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Targeted and Unknown Screening Approaches in Clinical Research and Forensic Toxicology Using LC/MSMS. New Acquisition and Processing Tools
Orbitrap instruments have gained in popularity, offering the possibility to identify new substances in complex matrixes. A new scan mode termed vDIA (variable data-independent acquisition) that provides higher selectivity and sensitivity will be described. Processing tools will also be discussed. TraceFinder 3.3 is used for targeted analysis using databases and libraries as well as the identification of unknown compounds. Library searching can also be performed using mzCloud a unique HRAM MSn spectral database. Structures can be confirmed using Mass Frontier that is capabable to automatically generate possible fragments at an expert level.
Combining Database Mining and Fragmentation Simulation for Facilitating Identification of Unknown Metabolites by UPLC-MS
Non-targeted metabolic profiling based on UPLC-MS opens new avenues to biomarker discovery. Although standardized UPLC-MS assays for measuring clinical samples are established, extracting valuable information of these large datasets is still challenging. To facilitate structural identification of metabolites in complex datasets, we developed a bioinformatic pipeline mining databases and performing in silico fragmentation using MetFrag. Discriminative metabolites between disease and control samples were first searched against HMDB, KEGG, Reactome and WikiPathways to identify specific pathways differentially regulated among sample groups. To further increase chances of identification, compounds in PubChem were fragmented in silico using MetFrag. By combining our bioinformatics tools with Progenesis QI, we demonstrate that our pipeline reinforces the identification of features of interest.
Probing Adrenal Insufficiency in Experimental Septic Shock Using Imaging Mass Spectrometry
Approximately half of the patients in septic shock have an inadequate cortisol response, described as critical illness related corticosteroid insufficiency (CIRCI). The underlying molecular mechanisms of CIRCI remain unclear. In this study, we probed the native distribution of lipids and metabolites in mouse models of sepsis and acute endotoxic shock using TOF-SIMS for high spatial resolution imaging.
We found elevated levels of cholesterol in the adrenal cortex in endotoxic shock indicating a progressing corticosterone synthesis. In late phase S. aureus sepsis, high vitamin E and low cholesterol in the adrenal cortex indicates oxidative stress. These findings may be of importance to resolve mechanisms underlying CIRCI.
An Investigation into Multi-Model Tissue Imaging on a Single Section by DESI and MALDI TOF Mass Spectrometry
DESI is an established technique in the field of mass spectrometry, and has been used for tissue imaging. DESI results are of very high-quality, for lipids and small molecules compared to the more established MALDI imaging technique. An additional advantage of DESI is that it can be performed without sample preparation, such as matrix deposition. Using optimized gas and solvent flow rates, DESI does not damage the tissue surface. Here we present work showing the potential to analyse a single tissue section by DESI imaging with subsequent analysis by MALDI imaging of the same tissue section.
Rapid Forensic Screening of 161 Drugs by Ultra-high Speed LC/MS/MS with Synchronized Survey Scanning
We developed rapid screening method for 161 drugs for forensic purposes using ultra-high speed LC/MS/MS with synchronized survey scanning that automatically performs product ion scan (PIS) when a precursor threshold is exceeded in multiple reaction monitoring (MRM). This approach enables the results both qualitative and quantitative in a single run. The combination of the developed method with ultra-high speed LC/MS/MS and modified QuEChERS enable to strengthen the efficiency of drug screening.
First Two Cases with Medium-chain Acyl-CoA Dehydrogenase Deficiency in Slovenia
Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inborn error of fatty acid metabolism. First two patients with MCAD deficiency in Slovenia were recently diagnosed. They had clinical features that were not characteristic for MCAD deficiency and were diagnosed only after the biochemical findings. In urine we measured elevated organic acids typical for fatty acid oxidation disorders. Acylcarnitines in dried blood spots, specific for MCAD deficiency, were also elevated. Confirmation with Sanger sequencing showed that both patients are compound heterozygotes, each harbouring one novel variant in ACADM.
Quantitative Analysis of Plasma Amino Acids on Tandem Mass Spectrometer
in Newly Diagnosed Pregnant Woman with Citrullinemia Type 1
A 20-year-old pregnant woman presented at 27th week of gestation with acute liver failure and hyperammonemia. Plasma amino acids were analyzed using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma citrulline (921 µmol/L; N 10-55 µmol/L) and glutamine (1141 µmol/L; N 340-740 µmol/L) were extremely high and arginine was unmeasurably low. There was no argininosuccinate in plasma. Based on these findings, citrullinemia type 1 was diagnosed. Routine weekly monitoring for ammonia and amino acid concentration continued until post partum period. The patient delivered a healthy baby. Women with newly diagnosed citrullinemia type 1 can have normal pregnancy outcomes if metabolic crisis is treated promptly and ammonia and amino acid levels are monitored regularly during pregnancy.
Influence of the Sample Collection Techniques on Human Blood Metabolome
In this study the impact of well-established techniques such as plasma (EDTA, heparin, citrate), serum and dried plasma spots (DBS) as well as novel techniques including DPS and Mitra™ on metabolite profile as well as short term stability were investigated.
Quantification of more than 180 endogenous metabolites including amino acids, biogenic amines, acylcarnitines, glycerophospholipids, sphingomyelins and hexose for all samples were performed employing the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences).
The quantitative metabolite profile of human blood obtained using different sampling techniques were comprehensively compared: The employed sampling methodologies have significant influence on quantitative metabolite profile as well as metabolite stability.
Effects of Carbohydrate Nutrients on Cytosolic Reducing Power and Argininosuccinate Synthetase Activities
This study aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of cytosolic NAD+ and NADH, and to investigate the effects of different types and amounts of carbohydrates in cell culture medium on cytosolic reducing power and the activities of argininosuccinate synthetase (ASS) in primary hepatocytes AML12. When replacing 18 mM glucose with 9 mM glucose or 9 mM glucose and galactose, increased lactate/pyruvate ratio and cytosolic NADH concentrations were found with the decrease of ASS activities. The ASS activities were related to cytosolic reducing power with a r value of 0.672 (p<0.05).
Quantitation of Serum Bile Acids by Liquid Chromatography-tandem Mass Spectrometry
Bile acids are recently recognized as hormones regulating various metabolic pathways and associated with many diseases. To study the changes of serum bile acid compositions in diseases, this study aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantitate serum bile acids. Using the established LC-MS/MS method, we measured 15 bile acids in serum samples with good precision and accuracy. The accuracy of the established method was further assessed by method comparison with an enzymatic method for total bile acids measurement. The correlation between the sums of 15 bile acids quantitates by LC-MS/MS and the total bile acids concentrations by an enzymatic assay showed great agreement with regression line y=0.95x+2.04 (r2=0.97).
Improved Performance of Targeted Metabolome Analysis with Waters Xevo® TQ-S and Xevo® TQ-S Micro Instruments
We present a standardized, quality controlled assay for the targeted quantitative analysis of >180 compounds of the blood metabolome based on combined UPLC-MS/MS and FIA-MS/MS. The developed and validated assay was successfully established on two high-performance Waters systems (two Acquity UPLC coupled to Xevo® TQ-S and Xevo® TQ-S micro MS) and the range of analytes and internal standards was extended by 20 compounds. Excellent intra-batch and inter-batch accuracy and precision were obtained in biological samples and three levels of quality control samples. Both platforms showed excellent analytical performance, whereas the Xevo® TQ-S instrument performed extremely well for low abundant metabolites.
Metabolomics in Paediatrics: Study of Differences in Maturation Grade of Piglets
The differences in the plasma metabolome between newborn and mature pigs are investigated in this pilot study using ultra high pressure liquid chromatography coupled to high resolution mass spectrometry. The endogenous metabolites were profiled in plasma samples collected from pigs of two days and eight weeks of age. Using multivariate data analysis both groups were differentiated according to age and putative relevant metabolites where selected and identified by comparison with online metabolite databases. Other aspects such as gender, anaesthesia or time of sample collection were also studied.
High-resolution Mass Spectrometry for Parasites in Stool Samples: A Metabolomics Approach
Intestinal parasites are a widely distributed class of pathogens worldwide. Current diagnostic methods rely on very limited and unspecific approaches that may delay the confirmation of the disease, causing increased severity and debilitating the patient. Our present contribution explores a metabolomics-based study of three major parasites: A. lumbricoides, G. lamblia and S. stercoralis, which are obtained directly from fresh stool samples. Preliminary findings demonstrate that principal component analysis (PCA) is a suitable statistical approach for differentiating the three parasites, helping elect chemical markers that can act as fingerprints of each species, thus leading towards a potential multi-parasitic detection method.
Exploratory Analysis of Bile Juice of Patients with Opisthorchiasis Felineus Infection
Opisthorchiasis is a form of foodborne trematodiasis which is caused by liver flukes. It has been shown that a chronic Opisthorchiasis infection increases a risk of cholagiocarcinoma of liver; the fact which turns this infection into a serious and largely overlooked threat to Global Health.One of the hypotheses postulates that a gradual change of homeostasis in a parasite microenvironment (bile) may lead to liver fluke-induced cancer.Here we present for the first time a cross-platform mass spectrometric analysis of bile juice collected from the patients infected with O.felineus. We show that shift in the metabolic composition of bile juice is detectable in the hydrophilic fraction of the metabolome and provide tentative identification of the most affected compounds.
Metabolomic Analysis by HDMS for the Study of the Frequent Wheezing
in Preschool Children
This study was meant to investigate upon the metabolic profiles which characterize children with frequent bronchospasm (wheezing) in preschool age. The wheezing is a common symptom for several respiratory diseases which occur in childhood, like respiratory infections, and it is also related to the diagnosis of asthma.
The purpose of this study is to extract, by analyzing urine samples with the high definition mass spectrometry associated to the UPLC, the compounds which characterize children affected by wheezing, and to appreciate whether they can be considered useful biomarkers in predicting the next development of asthma. The preliminary results of this part of the study have been encouraging: we obtained a clear distinction of the subjects affected by wheezing as compared to healthy children, described by robust OPLS-DA models, and we extracted some putative biomarkers.
MRM Based Assay for the Differential Diagnosis of Arylsulfatase A or Saposin-B Deficiency in Metachromatic Leukodystrophy
Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disease affecting the sphingolipid metabolism and leading to the accumulation of sulfatides in the body. MLD can be caused by a deficiency either of the enzyme arylsulfatase A or its activator protein saposin B. The latter would lead to an additional accumulation of globotriosylceramides (Gb3). We present a MRM-based mass-spectrometric assay for the simultaneous measurement of sulfatides and Gb3 from urine samples, which could be used for the differential diagnosis of arylsulfatase A or saposin B deficiency in MLD patients.
Quantification of Cholesterol and Related Metabolites in Serum and Dried Blood for the Screening of Smith-Lemli-Opitz Syndrome
Smith-Lemli-Opitz syndrome (SLOS) is an inherited metabolic disease in the cholesterol biosynthesis pathway characterized by accumulation of 7- and 8-dehydrocholesterol (DHC) and by reduced cholesterol concentrations in all tissues and body fluids. With this study, we developed a new, rapid, robust and high throughput tandem mass spectrometric method for the quantification of cholesterol and DHC as routine application for the selective SLOS screening and therapy monitoring in serum and dried blood spots.
Energy Metabolism Drift in the Ageing Brain
Brain function is highly dependent upon controlled energy metabolism, and a loss of control coincides with significant cognitive impairment in humans. This is particularly apparent in aging and age-related neurodegenerative diseases however it is still largely unknown how energy homeostasis is disrupted in the healthy aging brain. Here we applied brain region metabolome and proteome profiling at different stages of mouse lifespan. Global-untargeted metabolomics revealed a metabolome drift in the aged brain, depicted as a significant increase in metabolome deviation from the adult reference stage. Metabolome drift was characterized by the compromised cellular energy status (NAD+ decline, increased AMP/ATP), and markedly altered oxidative phosphorylation, glycolysis, nucleotide biosynthesis and degradation, amino acid biosynthesis, with parallels to Warburg reprogramming.
Development of a Real-time Breath Analysis Platform and Applications: Diagnosis in Humans and Drug Monitoring in Mice
We modified the entrance of a commercial mass spectrometer to allow for the real-time analysis of breath via secondary electrospray ionization-mass spectrometry. We have studied i) obstructive sleep apnea (OSA) in a randomized controlled trial; ii) breath levels of drugs in mice injected with these substances. We found a panel of breath metabolites that were significantly enhanced in breath after treatment withdrawal. We could also trace pharmacokinetic curves of ketamine injected in mice in real-time. The real-time mass spectrometric analysis of exhaled metabolites may contribute to address some of the most relevant clinical and pharmacological problems, which are currently investigated through the analysis of body fluids other than breath.
Validation of a Simple and Sensitive Method for the Quantification of the Bile Acid 7α-Hydroxy-4-cholesten-3-one (C4)
Bile acids malabsorption is encountered in numerous gastrointestinal pathologies and is a good example of treatable cause of watery diarrhea after ileal resection. Robust validated diagnostic methods are not routinely used in practice due to cost, logistics or laboratory equipment limitations. Here, we developed a simple and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to measure 7α-hydroxy-4-cholesten-3-one (C4) in human serum. 7α-hydroxy-4-cholesten-3-one was extracted from human serum with absorption chemistry based extraction plates, and separated by using reversed-phase chromatography with methanol and water (2 mM ammonium format) as mobile phase. The method was then fully validated.
A Metabolomics Approach to the Investigation of the Effects of Nitisinone on the Treatment of Alkaptonuria: A Preliminary Study.
Clinical Research
A metabolomics approach to investigate the effects of Nitisinone on urine samples with the Alkaptonuria (AKU) condition is presented. AKU is an inborn error of metabolism characterised by increased levels of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) due to a deficient enzyme, homogentisate 1,2-dioxygenase (HGD).
Six healthy urine samples and six urine samples before and after nitisinone treatment were analysed using a reversed phase UHPLC/QTOF MS method. Data were processed using a feature finding algorithm (Molecular Feature Extractor) and the resulting features were compared statistically across the three sample sets.
This comparison highlighted a number of compounds which were up- or down-regulated as a consequence of Nitisinone treatment, and compared to healthy samples.
Research use only. Not for use in diagnostic procedures.
LCMSMS Quantitative Analysis of Bile Acids in Serum and Evaluation and Comparison of Sample Preparation Techniques
An LC/MS/MS analytical method was developed for the quantitation of Bile acids by LCMSMS. Various sample preparation techniques were evaluated and compared. An Agilent 6460 tandem mass spectrometers in negative Electrospray mode with an Agilent Infinity 1260 HPLC system were evaluated for this analysis. 100 ul of serum were used with an Agilent Zorbax RRHD 100 x 2.1 mm, 1.8 um with a water:acetonitrile mixture containing 0.1% formic acid achieved baseline chromatographic separation within 6 minute. The limits of detection and limit of quantitation were determined to range from 0.1 to 0.5 nM with very good reproducibility observed for all compounds, techniques and configurations.
Development of an Assay of Erythrocyte Pyruvate Kinase Activity Using UPLC-MS/MS
We developed and evaluated a quantitative enzyme assay for pyruvate kinase deficiency using UPLC-MS/MS. All compounds were clearly separated within 4 min without ion suppression. Within-run coefficients of variation were 8.3% and 8.6% in low and high level, respectively. Calibration curves are linear over the range 0~4000 ¥ìg/mL. The amount of product obtained was proportional to the amount of hemolysates and increased linearly with the incubation period (0~60min). The enzyme activity in hemolysates from one patient with pyruvate kinase deficiency was about a half of those in the hemolysates of 20 normal individuals.
Clinical Diagnosis of Liquorice Induced Hypertension and Evaluation of 11 βHSD2 Activity in Patients by UPLC-MS/MS
Abundance of the vital hormone cortisol can cause hypertension and hypokalaemia. Conversion of the active hormone cortisol to the inactive hormone cortisone by the enzyme 11β-hydroxysteroid dehydrogenase (11βHSD2) is inhibited by glycyrrhetinic acid, the active substance in liquorice. Studies have shown that measurement of the ratio between cortisol and cortisone can be used to evaluate the enzymes activity. A reliable high through-put diagnostic method based on UPLC-MS/MS was developed for simultaneous quantification of cortisol, cortisone and glycyrrhetinic acid. This assay will facilitate clinical diagnosis of liquorice induced hypertension and evaluation of 11βHSD2 activity in patients.
Metabolomics: Advantages of Global (non-targeted) or Targeted Phospholipid Analysis
For metabolomics analysis targeted or non-targeted approaches based on Triple Quadrupole or QToF mass spectrometry are used to reveal significant differences between sets of samples (e.g., healthy vs. disease; before vs. after treatment) in the search for (bio)markers. While QTof scan an entire mass range of small molecules in very short time intervals and at high mass resolution, triple quads are more sensitive and specific mass transitions to quantify more reproducible. Here we present a comparison of high-end QToF and Triple Quadrupole analysis in terms of their ability to distinguish between sample sets. We focused in our comparison on phospholipids.
Targeted Metabolomics Approach Measuring Oxidative and Nitrosative Stress Using LC-MS/MS
Oxidative stress is an underlying pathogenic mechanism associated with the progression of several pathological conditions. The development of a targeted metabolomics approach for the simultaneous measurement of isoprostanes, nitro-fatty acids, prostanoids, lysosphingolipids, and lysophosphatidic acids, gives a glimpse into the cause and effect of inflammation and oxidative stress. Here we describe a fast, sensitive, and targeted UHPLC-MS/MS metabolomics method, enabling the simultaneous quantitative and qualitative determination of 17-isoprostanes as well as their respective isomeric prostanoid mediators, three nitro-fatty acids, four lysosphingolipid mediators, and 25 lysophosphatidic acid species from serum, for subsequent biological elucidation thereof.
This is a test
This is a test.
Measurement of Carbapenemase Activity in Enterobacterial Isolates by Means of an LC-MS/MS Approach
Carbapenems represent the last line of defense against Gram-negative strains resistant to all other classes of β-lactam antibiotics. Carbapenem resistance in enterobacteria is most commonly caused by expression of carbapenemases. An assay that could rapidly and quantitatively determine carbapenemase activity in these bacterial isolates from infected patients would be useful in the clinical practice. We developed a novel and simple method to measure carbapenemase activity from bacterial isolates by means of a straightforward Liquid Chromatography- tandem Mass Spectrometry (LC-MS/MS) approach.The method is currently under clinical evaluation for rapid assessment of carbapenemase related resistances in various clinical settings.
Rapid and Simple Differentiation of the Acinetobacter Calcoaceticus- Acinetobacter Baumanii Complex by MALDI TOF Using the VITEK MS Platform
The Acinetobacter calcoaceticus-Acinetobacter baumannii (AcB) complex comprises three species involved in human infections, A. baumannii, A.pittii, A. nosocomialis and one environmental species, A. calcoaceticus. The objective of this study was to set up a simple MALDI TOF MS method that could be implemented routinely allowing to distinguish them. 124 well-characterized strains of the four species of the complex were used to acquire 248 MALDI TOF MS spectra. An estimation of performance by cross-validation showed that all members of the Acb complex could be clearly distinguished. The validation of the prediction model showed that most of the strains could be identified to the species level.
Application of Dynamic Chemistry to Parasitemia Diagnosis
Protozoan parasites of the family Trypanosomatidae are the causative agent of devastating diseases in humans and livestock. Rapid and effective diagnosis would lead to better management of these diseases and relief of suffering.
The aim of this research project is to use nucleic acid analysis by dynamic chemistry in order to develop highly innovative products with a true diagnostic potential and utility for rapid detection of nucleic acids, offering benefits in terms of result consistency, time, cost and ease of use.
Application of Nanoflow LC-MS for Targeted Proteomic Quantification of CD39 and CD73 in Human Calcified and Non-Calcified Valves
CD39 and CD73 converts ATP to ADP and AMP and finally to adenosine. The expression of enzymes could be crucial for pathology of aortic valves. The aim of this study was to develop method for quantification of ecto-enzymes in human aortic valves and to correlate results with immunohistochemistry assays. The contents of proteins in calcified valves were: 0.72±0.19 (CD39) and 0.98±0.24 (CD73) pmol/mg, whereas, in the non-calcified valves were: 1.03±0.44 (CD39) and 1.29±0.16 (CD73) pmol/mg. This results are in agreement with immunohistochemistry. We conclude that the method is suitable for quantification of CD39 and CD73 in human aortic valves.
Mass Spectrometry-based Proteomic/Metabolic Studies of Hearts from ApoE-/-/LDLR-/- Mice Revealed Potential Mechanism for Its Increased Susceptibility to Hypoxia
The aim of this study was to characterize global cardiac proteome changes in atherosclerotic ApoE-/-/LDLR-/- mice and correlate these findings with changes in metabolic substrate preference and response to reduced oxygen tension. Hypoxia in ApoE-/-/LDLR-/- mice caused maximum decrease of ST segment of ECG by 0.14 ± 0.02 mV while there was no change in ST segment (<0.01 mV) in control mice. Differential analysis of cardiac proteome revealed coordinated increased expression of mitochondrial proteins including those responsible for fatty acid oxidation. Metabolic experiments with [1-13C]glucose were consistent with increased use of fatty acids in atherosclerotic hearts.
First-trimester Multimarker Prediction of Gestational Diabetes Mellitus
Using Multiple-Reaction-Monitoring Mass Spectrometry
Gestational diabetes mellitus (GDM) is a common complication of pregnancy associated with increased risk of adverse outcome. While early and accurate prediction of GDM is needed to allow intervention and improve perinatal outcome, no single protein biomarker has yet proven useful for this purpose.
Here we have evaluated a 25-plex panel of candidate serum biomarkers for prediction of GDM using multiple-reaction-monitoring mass spectrometry (MRM-MS). Multimarker models were constructed combining protein levels and clinical data and evaluated by receiver operating characteristic (ROC) analysis. For both obese (n = 274) and non-obese (n=133) woman these models achieved a higher area under the curve (AUC) than for the best single predictor adiponectin. We conclude that multimarker models combining protein markers and clinical data have the potential to predict women at high risk of GDM.
High-throughput Automated Analysis of Serum N-glycans to Study Pregnancy-associated Improvement of Rheumatoid Arthritis
Improvement of RA during pregnancy has been associated with changes in glycosylation. In this study the N-glycome of total serum was analyzed using an automated sample preparation system followed by MALDI-TOF-MS. The preliminary results of this study confirmed the findings of previous studies, as well as added new information on the N-glycome of serum from pregnant RA patients and healthy volunteers. Linkage specific differences were observed between RA patients and healthy volunteers, as well as pregnancy induced changes. In-depth analysis of this data should result in additional information on pregnancy- and RA-associated differences, likely providing insight in pregnancy-associated improvement of RA.
Quantitative Strategies for Multiplexed High-Throughput Protein Analysis
New avenues such as personalized medicine in clinical laboratory settings require robust platforms to assess quantitative read-outs for diagnostic purpose and individualized therapies. In this respect, use of quantotypic peptides unparallels protein quantification by absolute means in a singular LC-MS run. Quantotypic peptides are represented as an array of concatenated proteotypic peptides, which are synthesized from tryptic fragments, and termed as a QconCAT protein. Concomitant detection of up to 100 peptides, and thus the ease of multiplexing absolute protein quantification as high-throughput fashion will be presented highlighting current reference examples applied for instance in human health proteomics.
Comparison of Nanoscale/Microfluidic LC Platforms and High/Low Resolution Mass Spectrometers for Quantitative MRM Analysis of Candidate Peptide Biomarkers
Biomarker discovery and validation are the first steps in understanding disease and drug development. Validation is challenging as it requires analyzing large numbers of samples with high-throughput, high sensitivity, high resolution, large dynamic range and excellent selectivity. Targeted LC-MS based assays afford protein quantification with the reproducibility and throughput required in order to improve biomarker acceptance. MRM, using both low, high and ion mobility enabled high resolution mass spectrometry, are enabling technologies. Miniaturized LC systems offer improved mass-sensitivity but often lack throughput, robustness and reproducibility. Here, we compare the application of an integrated microfluidic device with nanoscale LC, using different MS platforms, for the quantification of marker peptides, considering throughput, sensitivity, selectivity and reproducibility.
Towards Robust Plasma Sample Preparation for Clinical Relevant Proteomes
Mass spectrometry (MS)-based proteomics is potentially an ideal technology to discover disease indicators in the blood plasma proteome. We show that recent developments in our group such as the ‘in StageTip’ (iST) sample-preparation workflow and optimized high-throughput LC-MS measurement technologies enable fast and multiplexed sample processing with very high reproducibility (R2 ≥ 0.99) and low coefficients of variation (CV < 20 %). In very short, automated LC MS/MS runs we already quantified more than 400 proteins, including 50 FDA-approved biomarkers. These developments will contribute to the development of MS-based plasma proteomics for routine, quantitative analysis of patient samples in clinical settings.
SRM Analysis of Natural Genetic Variation on Protein Abundances in C. elegans Signalling Pathways
Complex diseases (e.g. cancer) are caused by a combination of genetic and environmental factors, including lifestyle. Many of the signalling pathways involved in these diseases are conserved among species and also present in the small nematode Caenorhabditis elegans. To study the influence of natural genetic variation on the protein abundance of signalling pathway components, we used selected reaction monitoring and quantitative trait loci mapping. We analysed recombinant inbred lines generated from the two genetically divergent C. elegans wild-type strains Bristol N2 and Hawaii CB4856. We found a weak trans-QTL for the phosphatidylserine receptor protein PSR-1. Our data suggest that protein levels of selected signalling pathway components are under strong evolutionary control than transcript levels.
Comparative Analysis of Serum Samples from Cervical Cancer Patients for Biomarker Discovery Using iTRAQ Labelling
We performed a quantitative study of serum samples from patients related to pre-cancer, early and advanced stages of the disease versus a healthy control group. We used a ''pooling'' approach per group for sample preparation prior to LC-MS/MS. 2 replicates of 4 pooled samples (~50 samples each) were immune-depleted, trypsin digested, labeled with stable isotope reagents (iTRAQ®, 4-plex), pre-fractionated on an ERLIC column, followed by LC-MS/MS on a Q-Exactive Plus mass-spectrometer. Approximately 1600 proteins were identified per sample. The six most promising candidates were selected for validation in individual patients from different groups including CIN, early and advanced cervical cancer.
Improvement in Speed and Reproducibility of Protein Digestion and Peptide Quantitation, Utilizing Sample Preparation Technology in a Full Solution Workflow
We describe a workflow including novel, rapid and precise digestion of Cytochrome C, followed by micro-elution solid phase extraction (SPE) clean-up and analysis with next-generation UHPLC and high resolution mass spectrometry detection (UHPLC-HRMS). Four well characterized peptides, derived from cytochrome C, were used for assessment of the novel digestion procedure. Four exogenous peptides were also spiked in post digest, which allowed assessment of the reproducibility of the digest along with an independent assessment of the clean-up procedures.
The workflow presented supports fast, generic, and robust analytical methods suitable for a high throughput environment.
Detection and Quantitation of Yersinia pestis Protein Biomarkers by Dried Matrix Spot (DMS) and Liquid Chromatography-Tandem Mass Spectrometry
Dried Matrix Spot (DMS) or Dried Blood Spot (DBS) offer many advantages as a sample format including ease and safety of storage and transport. Recent studies started to take a growing interest in this sampling protocol for proteomic. Within this context, we’re developing a DMS extraction coupled to liquid chromatography-tandem mass spectrometry approach for the quantitation of Yersinia pestis biomarkers in biological fluids. We have obtained promising results for Y. pestis F1 antigen detection in human serum after protocol optimization and additional improvements are required to increase the targeted quantitation sensitivity in different complex matrices (whole blood and bubonic-like content) and monitor simultaneously others protein biomarkers of plague infection.
Characterization of Stable Isotope Labeled Recombinant Proteins for Use as Internal Standards in Quantitative MS Workflows
Methods for quantification of clinical protein biomarkers by MS must be developed to minimize and control experimental variations in all steps of the workflow. We have characterized stable isotope labeled (SIL) full length proteins for utility as internal standards in quantititative MS workflows. Characterization of clinically-relevant SIL proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin and APOA1, are reported in this study. SIL proteins were analyzed by HPLC-UV-MS or SDS-PAGE for purity, intact mass analysis to determine protein processing, and by bottom-up proteomics for sequence confirmation, heavy isotope incorporation and digestion kinetics.
Robust and Reproducible Experimental Methods for Large-scale Translational Proteomics Studies
Translational proteomics research is used to stratify proteins for plausible inclusion into biomarker panels to differentiate disease states. The primary issue attributed to the workflow is identifying representative proteins for inclusion into targeted groups. New strategies employ global differential expression analysis across large-scale studies to determine biological variance and strengthen statistical measures used to stratify protein markers. To successfully achieve large-scale studies, new experimental methods are used, including UHPLC separations, analytical flow rates, and pSMART data acquisition. In addition, novel software approach are used to automatically filter key proteins.
The Day has Come: Immunoenrichment, Mass Spectrometry and the Clinical Lab — An Application for Diabetes Clinical Care.
Western analysis and ELISA techniques have problems providing high throughput, multiplexing and specific assays to quantify multiple forms of the same protein from clinical specimens. In brief, the clinical utility of insulin and proinsulin has been low given the variability between assays and lack of standardization. This has lead to problems in the clinical application and adoption of a robust insulin/proinsulin assay in the clinical lab. The establishment of an accurate and standardized approach would have clinical value in the assessment of endogenous compared to exogenous insulin. To address this shortcoming, we have developed and validated a multiplexed assay for intact proinsulin, insulin and its analogues that couples immunoenrichment with high resolution and accurate mass spectrometric detection across the clinical range.
Determination of Etonogestrel in Blood Plasma by High Performance Liquid Chromatography - Mass Spectrometry
Development and validation of methods for determining plasma concentrations picogram etonogestrel - active metabolite of desogestrel. The method used - HPLC/MS. Analytical equipment - liquid chromatograph Shimadzu and a mass spectrometer with a triple quadrupole ABSciex QTrap 4500. We used precolumn and column, flow rate 0.4 ml/min, eluting with a gradient system (acetonitrile/water). For the selective detection mode is MRM. Sample preparation is a liquid-liquid extraction with diethyl ether. Calibration curve is linear in the defined range of values (40-4000 pg/ml). Methods of analysis it is necessary to research on the pharmacokinetics and bioequivalence of drugs containing it.
Determination of Human Reference Values for Serum Total 1,25-dihydroxyvitamin D3 Using an Extensively Validated 2D ID-UPLC-MS/MS Method.
Measurement of 1,25-dihydroxyvitamin D is required when disorders of 1α-hydroxylation, extrarenal 1α-hydroxylation, or vitamin D receptor defects are suspected. This study aimed to determine reference values for 1,25-dihydroxyvitamin D3 and D2 using a 2D ID-UPLC-MS/MS method. It was able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D3 and D2 in human serum and was extensively validated. The major finding of the present study is a reference interval of 59-159 pmol/L for 1,25-dihydroxyvitamin D3 determined with a highly sensitive and precise LC-MS/MS method.
Fast and Quantitative Method for the Analysis of 25 Antipsychotics and Metabolites in Plasma with UHPLC-MS/MS
A fast and accurate UHPLC-MS/MS method was developed and validated for the simultaneous quantification of 25 antipsychotics in 100 µL of plasma. After protein precipitation with 0.5 mL of acetonitrile, the samples were centrifugated and the organic phase transferred to the vials for direct injection in the UHPLC-MS/MS system. The analytes were separated on a BEH C18 analytical column with gradient elution in a total run time of 3.5 min. Limits of quantification were selected according to their therapeutic concentration and ranged from 0.5-25 ng/mL. The method has been validated according to the recent international validation guidelines. The method was successfully applied to the analysis of authentic samples from forensic toxicology cases.
High-sensitivity Quantitation of Catecholamines in Plasma by Triple-quadrupole Mass Spectrometry
Catecholamines in plasma, namely norepinephrine (NE), epinephrine (EP) and dopamine (DA), are routinely measured as biomarkers for diseases such as hypertension or neuroblastoma. However, the conventional assay for catecholamines based on fluorescence HPLC lack sensitivity and throughput. Here we developed two types of LCMS methods for determining the plasma concentration of catecholamines at low pg/mL sensitivity in a short analysis time.
Comparison of Saponification and Protein Precipitation Methods for the Extraction of Vitamin D in Breast Milk
In milk, vitamin D is bound to proteins and lipids therefore needs to be released before analysis. Saponification has generally been the method of extraction of vitamin D from foods while protein precipitation has been used for plasma/serum. In this study, we compared the efficiencies of saponification and protein precipitation in releasing four key vitamin D compounds, D2, D3, 25(OH)D2 and 25(OH)D3, from breast milk. The extractions were followed by derivatisation with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis by liquid chromatography-tandem mass spectrometry. The protein precipitation method was found to be the more efficient extraction method.
The Future of Therapeutic Drug Monitoring by Clinical Mass Spectrometry. the Quest to Improve the Patient Experience
Mass spectrometry has been used in UK clinical diagnostics for over 15 years. Most laboratories implementing this technology concentrate on mainstream TDM applications but should we restrict quantitation to the parent molecule? Examples will be presented to illustrate how significant improvements in safety and efficacy can be made for drugs introduced >25 years ago, how selected metabolite monitoring can identify patients at risk from potentially toxic side effects and potential new monitoring strategies. These approaches may not be without difficulties requiring population screening to establish their significance, but they can be achieved with the latest advances in technology.
Diagnosis of Phenylketonuria by MALDI-TOF Mass Spectrometry
Using Parylene Matrix Chip
The diagnosis of Phenylketonuria, an inborn metabolic disease, is generally performed with various detection methods. In this study, parylene matrix chip was developed for the qualitative and quantitative analyses of phenylalanine in water and serum using MALDI-TOF mass spectrometry by reducing interference of conventional organic matrix. Parylene-N thin film was deposited on dried organic matrix (CHCA) spots with the thickness of 50~80 nm. Phenylalanine in serum was analyzed after methanol treatment. As a result, interference was significantly reduced and quantitative detections of phenylalanine in water and serum were feasible using parylene matrix chip.
Automated Procedure for Therapeutic Drug Monitoring of Immunosuppressants by LC-MS/MS
The implementation of a fully automated procedure based on LC-MS/MS for simultaneous routine determination of cyclosporine A, everolimus, sirolimus, and tacrolimus is presented. The procedure is based on the IVD-CE certified MassTox Immunsuppressiva – Oneminute test from Chromsystems, automated on a Hamilton MassSTAR. Critical parameters such as turn-around time, the proper handling of whole blood samples (homogeneity, barcode-reading, pipetting, logistics…), and integrating the HPLC-System (Ultimate 3000, Thermo) as well as the MS-System (TripleQuad 4500, ABSciex) into the Laboratory Information Management System (Molis) are discussed.
Development and Validation of an Isotope Dilution Mass Spectrometric Assay for Urinary 6-sulfatoxymelatonin and Establishment of Reference Ranges
Melatonin is an endogenous marker of circadian rhythm in humans and is best known for its role as signaling molecule for the length of day and night. The major urinary metabolite of melatonin in urine is 6-sulfatoxymelatonin and can be used for the total output of melatonin over 24 hours (24h). As melatonin is associated with several disease states such as depression and metabolic syndrome, we developed and validated a high-throughput isotope dilution mass spectrometry assay for 6-sulfatoxymelatonin in urine. We will present validation data and age-dependent reference ranges for 6-sulfatoxymelatonin in 24-h urine.
Simultaneous Measurement of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization
Our objective was to develop a very sensitive LC-MS/MS assay for both testosterone and estradiol in serum in a single analysis without the need for chemical derivatization. Serum samples were prepared by the addition of deuterated internal standards followed by a liquid-liquid extraction and LC-MS/MS in both positive and negative ion modes. The assay was very sensitive, had wide analytical measurement ranges and was precise. The accuracy was assessed by comparison with established LC-MS/MS assays, recovery studies, and comparison with several different reference materials. We have successfully developed an accurate and highly sensitive assay to simultaneously measure testosterone and estradiol levels in serum.
Development and Validation of an LC-MS/MS Assay for the Simultaneous Measurement of 17-hydroxyprogesterone, Testosterone, Androstenedione and DHEAS
A novel LC-MS/MS method for simultaneous measurement of serum 17-hydroxyprogesterone (17-OHP), androstenedione (A4), testosterone and DHEAS was developed and validated for use in clinical diagnostics. Standard curves were linear (r2>0.99) across the calibration ranges of all analytes. The lower limits of quantification were 1.7 nmol/l for 17-OHP, 0.5 nmol/l for A4, 1 nmol/l for testosterone, and 0.17 umol/l for DHEAS. The intra- and inter-assay imprecision were <10% and inaccuracy was <15% for all four analytes across the analytical range. No significant ion suppression or enhancement was observed and the isobaric interferent 11-deoxycorticosterone was chromatographically resolved from 17-OHP. Run time was 7.2 minutes per injection.Comparison of measurement of the EQA samples for A4, testosterone and DHEAS by our LC-MS/MS method with other group peers showed no significant difference.
The Development, Validation and Application of a Novel Non-contact Method for the Hematocrit Prediction of Dried Blood Spots
Varying hematocrit values are known to impact dried blood spot (DBS) based quantitation. To evaluate the extent of this effect on a certain DBS result, it is crucial to know the hematocrit value of that DBS. Since the only existing method for hematocrit estimation of DBS suffered from some practical drawbacks, a more convenient, non-contact method was developed, which allows hematocrit estimation in mere seconds. Application to 288 patient samples with varying hematocrits (0.20 - 0.50) revealed a strong correlation between the estimated hematocrit values determined on DBS and the true hematocrit values measured on the corresponding whole blood samples.
A New Approach to Simplify LC Tandem-MS Workflow Complexity with a New Four Channel HPLC
Today's clinical laboratories are challenged to increase sample throughput with method flexibility and reduce sample turnaround time. When analytes elute for only a portion of an HPLC run, mass spectrometers configured in a single channel LC-MS workflow may only be in use in a fraction of the method time. A new HPLC brings the productivity of up to four HPLC channels to a single mass spectrometer, four times the samples can be run, maximizing the LC-MS output. With built-in multichannel optimization software, up to four identical or different test methods can run, and simplify workflow.
For in vitro diagnostic use. Not available in all countries.
Does Volumetric Absorptive Microsampling Eliminate the Hematocrit Bias for Caffeine and Paraxanthine in Dried Blood Samples? A Comparative Study
Dried blood microsampling is increasingly receiving attention in different fields. However, progress in the field of dried blood spot (DBS) sampling has greatly been hampered by the hematocrit issue, in which blood with different hematocrit spreads differently on filter paper, resulting in deviating results. One of the proposed solutions to cope with this issue is so-called volumetric absorptive microsampling (VAMS). Here, using real patient samples, we evaluated whether VAMS effectively overcomes the DBS-associated hematocrit issue. To this end, validated methods were set up to compare caffeine and paraxanthine concentrations in paired blood-DBS-VAMS samples. We conclude that VAMS effectively overcomes the hematocrit issue.
Development of a Diagnostic and Interpretative Laboratory Service for the Measurement of Trace Elements Using ICP-MS
Trace element analysis is a specialized area of clinical diagnostics used to identify both deficiencies and excesses of endogenous essential elements and also to detect potential poisoning from exogenous toxic metals. Analysis has classically been performed in specialist laboratories due to the high technical demands required to deliver a quality service. With advances in ICP-MS technology, sensitive, specific and robust trace element analysis has become available to a larger number of clinical laboratories. Here we report on the development, validation and implementation of a diagnostic and interpretative laboratory service for the measurement of trace elements using ICP-MS.
Measurement of Methylguanidine and Guanidinesuccinate by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS)
Guanidino compounds (GC) are considered neurotoxic and are significantly increased in renal failure. The measurement of methylguanidine (MeG) and guanidinesuccinate (GSA) in serum is potentially useful as a measure of dialysis efficacy. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed with online solid phase extraction (SPE) for the determination of MeG and GSA in serum. The analytical run-time was 5 minutes per sample with standard curves for both assays linear up to 50 µmol/L. Intra and inter-assay CV’s for MeG and GSA were <7%. A simple, high-throughput UPLC-MS/MS method was developed for MeG and GSA without pre-derivatisation.
High-throughput Therapeutic Drug Monitoring (TDM) with LC-MS/MS
Using Either Manual Sample Preparation or Fully Automated On-line SPE
For accurate, sensitive and fast therapeutic drug monitoring (TDM) in clinical routine analysis, RECIPE has developed its ClinMass® TDM Kit-Systems MS9000 and MS9050. The systems work with just one analytical column and the same solvents for all analytes, so high throughput with no change in hardware between the methods can be realized. The MS9000 Kit-System offers a fast sample preparation technique with fast LC-MS/MS analysis, the MS9050 Kit-System is working with fully automated sample preparation without the need of a liquid handling system. Both Kit-Systems show outstanding validation results for Tricyclic Antidepressants and Benzodiazepines with more drug classes in progress.
The Analysis of Common Antiepileptic Drugs in Human Urine by LC-MS/MS
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) in therapeutic drug monitoring and toxicology labs has increased significantly over the years. LC-MS provides sensitivity, speed, and the ability to simplify sample preparation. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles (SPP) with the resolution of Ultra Selective Liquid Chromatography™ (USLC™) technology. In this example, a simple dilute and shoot method was developed for 14 common antiepileptic drugs in urine using a Raptor™ Biphenyl column.
A Multi-Class Drug and Metabolite Screen of 231 Analytes by LC-MS/MS
Therapeutic drug monitoring can be challenging due to the low cut-off levels, potential matrix interferences and isobaric drug compounds. To address these challenges, many drug testing facilities are turning to liquid chromatography coupled with mass spectrometry (LC-MS/MS) for its increased speed, sensitivity, and specificity. The Raptor™ Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles with the resolution of Ultra Selective Liquid Chromatography™ column technology. In this example, a method was developed for a 231 compound multi-class drug and metabolite screen.
A Rapid and Sensitive LC-MS/MS Method for the Analysis of Free Thyroid Hormones
The unbound or “free” thyroxin (T4) and tri-iodothyronine (T3) are the active forms of thyroid hormones which only exist at low levels (pg/mL) in the circulation. The measurement of these free hormones is necessary to assess thyroid function for both veterinary and human clinicians. A fast, accurate, and sensitive method was developed for the analysis of thyroid hormones at the free form levels using the highly efficient and selective Raptor™ Biphenyl column. The clinical applicability of the method was demonstrated by analyzing the fortified thyroid hormone in phosphate buffer saline containing 4% human albumin.
Method Development for Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots by UHPLC-MS/MS
Newborn screening for congenital adrenal hyperplasia (CAH) has high false-positive rates, necessitating confirmation of primary results. We developed a single LC-MS/MS method for dried blood spots (DBS) that allows concurrent confirmation and differential diagnosis of CAH. All five steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone) were baseline resolved and reliably determined (UHPLC: PerkinElmer Flexar FX-10; MS/MS: ABSCIEX QTRAP 5500; column: Phenomenex Kinetex XB-C18). In cross-border cooperation (HU-RO Project 0802/008 SCREENGEN), CAH was confirmed in one of a total of 163 samples tested positive in primary screening. Our validated assay can use the same DBS as in primary screening (2nd-tier test), eliminating the need for repeated blood sampling and accelerating diagnosis.
Approaching the Aldosterone Challenge Two-dimensionally
We developed a 2D LC-MSMS method for the determination of aldosterone in human plasma (heparinized plasma, EDTA plasma and serum) and urine with a very simple and fast Supported Liquid Extraction sample pre-treatment. Coefficients of variation ranged from 12% at 0.06 nmol/L to 7% at 2.4 nmol/L. The method was linear to 15 nmol/L. There was no carry-over, extraction recovery was 91% on average and the matrix effect -44%. The accuracy of the method was tested against Cerilliant certified reference material. Aldosterone reference values for plasma were <0.54 nmol/L (seated) and < 0.86 nmol/L (standing) and for urine was <38 nmol/24hr.
Adherence Assessment to Cardiovascular Pharmacotherapy Using Quantitative LC-HRMS Analysis of Dried Blood Spot Samples
The analysis of dried blood samples (DBS) by liquid chromatography – high resolution mass spectrometry (LC-HRMS) for assessing medication adherence to candidate therapeutic drugs used in cardiovascular therapy was investigated. To evaluate the method 8 mm discs were punched from each DBS and extracted followed by subjecting to LC-HRMS analysis. Trials on top UK used cardiovascular drugs are reported demonstrating the ability of the system to detect the target analytes during the 24 hour repeat prescription cycle. The system responded successfully to challenges by samples from volunteers either of known adherence or receiving no medication.
High Resolution LC-ToF Mass Spectrometry Data Used for Drug Monitoring in Clinical Applications Using Dried Blood Spot Samples
Quantitative dried blood spot (DBS) analysis was used to investigate if the accurate mass capability (±2ppm m/z) of a ToF mass spectrometer would provide compound specificity comparable to the multiple reaction monitoring (mrm) approach used with a triple quadrupole mass spectrometer. Dried blood spots are now widely used for micro-sampling in clinical investigations and in this study the determination of atenolol, caffeine, captopril and dexamethasone in DBS samples was carried out using liquid chromatography-high resolution mass spectrometry. Some initial pharmacokinetic results from therapeutic drug monitoring data obtained from the DBS samples, is reported.
Steroid Profile in Heel Prick Blood as Second-tier Test for the Neonatal Screening on Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia (CAH) is an inherited disorder of adrenal steroid synthesis leading to deficient production of cortisol and aldosterone and an excessive production of androgens. In the Dutch neonatal CAH screening program, based on the measurement of 17-hydroxyprogesterone in dried blood spots (DBS) by immunoassay, the recall rate is ± 0.1%. As a second-tier test we measured the steroid profile in DBS using UPLC-MSMS, determined the reference interval of the steroids based on gestational age and analyzed DBS from 94 newborns with an aberrant CAH screening for 17-hydroxyprogesterone. We found 21-deoxycortisol as discriminative for CAH.
Quantification of Dihydrotestosterone in a Multiple Hormone Assay in Serum by LC-MS/MS
One of more specific clinical applications for measurement of dihydrotestosterone is monitoring of treatment of boys with 5á- reductase or other forms of deficiencies of sex development. An LC-MS/MS analytical method has been developed for quantification of four endogenous steroid hormones: 4-androstenedione and 17-hydroxyprogesterone, testosterone and also recently dihydrotestosterone in serum in our routine Laboratory.
A dilution and an SPE procedure using a robotic liquid-handling followed by UHPLC-ESE-MS/MS are used for quantification of the steroids. The Total CV of the assay was < 7%. A good linearity was obtained with R2 > 0.99 for each compound within its calibration range: 0.3-500 nmol/L for 17-hydroxyprogesterone, 0.3-100 nmol/L for androstenedione, 0.1-100 nmol/L for testosterone and 0.5-100 nmol/L for dihydrotestosterone.
Using the Waters® Forensic Toxicology Application Solution with UNIFI® to Screen for Cannabinoids
The Waters Forensic Toxicology Application Solution with UNIFI® comprises acquisition of accurate mass MSE data on a time-of-flight mass spectrometer, operating in electrospray positive ionisation mode, followed by comparison of the data with a comprehensive library containing more than 1000 toxicologically-relevant substances. Cannabinoids can also ionise in negative electrospray mode and the aim of recent work has been to extend the Forensic Application Solution to include those compounds. The new method was used to determine the presence of urinary cannabinoids, particularly at concentrations below current EWDTS screening cut-off, and to compare the values obtained with a recently published UPLC-MS/MS assay.
Analysis of Thiamine Pyrophosphate and Pyridoxal-5’-phosphate in Whole Blood Using the LCMS-8050 and the Magnamedics MagSi-VitBPREP Kit
To this day Thiaminepyrophosphate and Pyridoxal-5’-phosphate in whole blood are predominantly analysed with HPLC and fluorescence detection with excessive sample preparation including derivatisation. The aim of this study was to set up a simple and fast UHPLC method with mass spectrometric detection, a total solution. Both compounds were prepared using a MagSi-VitBPREP kit (Magnamedics Diagnostics B.V.), the supernatant was injected into the Nexera X2 UHPLC coupled to an LCMS-8050 LC-MS/MS (Shimadzu Corporation). Both compounds showed good linearity (r2> 0.999) in a clinically relevant concentration range with a LOD and LOQ of 2.7 nmol/L and 8.2 nmol/L for TPP and 0.6 nmol/L and 1.8 nmol/L for PLP.
Liquid Chromatography-tandem Mass Spectrometry as an Alternative Pertinent Method for the Falsely Elevated Cyclosporin a Concentration by Immunoassay
We reported the case of a 26-year-old male, who had received kidney transplantation. Four weeks after Cyclosporin A (CsA) discontinuance, the CsA concentration of the patient determined as 343.8 ng/mL by the Siemens Dimension ExL immunoassay. We suspected a falsely elevated CsA result due to interfering antibodies such as heterophil antibodies or antibodies against antibody-enzyme complex. When we repeated CsA analysis of the sample LC-MS/MS method, the CsA concentration was <26.1 ng/mL, which consistent with the drug dosing. A LC-MS/MS method can be preferable for therapeutic drug monitoring of CsA immunosuppressive drugs, especially for the cases showed interference in immunoassay.
Automated Sample Preparation for Immunosuppressant Analysis
Immunosuppressive drugs are applied to prevent the body from rejecting an organ transplant or to treat autoimmune diseases. As each patient’s absorption and metabolism of the drugs varies, correct dosing is crucial to avoid toxic reactions but still keep the drug at effective levels.
Therapeutic drug monitoring of the immunosuppressive drugs, cyclosporin A, everolimus, sirolimus and tacrolimus, is common in clinical laboratories. Current automation solutions for sample preparation for LC-MS/MS analysis focus on the processing part but neglect the monitoring and audit trail, thus no CE-IVD compliant solution has been available so far.
Here we present an automated CE-IVD compliant sample preparation solution for LC-MS/MS analysis of immunosuppressant sample from blood.
A Quest for Accuracy: Evaluation of a Reagent Kit Under Development for the Monitoring of Vitamin D in Serum by UPLC-MS/MS
An assay for the quantification of 25OHD3, 25OHD2 and total 25OHD measurement in human serum is described using the MassTrak™ Vitamin D Kit, currently under development. Feasibility studies indicate the MassTrak Vitamin D Kit provides an accurate, precise and robust assay for the measurement of 25OHD3 and 25OHD2, and thereby total 25OHD measurement in human serum. The assay uses an automated off-line sample preparation liquid handling system with the Waters ACQUITY® I-Class/Xevo TQD UPLC-MS/MS analytical system, for detection and quantification by multiple reaction monitoring (MRM), whilst providing sample tracking capabilities.
A Method for the Quantitation of Multiple Drugs in Urine by QTRAP Mass Spectrometry Utilizing Cliquid® 3.3.software, with Confirmation via MS/MS Library Search
Here we demonstrate a drug screening (as well as simultaneous screening and quantitation) method using a SCIEX QTRAP® 4500 hybrid triple quadrupole/ linear ion trap mass spectrometer, which provides simultaneous MRM detection and ‘on-the-fly’ acquisition of a full-scan MS/MS spectrum for every detected compound. The acquisition of MS/MS spectra are triggered when the intensity of the MRM exceeds a pre-defined threshold, and the acquired spectra are searched against a spectral reference library to provide unambiguous compound identification.
Analysis of Ethanol Metabolites in Urine Using Hydrophilic Interaction Liquid Chromatography Coupled to Mass Spectrometry
The analysis of ethanol metabolites such as ethyl glucuronide and ethyl sulfate is critical for monitoring of alcohol consumption. Hydrophobic stationary phases that are typically employed provide little retention of the polar analytes even at very high aqueous mobile phase content. The low retention results in a high probability of co-eluting matrix interference that may cause ion suppression or enhancement leading to irreproducible results. In this study, hydrophilic interaction liquid chromatography (HILIC) was investigated for the retention and selectivity of ethanol metabolites. The proposed method exhibits several advantages over existing reversed phase procedures.
A Rapid Research Method for the Determination of Cortisol and Cortisone in Urine Using UHPLC Coupled to APCI High Resolution Accurate Mass Spectrometry
Clinical researchers are interested in investigating cortisol as a potential clinical marker that may indicate adrenal or pituitary disorders. Cortisone, and the ratio of cortisol to cortisone, may add further diagnostic value. A method for the determination of cortisol and cortisone in urine has been developed. The method is based on protein precipitation followed by reversed phase chromatography and quantitation using area response of extracted ion chromatograms from the intact compound on a Thermo Scientific™ Exactive Plus™ High Resolution Accurate Mass (HRAM) mass spectrometer. The method has been validated and found suitable for quantitation of cortisol and cortisone in urine.
Free Metanephrines in Plasma by Liquid Chromatography Tandem Mass Spectrometry
Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, exhibit excessive catecholamines production. 3-O-methylated catecholamine metabolites, metanephrine (MN) and normetanephrine (NMN), are prioritized for diagnosis of pheochromocytoma due to their highest diagnostic sensitivity and specificity. The aim of this work was to develop the LC-MS/MS method based on hydrophilic interaction liquid chromatography (HILIC) which overcomes inconveniences related with classical HPLC, GC and immunoassay approaches, like extensive sample preparation, the step of derivatization or cross-reactivity. This method fulfilled validation requirements and provides simple sample preparation, short runtime, high analytical sensitivity, specificity and accuracy.
A Novel Fast Quantification Method for Amino Acids in Human Plasma by LC-MS/MS, without Ion Pairing or Derivatization
Amino acids are routinely assayed to diagnose inherited metabolism disorders. As highly polar compounds, they are hardly retained onto reverse phase. Derivatization or use of ion pairing reagent is required. For rugged analysis, there was a need for a new solution not using mentioned reagents. A new LC-MS/MS method was then developed, for the simultaneous high sensitive quantification of 49 amino acids, in 15 min, using a mixed-mode column, typical volatile mobile phase and very simple sample preparation. Total cost per sample is reduced from 13€ to 2€ compare to current methods. Analyses of plasma controls showed good accuracies.
A High Efficiency Workflow for Separation and Quantitation of Plasma Vitamin D Metabolites Using a New Four Channel HPLC and a Mass Spectrometer
A new four channel HPLC interfaced to a triple quadrupole MS was tested for separation and quantitation of 25-OH Vitamin D2, 25-OH Vitamin D3 and 3-epi-25-OH in human plasma. Baseline chromatographic separation of the 25-OH Vitamin D3 epimers was accomplished using a PFP column, avoiding the overestimation of 25-OH Vitamin D3 level. The new HPLC reduced solvent consumption by at least 60% by using positive-displacement pumps. It maximized utilization of MS instrument by connecting up to four HPLC channels to a single mass spectrometer, permitting high efficiency analysis of 40 samplers per hour, 960 samples per day. The study demonstrated good accuracy, reproducibility, and linearity over a range of 2-100 ng/mL for each analyte.
Online Analysis of Immunosuppressants in Whole Blood Using the EVOQ LC-Tandem Quadrupole Mass Spectrometer
A robust and reliable research method to quantitate the immunosuppressants cyclosporine A, tacrolimus, sirolimus and everolimus in whole blood samples using the online sample extraction coupled to a UHPLC-tandem quadrupole mass spectrometer is demonstrated. Exploitation of integrated online extraction with the ClinMass® LC-MS/MS complete immunosuppressant kit provides fast and easy sample cleanup. Interlacing the online extraction and chromatographic separation reduces the overall run time to 3 minutes per sample. The calibrations showed excellent linearity (r²≥0.997) and the assay had a very good precision with an RSD <9% as well as high accuracy with a bias of <±6.5%.
Determination of Tetrahydrocannabinol (THC) and Its Main Metabolites Using GC Triple Quadrupole Mass Spectrometry
Purpose: To introduce a novel gas chromatography-triple quadrupole MS system for the determination of the analytes of interest in THC use screening.
Methods: Gas chromatography tandem mass spectrometry are applied to the samples prepared by liquid /liquid extraction.
Results: This work will show the excellent sensitivity and linearity that such a system can provide for this type of analysis.
Methods for Screening and Verification of Anabolic Androgenic Steroids and Some of their Metabolites Using an Orbitrap Mass Spectrometer
Anabolic androgenic steroids are usually associated with sports doping. Due to the severe side effects associated with androgenic anabolic steroids there is a need for analytical methods not only in sports doping but also in society. Therefore two methods were developed, one for screening and one for verification of 17 analytes, anabolic steroids and metabolites. Both methods are based on Solid Phase Extraction followed by LC-MS using Thermo Scientific™ QExactive Focus™ mass spectrometer. The methods were developed to replace a GC-MS method currently in routine use.
Quantitation of Benzodiazepine and Z-drug Hypnotics Using High Resolution, Liquid Chromatography-Quadrupole Time-Of-Flight Mass Spectrometry
Benzodiazepines and Z-drug hypnotics are psychoactive drugs that are used in various clinical therapies. Due to their highly addictive nature and a frequently misuse they are regulated. Therefore, rapid detection and quantification methods are needed.
The goal of this study is to evaluate the suitability of high resolution, accurate mass Quadrupole Time of Flight (QTOF) technology for the simultaneous detection, confirmation and quantification of these drugs in human serum.
Here we report the quantitation of 13 compounds in serum as well as the measurement of spiked serum samples. The verification of found positives is based on retention time, exact mass, diagnostic ions and isotope pattern fit. The linear working range for most analytes is between 2.5-1000 µg/L.
Fast and Cost-effective Sample Preparation Based on Paramagnetic Beads for LC-MS/MS Measurements of Immunosuppressants and Steroids in Clinical Samples
MagnaMedics develops sample preparation methods based on magnetic beads. Proteins and impurities are precipitated on a magnetic silica based solid support and removed from the sample by magnetic separation. This makes the use of a centrifuge obsolete and opens up high throughput automation possibilities and fast sample preparation of clinical samples. MagnaMedics sample preparation kits were used to clean-up samples for LC-MS/MS measurement of four immunosuppressants and four steroids. Results showed good reproducibility and linearity over a clinically relevant concentration range.
MS Binding Assays for the Three Monoamine Transporters Employing the Triple Reuptake Inhibitor Indatraline as Native Marker
We herein describe the development of MS Binding Assays addressing the human monoamine transporters, as non-labeled alternative to the conventionally used radio-ligand binding assays. For this purpose indatraline, a so called triple reuptake inhibitor, was chosen as native marker. First, a sensitive LC-ESI-MS/MS method for quantification of indatraline in biological matrices using a deuterated internal standard, indatraline-d7, was developed, followed by a validation according to the FDA guidance. The method evolved enabled us to investigate the binding behavior of indatraline at the three targets (i.e. DAT, NET, and SERT) and its application as native marker in competitive experiments.
Rapid Measurement of Tacrolimus, Cyclosporin a and Sirolimus in Blood by Paper Spray-tandem Mass Spectrometry (PS-MS/MS)
therapeutic drug monitoring (TDM) of immunosuppressant(s) is critical in preventing organ rejection after transplant. Automated immunoassays provide quick turnaround time but are costly and lacking standardization. Conventional tandem mass spectrometry (MS/MS) requires a liquid chromatography (LC) system and assay requires pre-analytical manipulation which is not amenable to random access testing. Paper spray (PS) ionization is a technique that generates gas phase analyte ions directly from dried blood spots for quantitative MS/MS analysis, without complex sample preparation, nor a LC system.
We evaluated PS-MS/MS for simultaneous tacrolimus, cyclosporin A and sirolimus TDM in a clinical diagnostic laboratory, the results showed that PS-MS/MS method has acceptable AMR and precision, and results correlate well with those of a FDA approved immunoassay currently used in clinical lab.
Analysis of Serum Testosterone, Androstenedione, 17-Hydroxyprogesterone and DHEAS for Clinical Research
Here we evaluate a LC-MS/MS method for the measurement of serum testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone sulfate (DHEAS) enabling research of metabolic dysfunction. Sample preparation was performed using the Waters® Oasis® PRiME HLB µElution 96-well plate on the Tecan Freedom Evo 100 liquid handling system. Using an ACQUITY UPLC I-Class system, samples were injected onto a 2.1 x 50 mm Waters ACQUITY UPLC HSS T3 column using a water/methanol/ammonium acetate gradient and quantified with a Waters Xevo TQ-S mass spectrometer to obtain optimal analytical sensitivity for the analytes. This offline automated method demonstrates excellent linearity, precision and accuracy, while providing high sample throughput capabilities.
We Have the Analyte – But Where Is the Dross? A Systematic Approach to Investigate the Matrix Removal During Sample Preparation
Investigations into sample preparation procedures usually focus on analyte recovery with no information provided about the fate of other components of the sample (matrix). However, for many analyses, particularly those using LC-MS, quantitative measurements are greatly influenced by sample matrix. Using the example of the drug amitriptyline and three of its metabolites in serum, we track the fate of these trace analytes during nine sample preparation procedures commonly used for serum analysis while monitoring the undesired matrix compounds using a combination of charged aerosol detection (CAD), LC-CAD, and a metabolomics-based LC-MS/MS approach.
A Method for the Quantification of PEth 16:0/18:1 in Human Blood Based on Parallel Reaction Monitoring Using Orbitrap High Resolution Accurate Mass Spectrometry
Phosphatidylethanol (PEth) is an abnormal phospholipid that is only formed in red blood cells membrane in the presence of ethanol and its usage as a potential alcohol biomarker is being investigated by the Swedish Healthcare system; PEth 16:0/18:1 is the most abundant form. An analytical research method based on liquid chromatography high resolution mass spectrometry for the quantification of PEth 16:0/18:1 in human blood is described. Mass spectrometric detection was performed by parallel reaction monitoring (PRM) on a Thermo Scientific™ Q Exactive™ Focus high resolution mass spectrometer using heated electrospray ionization.
Fully Automated Direct Extraction and Analysis of Dried Blood Spots for the Determination of Four Anti-epileptic Drugs and Two Active Metabolites
Dosage adjustment of anti-epileptic drugs by therapeutic drug monitoring is very useful, especially for children. Considering the benefits of dried blood spots (DBS), this matrix could be an alternative to conventional venous sampling for this purpose. Since manual punching and off-line extraction slow down DBS analysis, an automated direct extraction and analysis of DBS can be advantageous. Therefore, we developed and applied a method for the determination and quantification of six anti-epileptic drugs, using a prototype device for on-line extraction of DBS samples followed by automated on-line solid phase extraction prior to liquid chromatography-tandem mass spectrometry.
Performance Comparison of a New LC-MS/MS Kit for Immunosuppressant Analysis with Established Methods
Rapid turnaround of immunosuppressant samples, as with all therapeutic drug monitoring, is necessary in a clinical laboratory. The new kit employs simple, automatable sample preparation coupled with on line extraction techniques coupled with LC-MS/MS analysis. Comparison data with established techniques such as immunoassay are presented here.
Improved Recovery, Reproducibility and Matrix Effects with an Advanced Technology in Solid Phase Extraction (SPE) – Oasis PRiME HLB
Oasis PRiME HLB is a novel reversed-phase solid phase extraction (SPE) sorbent developed to enable simpler and faster SPE protocols, while at the same time generating cleaner extracts than other sample preparation methods. In this application, a 3 step load-wash-elute SPE protocol, eliminating conditioning and equilibration, was successfully employed to extract 22 synthetic cannabinoids and metabolites from whole blood samples using Waters Oasis PRiME HLB 10 mg plates. Superior analyte recoveries, low %RSDs and modest matrix effects (ME) were achieved across the entire panel of compounds. At the same time, parallel extractions were conducted with other reversed phase (RP) SPE devices using recommended 5 step SPE protocols. Lower recoveries, higher %RSDs and higher matrix effects were obtained.
A Unified Sample Preparation Procedure for General Unknown Screening (GUS) of Compounds in Whole Blood Samples
A General Unknown Screening (GUS) is an ideal procedure to investigate intentional or accidental drug overdose, especially from incapacitated individuals. To achieve this task, a simple method is presented that requires small amount of whole blood sample. The sample is first subjected to protein precipitation and then phospholipid removal procedures. This supernatant is then injected on a biphenyl reversed-phased column fitted with a small internal volume in-line mixer. A high resolution Q-TOF system provides the data collection in MS (and MS/MS) analysis mode. The acquired data is then matched against a library containing 7,000 plus compounds.
Chiral Analysis of Methamphetamine and Related Compounds by LC-MS
Methamphetamine is a chiral compound that is often associated with recreational abuse. Methamphetamine however, may also be legally present in a sample as the L isomer is used in several over-the-counter (OTC) medicines. Immunoassay and LC/MS screening methods do not differentiate between the legal and illicit versions of the drug and therefore will report a positive finding if either are detected. Herein a chiral LC-MS method is proposed that provides rapid and effective separation of the enantiomers. Spiked urine samples containing methamphetamine were extracted and analyzed using the developed conditions. Excellent recoveries, detection limits and reproducibility have been established.
Rapid Determination of Drug Protein Binding Affinities Using Solid Phase Micro Extraction
Determination of free circulating drug is important in establishing the pharmacokinetic activity. Techniques used for determining drug protein binding levels consist of ultrafiltration, ultracentrifugation and micro dialysis. In the case of micro dialysis, processing may be greater than 6 hours. In this study, a novel BioSPME micro extraction device is evaluated as a rapid means of determining drug protein binding affinities from plasma. Initial studies demonstrate that drug binding affinities can be determined for a model set of compounds in less than 30 minutes using the micro extraction technique.
Comparison of Validation Results of the Manual Sample Preparation and Automated Sample Preparation for 25-hydroxyvitamin D2-D3 LC-MS/MS Analysis
An automated 25-OH Vitamin D2-D3 LC-MS/MS Analysis Kit was developed for rapid, sensitive and reliable quantitative detection of the 25-OH Vitamin D2-D3 concentrations in human serum samples and it gives results in 1.7 minutes with full automated Zivak MULTITASKER sample preparation and injection system. In this experiment, we compared the validation results between automated and manual sample preparation methods for 25-OH Vitamin D2-D3 analysis. According to results, the automated sample preparation and injection system has significantly better repeatibility and reproproducibility.
Things We Don’t Need in SPE - Plasma Corticosteroid Analysis Using a Novel Reversed-phase Solid Phase Extraction Sorbent that Removes Residual Phospholipids
A novel reversed-phase solid phase extraction (SPE) sorbent has been used to extract a panel of corticosteroids from plasma. Pretreated samples were loaded directly onto the SPE sorbent without conditioning and equilibration. The three-step protocol (load-wash-elute) eliminated >97% of phospholipids compared to protein precipitation. The resultant method demonstrated consistent recovery and minimal matrix effects. Quantitative results were linear, accurate and precise, with %CV and bias values under 10% for all compounds at all QC levels. This represents an advance in reversed-phase SPE enabling simpler, faster and cleaner procedures.
Identification of Active Compounds and Chemical Standardization of Botanical Dietary Supplements
To enable clinical studies that establish the safety and health benefits of botanical dietary supplements, active compounds need to be identified, and chemical assays developed and validated for their chemical standardization. Using hops and licorice supplements as examples, the mass spectrometry-based assays pulsed ultrafiltration and magnetic microbead affinity selection screening were used to facilitate the rapid identification of estrogenic and chemopreventive natural products. Then, fast assays were validated for the quantitative analysis of these compounds based on UHPLC-MS/MS. The assays included identification and measurement of xanthohumol, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin in hops and isoliquiritigenin and liquiritigenin in licorice.
Quantification of Serum 1,25-(OH)2D3 by UPLC-MS/MS: Comparison of Methods Involving Liquid-liquid Extraction, Immuno-extraction and Chemical Derivatization
A challenge facing serum 1,25-dihydroxyvitamin D [1,25-(OH)2D3] quantification is low circulating levels (15-60 pg/mL), requiring a selective extraction technique coupled with derivatization prior to LC-MS/MS. We compared liquid-liquid (LLE)- and immuno-extraction (IE) methods with derivatization using PTAD or DMEQ-TAD, and analyzed samples on ACQUITY/TQ-S instruments. We observed that all methods provided quantification of 20-30 pg/mL 1,25-(OH)2D3, however IE/DMEQ-TAD offered the greatest sensitivity and selectivity with an estimated LLOQ of 10 pg/mL. LLE offered the advantage of simultaneously detecting other metabolites including 25-OH-D3 and 24,25-(OH)2D3. We conclude that the choice of technique used to measure 1,25-(OH)2D3 depends on level of sensitivity required, and interest in measuring other metabolites.
A Novel Method for the Unambiguous Measurement of Low-level Testosterone in Human Serum Using Differential Ion Mobility Spectrometry-tandem Mass Spectrometry
Uncharacterized, endogenous components in biological fluids have the potential to interfere with the measurement of low-level steroids such as testosterone by LC-MS/MS. In this work, we present a novel method employing differential ion mobility spectrometry in conjunction with LC-MS/MS analysis to eliminate potential interferences, thereby simplifying sample pre-treatment and enabling reduced LC run-times. The method described here provided an LOQ of 0.1 pg/mL testosterone in serum, using an injection volume of 50uL of the prepared sample.
Measurement of a Panel of Steroids by LC-MS/MS, Employing Rapid Polarity Switching
In this work we have applied LC-MS/MS to the measurement of a panel of steroids including: Aldosterone, Estradiol, Testosterone, Androstenedione, Progesterone, 17-alpha-OH-Progesterone, Corticosterone, Cortisol, 11-Deoxycortisol, 21-Deoxycortisol, 5-alpha-Dihydrotestosterone (DHT), 11-Deoxycorticosterone, DHEA, DHEAS, Pregnenolone, Pregnenolone Sulfate, and 17-alpha-OH-Pregnenolone. We have employed rapid polarity switching, using electrospray ionization, to ensure maximum sensitivity for all analytes. The method provided LOQ ranging from 0.1 pg/mL to 10 pg/mL for the various steroid analytes.
Development of an Automated High Throughput HPLC-ESI-MS Method for Determination of 25-OH-vitamin D2 and D3 in Serum
A fully automated LCMS method for measuring 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 has been developed. An automated liquid-liquid extraction was performed on a robot before the LCMS analysis. The separation was performed on the new biphenyl phase and the to forms was separated under one minute. The method is validated and it has been in routine use for over six month. On one instrument we run up to 600 samples per day. The method has replaced three different immunological methods that were run on three different locations.
Sensitive and Specific Analysis of Plasma Free Metanephrines Using Automated On-line Solid-phase Extraction LC-MS/MS
RECIPE’s CE IVD certified ClinMass® LC-MS/MS Complete Kit – Free Metanephrines in Plasma (MS11000) allows the automated, highly sensitive and selective quantitation of metanephrine, normetanephrine and 3-methoxytyramine in human plasma. After simple and effective protein precipitation, simultaneous extraction, concentration, separation and mass selective detection is achieved using automated on-line SPE LC-MS/MS. The method shows outstanding validation results for all analytes. Close agreement of analyte concentrations with results reported for RCPAQAP proficiency testing scheme could be obtained. With RECIPE’s MS11000 a fast, easy and reliable analysis of plasma free metanephrines has been realized which is very well suited for clinical routine analysis.
Catecholamine Analysis: Evaluation of Method Optimization to Improve Sensitivity and Reduce Limits of Quantitation Using LC-MS/MS
Catecholamines are classic biomarkers for the detection of diseases like hypertension, pheochromocytoma and neuroblastoma. The main target analytes are epinephrine, norepinephrine and dopamine and are traditionally analyzed using liquid chromatography with electrochemical detection. This poster investigates various parts of the method development process to evaluate the sensitivity of LC-MS/MS analysis. A highly sensitive LC/MS system, a Shimadzu Nexera UHPLC coupled to an AB SCIEX 5500 triple quadrupole MS was used for analysis. Method sensitivity in terms of pre-cursor ion selection and MRM transitions, chromatography and solid phase extraction protocols were all evaluated for increased sensitivity.
Comparison of 25-hydroxy Vitamin D Extraction Using Supported Liquid Extraction and Phospholipid Depletion Plate Technology Prior to LC-MS/MS Analysis
Vitamin D analysis has extremely important clinical relevance. Many sample preparation approaches to the extraction of 25-hydroxy vitamin D have been employed prior to LC-MS/MS analysis. This poster compares the use of supported liquid extraction and a novel protein and phospholipid depletion plate, for the extraction of 25-hydroxy-vitamin D from serum. Optimum protocols were compared for recoveries, extract cleanliness, calibration line performance using PBS/BSA, calibrated serum samples and DEQAS external quality control samples. The extraction protocol was ultimately transferred to an SPE automation platform and method performance versus manual processing compared.
A Novel UHPLC-MS/MS Method for the Simultaneous Quantification of 10 Antihypertensive Drugs in Human Plasma
To date the management of resistant hypertension is a critical health problem.
The aim of this work was to validate a UHPLC-MS/MS method of anti-hypertensive drugs (clonidine, atenolol, telmisartan, olmesartan, hydrochlorothiazide, doxazosine, chlorthalidone, ramipril, amlodipine and nifedipine) following FDA guidelines.
After protein-precipitation, supernatants were dried and resuspended. 4 μL of these extracts was injected in Shimadzu Nexera-X2 UHPLC coupled with MS-8050 tandem-mass detector.
Accuracy and precision, and all requested parameters fitted the limits indicated by FDA guidelines.
The validated method is now entering the use in the clinical practice and in a study on the resistant hypertension with encouraging results.
Development of high-throughput Reference Measurement Procedure for Determination of Estradiol-17β with Human Serum using ID-MS
Reference measurement of estradiol (E2) can provide a true value compared with routine methods. At present, there are only three Reference Measurement Procedures (RMP) for serum E2 approved by The Joint Committee for Traceability in Laboratory Medicine. We developed a high-throughput RMP for serum E2.this method is qualifies as RMP and can be used to provide an accurate base to routine methods for E2 providing higher order measurement traceability.
A Rapid and Simple LC-MS/MS Diagnostic Test for the Exclusion of Methylmalonic Acidemia
Methylmalonic acidemia is a congenital metabolic disorder (incidence 1:50,000) that is characterized by an incomplete breakdown of several amino acids and odd-chain fatty acids. As a consequence the metabolite methylmalonic acid (MMA) is elevated in urine and can, therefore, be used for diagnosis. Data demonstrate the suitability of our simple and rapid LC-MS/MS method for the analysis of MMA from urine for identifying methylmalonic acidemia with a quality that is comparable to the gas chromatographic reference method.
Detecting Multiple Lysosomale Storage Disease by a Tandem Mass Spectrometry from Dried Blood Spot: Method Validation in Rouen
The MS/MS based diagnosis 6 Lysosomal Storage Diseases on DBS (Niemann-Pick-A/B, Pompe, Fabry, Gaucher, Krabbe and mucopolysaccharidosis type I diseases) was validated in Rouen. Samples were analyzed on an API 4000 Q-Trap LC-MS/MS system. The electrospray source operated in positive mode, and the analytes were interrogated in multiple reaction–monitoring mode. Data were acquired and processed by Analyst. To support the quality of this assay we use Quality Controls materials provided by the Center for Disease Control (Atlanta, USA). The results showed a robust analytical performance and that this multiplex assay made diagnosis of these diseases more accessible post-natal.
Singulus Pulpitum: Microfluidics Coupled with Mass Spectrometry for Multi-omics and Targeted Assays in Translational Research
Translation medicine is an interdisciplinary science that aims at combining the information taken from bench to bedside. In this process molecules are isolated and identified in discovery and then utilized in the clinical setting as biomarkers of health and disease to better develop therapies. It has become recently apparent that proteomics, metabonomics, lipidomics, and glycomics data combined are necessary to address the challenge of translational research which places strain on available sample and instrument utilization. Due to the complexity of deriving meaningful information from these studies, the development of new analytical technologies is critical. Here we present the utilization of a microfluidic LC coupled with mass spectrometry for both discovery and targeted studies in translational research.
Combination of Raman Spectroscopy and Rapid Evaporative Ionization Mass Spectrometry (REIMS) for the In-situ Identification of Brain Tumors During Surgery
Brain cancers are one of the leading causes of cancer-related deaths in children. In this study, we aimed to create a workflow using an optical method (Raman spectroscopy) followed by an MS-based method (REIMS) for tissue identification within the operating theatre. The workflow also included Sonowand ultrasonic neuronavigational system and conventional histopathology to validate the samples. We have successfully implemented a novel protocol for intraoperative tissue identification and validation. Both REIMS and Raman method showed tissue specificity in the phospholipid region, and proved to be feasible for the distinction of healthy tissue and different brain cancers during the operation. Combining these technologies has the potential for providing important information to brain surgeons in the assessment of tumor margins, leading to an increase in the survival rate of the patients.
Fast Determination of 17-Hydroxyprogesterone by LC-MS/MS for Diagnosis of Congenital Adrenal Hyperplasia
STAT 17-hydroxyprogesterone [17-OHP] is commonly ordered for confirmation of congenital adrenal hyperplasia [CAH] mostly in neonates and pediatric patients. Rapid test turnaround time [TAT] is critical, especially in life-threatening salt-wasting cases. Since conventional LC-MS/MS requires a time consuming manual sample preparation step, we investigated commercially available dispersive pipette extraction tips [DPX tips] to shorten the sample preparation time with the aim of building a STAT test to meet a clinical diagnostic need. Results indicate that DPX tips are effective for 17-OHP analysis, and may reduce sample preparation time by 75%. This rapid method is able to meet the STAT test requirement for CAH confirmation in children.
Quantitative Method of Protein Drugs by Isotope Dilution Inductively Coupled Plasma Mass Spectrometry
We have developed an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. The method was validated and applied to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analyzing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, SI-traceable certification value was obtained.
SpheriCal® - Monodisperse Polyester Dendrimers as Universal Mass Calibrants in Mass Spectrometry
A library of polyester dendrimers were prepared according to previously reported techniques with molecular weights ranging from 700 to 30000 u. MALDI-TOF-MS was used to characterize crude reaction mixtures to verify that each step in the synthesis was driven to completion, and to confirm monodispersity. MALDI-TOF-MS sample preparation utilized 9-nitroanthracene as the matrix, sodium trifluoroacetate as the counter ion and tetrahydrofuran as the solvent.
The dendrimer calibrants exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700-30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.
Evaluation of the Contamination of Trace Elements from Blood Collection Tubes
We evaluated the results of trace elements in serum and whole blood according to using general plain tubes and trace elements free tubes. Whole blood samples and serum samples were collected from 50 healthy persons. Cd, Hg, and Pb in the whole blood samples were not significantly different between trace element free EDTA tube and general EDTA tube. However, all trace elements except Mn and Mo in the serum samples were significantly different between trace element free plain tube and general plain tube. Therefore, the trace element free blood collection tube is essential for the accurate analysis of trace elements.
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Corporate Workshops
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Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thermo Scientific Time Matters. Simplify Workflow Routines Thursday from 8:00 - 8:40 AM in Papageno Hall Discover four medical devices for general clinical use: Thermo Scientific™ Prelude MD™ HPLC, Thermo Scientific™ Prelude LX-4 MD™ HPLC, Thermo Scientific™ Endura MD™ mass spectrometer, and Thermo Scientific™ ClinQuan MD™ Software. Clinical laboratories can use these devices to build their own lab developed tests (LDT). Various example compounds and workflows will be used to demonstrate the robustness and time efficiency of these new medical devices. For in vitro diagnostic use. Not available in all countries.
Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Shimadzu TDMPREP - Centrifugation Free Sample Preparation with Magnetic Beads! Thursday from 2:30 - 3:30 PM in Mozart 1-3 Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Shimadzu New Developments in NBS using DBS for Metabolic Profiling including Steroids Thursday from 2:30 - 3:30 PM in Mozart 1-3 Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Shimadzu New LCMS-8060: Opens Up the Way for New Applications & Fully Automated System including Sample Preparation Thursday from 2:30 - 3:30 PM in Mozart 1-3 Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Thermo Scientific Identifying Unknown Unknowns with High Resolution Accurate Mass MS in Toxicology Thursday from 2:30 - 3:30 PM in Mozart 4-5 Despite the increasing use of modern high resolution mass spectrometers, toxicological analysis is hindered by an inability to identify detected compounds effectively. We will present an advanced computational and database framework leading to the much anticipated increase in coverage of toxicologically relevant compounds and their transformation products, taking into account all the important experimental and calculated information necessary for efficient and reliable identifications. Those methods are exploiting combination of library searching methods, both spectral (mzCloud) and structural (PubChem, ChemSpider), with computational techniques like quantum chemical methods, precursor ion fingerprinting, fragment ion search and others. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Agilent Technologies Introducing the New Agilent StreamSelect LC/MS System Thursday from 2:30 - 3:30 PM in Papageno Hall Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Agilent Technologies Metabolomic Workflows for Clinical Research - Demonstrating a Tool to Address Investigation of Inborn Metabolic Errors Thursday from 2:30 - 3:30 PM in Papageno Hall Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Agilent Technologies Rethink your Protein Sample Preparation Strategy with Agilent AssayMAP Bravo Thursday from 2:30 - 3:30 PM in Papageno Hall
Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Spark Holland Scientific and Instrumental Advances in Dried Blood Spot Analysis Thursday from 2:30 - 3:30 PM in Paracelsus Hall Dried blood spot analysis is emerging as a useful tool for quantitative bioanalysis, particularly for micro volumes of blood or plasma. DBS enables remote sampling in low resource settings or at-home sampling for out-patients. The micro-volume concept helps to reduce lab-animal use and allows use of finger prick instead of venous puncture, making the technique easier applicable for children. However, issues such as the effect of hematocrit on spot size have hindered broad acceptance of DBS for routine bioanalysis. Recent innovations have addressed these issues and will be presented and discussed in this workshop by professor Christophe Stove (Gent University). An introduction to the new DBS autosampler from Spark Holland will be presented by Bert Ooms, principal scientist(Spark Holland). Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Spark Holland Scientific and Instrumental Advances in Dried Blood Spot Analysis Thursday from 2:30 - 3:30 PM in Paracelsus Hall Dried blood spot analysis is emerging as a useful tool for quantitative bioanalysis, particularly for micro volumes of blood or plasma. DBS enables remote sampling in low resource settings or at-home sampling for out-patients. The micro-volume concept helps to reduce lab-animal use and allows use of finger prick instead of venous puncture, making the technique easier applicable for children. However, issues such as the effect of hematocrit on spot size have hindered broad acceptance of DBS for routine bioanalysis. Recent innovations have addressed these issues and will be presented and discussed in this workshop by professor Christophe Stove (Gent University). Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Spark Holland Scientific and Instrumental Advances in Dried Blood Spot Analysis Thursday from 2:30 - 3:30 PM in Paracelsus Hall An introduction to the new DBS autosampler from Spark Holland will be presented by Bert Ooms, principal scientist(Spark Holland). Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Waters UPLC-MS/MS-Based Studies of Vitamin D Metabolism: Lessons Learned from CYP24A1 Knockout Mice and Men Friday from 2:30 - 3:30 PM in Mozart 1-3 Vitamin D function in the body is partly regulated by catabolism of 1,25-(OH)2D and 25-OH-D by CYP24A1. We have developed a sensitive assay using ACQUITY/TQ-S instruments to quantitate metabolites formed by CYP24A1 including 24,25-(OH)2D3, in addition to 25-OH-D. The talk will focus on the application of our assay to a unique combination of samples from CYP24A1-null mice and patients with inactivating mutations to CYP24A1 (Idiopathic Infantile Hypercalcemia) as well as subjects taking vitamin D supplements. We reveal that the ratio of 25-OH-D3:24,25-(OH)2D3 is a useful tool to predict the presence of CYP24A1 mutations as well vitamin D deficiency. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: SCIEX Mass Spectrometry Applications for Clinical Research Friday from 2:30 - 3:30 PM in Mozart 4-5 In this workshop we bring together scientists, clinicians and biochemists that hold an interest in the use of Mass Spectrometry in the areas of Clinical Research as we discuss what can achieved with today’s technology and what we can expect in the future. We have two speakers delivering this workshop. Dr Robert Graham is Senior Lecturer in Clinical Proteomics at the University of Manchester, UK. Dr Graham will discuss the use of advanced accurate mass MS technologies in ovarian cancer biomarker research We also have Dr Michael Wright, from the Prince of Wales Hospital, Sydney, Australia Dr Wright is Senior Leader of clinical Chemistry and Endocrinology. In the workshop he will discuss novel and advanced MS technologies specifically with respect to their use in reducing background and matrix effects in sample analysis. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Phenomenex Workshops on Navigating Sample Prep Techniques & Leveraging the Power of Microsampling Friday from 2:30 - 3:30 PM in Papageno Hall 1. Navigating Sample Preparation Techniques in the Clinical LCMS Laboratory
2. Leverage the Power of Microsampling to Benefit Both Your Patient and Your Laboratory
Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Tecan Workshops on Method Automation Friday from 2:30 - 3:30 PM in Paracelsus Hall 1. Automation and Validation of a SPE Method for the Analysis of 23 Opioids, Cocaine, and Metabolites in Urine with UHPLC-MS/MS
2. Automated LC-MS/MS methods for Clinical Diagnostics - Realized for Therapeutic Drug Monitoring and Vitamins
3. Automated SPE Method Development using StrataTM – X 96-Well SPE Method Development Plates in Conjunction with a Tecan Freedom EVO® Liquid Handling Platform Sean Orlowicz, Manager- Phenomenex Customer experience and commercial insights will be presented for applying automated MS sample preparation in areas of clinical and forensic sample testing, such as therapeutic drug monitoring, DOA- and Vitamin testing. We have pulled together speakers that will present most relevant automated methods and provide guidance on successfully implementing lab established and kit based methodologies. Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Tecan Automation and Validation of a SPE Method for the Analysis of 23 Opioids, Cocaine, and Metabolites in Urine with UHPLC-MS/MS Friday from 2:30 - 3:30 PM in Paracelsus Hall Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Tecan Automated LC-MS/MS methods for Clinical Diagnostics - Realized for Therapeutic Drug Monitoring and Vitamins Friday from 2:30 - 3:30 PM in Paracelsus Hall Notice: Undefined index: abstract_title in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 318 Notice: Undefined index: abstract_body in /home/msacl0017/public_html/include/webpage_structure/include_subtab_content_nextgen.php(1170) : eval()'d code on line 319 Hosted by: Tecan Automated SPE Method Development using StrataTM – X 96-Well SPE Method Development Plates in Conjunction with a Tecan Freedom EVO® Liquid Handling Platform Friday from 2:30 - 3:30 PM in Paracelsus Hall
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