with a range of materials science applications because of the limited depth range, the loss of intensity in the subsurface and the loss of depth resolution at large distances from the original surface. Therefore, the purpose of this paper is to develop a complementary approach for the molecular 3D analysis at large depth, using a combination of ultra-low angle microtomy (ULAM) and surface analysis of the sectioned material with ToF-S-SIMS. Single inkjet dots with a diameter of 100 μm and height of 22 μm on a PET substrate have been used as a test system for the methodology. It is demonstrated that the use of a diamond knife allows the molecular composition and distribution of components within the microscopic feature to be probed with a lateral resolution of 300 nm. Hence the methodology approaches the physical limit for ion imaging of organic components with local concentrations in the % range. In practice, the achievable depth resolution with ULAM-S-SIMS is ultimately limited by the surface roughness of the section. Careful optimisation of the ULAM step has resulted in a surface roughness within 6 nm (R a value) at a depth of 21 μm. This offers perspective to achieve 3D analysis with a depth resolution as good as 18 nm at such a large distance from the surface. Furthermore, the ULAM-S-SIMS approach is applicable to materials unamenable to ion beam erosion. However, the method is limited to dealing with, for instance, Si or glass substrates that cannot be sectioned with a microtomy knife. Furthermore, sufficient adhesion between stacked layers or between the coating and substrate is required. However, it is found that the approach is applicable to a wide variety of industrially important (multi)layers of polymers on a polymer substrate.
Keywords: Polymers; Interface; Surface analysis; Mass spectrometry; ICP-M
Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore by Ryan M. Rich; Dorota L. Stankowska; Badri P. Maliwal; Thomas Just Sørensen; Bo W. Laursen; Raghu R. Krishnamoorthy; Zygmunt Gryczynski; Julian Borejdo; Ignacy Gryczynski; Rafal Fudala (2065-2075).
Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.
Keywords: Fluorescence; Luminescence; Spectroscopy theory; Spectroscopy instrumentation; Bioanalytical methods
Sensitive determination of organic acid preservatives in juices and soft drinks treated by monolith-based stir cake sorptive extraction and liquid chromatography analysis by Fuhua Lin; Shuyu Nong; Xiaojia Huang; Dongxing Yuan (2077-2081).
A simple, efficient, and sensitive method for simultaneous determination of sorbic acid (SA), benzoic acid (BA), and cinnamic acid (CA) in juices and soft drinks was developed by stir cake sorptive extraction (SCSE) coupling to high-performance liquid chromatography with diode array detection. The SCSE based on polymeric ionic liquid-based monolith (PILM) as extractive medium was used to concentrate these three organic acid preservatives. Because hydrophobic and ion-exchange interactions co-contributed to the extraction, the PILM-SCSE exhibited a high extractive capability towards analytes. To obtain optimum extraction performance, several SCSE parameters were investigated and discussed, including desorption solvent, pH value, ionic strength in the sample matrix, and the extraction and desorption time. Under the optimized extraction conditions, limits of detection of 0.16, 1.08, and 0.18 μg/L (S/N = 3) and quantification limits of 0.52, 3.42, and 0.61 (S/N = 10) were obtained for SA, BA, and CA, respectively. The method also showed good linearity and reproducibility, as well as advantages such as simplicity, low cost, and high feasibility. Finally, the proposed method was successfully applied to the determination of SA, BA, and CA in real juices and soft drinks, and the recoveries ranged from 63.0 to 107 %.
Keywords: Stir cake sorptive extraction; Polymeric ionic liquid-based monolith; Preservative; Juices; Soft drinks; HPLC
Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry by Hailei Lang; Sheng Wang; Qidong Zhang; Beibei Zhao; Lei Wang; Baojun Cao; Juan Wang; Jian Mao; Jianxun Zhang (2083-2089).
A hydrophilic interaction liquid chromatographic–tandem mass spectrometric (HILIC–MS–MS) method for investigation of the in vivo metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen, in rabbit blood has been developed and validated. This method achieved excellent repeatability and accuracy. Recovery ranged from 76.9 to 116.3 % and precision (as RSD) between 0.53 and 6.52 %. Linearity was good for all compounds (R 2 > 0.9990) and the limit of detection (LOD) ranged from 0.016 to 0.082 ng mL−1. Pharmacokinetic analysis indicated that NNK was rapidly eliminated in vivo in rabbit blood and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite. The hydroxy acid, keto acid, and NNAL-N-oxide were also important metabolites in rabbit blood. It is probable that α-methylene hydroxylation was the major pathway of α-hydroxylation of NNK and NNAL in the rabbit. Figure The process of the experiment in this study. NNK solution was injected into rabbit body. Blood samples were obtained and processed, and then transferred into vials. NNK and its metabolites were separated by HILIC column. The ion source of MS is ESI and MRM mode was employed for monitoring ion pairs. The chromatogram of NNK and its metabolites was obtained.
Keywords: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); HILIC–MS–MS; Metabolites; Pharmacokinetics
Label-Free Technologies 2012: Advances and Applications by Melanie Ewald; Oliver Bleher (2091-2092).