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High Sensitivity Measurement of Pancreatic Polypeptide and Its Variant in Serum and Plasma by LC-MS/MS
Aliquots of serum or plasma samples are combined with stable isotope labeled internal standard. Pancreatic polypeptide (PP) and its truncated variant PP3-36 are enriched by incubation with anti-PP antibody conjugated to magnetic beads. Peptides are eluted from beads in acidic buffer and the samples analyzed using liquid chromatography coupled with tandem mass spectrometry. Instrumental analysis of PP and PP3-36 is performed using electrospray ionization ESI in positive ion mode and multiple reaction monitoring (MRM) acquisition. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitative Organic Acids in Urine by Two Dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS)
Seventy-six organic acids in urine specimens are determined with quantitative two dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS). The specimen is treated with urease to remove urea then derivatized to form pentafluorobenzyl oximes (PFBO) of oxoacids. The sample is then treated with ethyl alcohol to precipitate proteins and centrifuged. After drying the supernatant, the organic acids are derivatized to form volatile trimethylsilyl (TMS) derivatives for separation by capillary two dimensional Gas Chromatography (GCxGC) with temperature programming and modulation. Detection is by Time of Flight ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Mass Spectrometry in Clinical Laboratory: Applications in Biomolecular Analysis
Mass spectrometry (MS) is a technique that can identify analytes on the basis of mass-to-charge (m/z) ratio. Although this technique has been used in research and specialized clinical laboratories for decades, however, in recent years, MS has been increasingly used in routine clinical laboratories. MS, especially when coupled to gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of MS include simultaneous analysis of multiple analytes (>100) and generally without need for specialized reagents. Commonly measured analytes by MS include drugs,...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine by LC-MS/MS. Method for Analyzing Isomers Without Chromatographic Separation
Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentati...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Metanephrine and Normetanephrine in Urine Using Liquid Chromatography-Tandem Mass Spectrometry
Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas–catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/M...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry
We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Insulin-Like Growth Factor 1 in Serum by Liquid Chromatography High Resolution Accurate-Mass Mass Spectrometry
Insulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide hormone which acts as the principal mediator of the effects of growth hormone (GH). Due to a wide variability in circulating concentration of GH, IGF-1 quantitation is the first step in the diagnosis of GH excess or deficiency. Majority (>95 %) of IGF-1 circulates as a ternary complex along with its principle binding protein insulin-like growth factor 1 binding protein 3 (IGFBP-3) and acid labile subunit. The assay design approach for IGF-1 quantitation has to include a step to dissociate IGF-1 from its ternary complex. Several commercial assays employ a bu...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantitation of Insulin Analogues in Serum Using Immunoaffinity Extraction, Liquid Chromatography, and Tandem Mass Spectrometry
We present an immunoaffinity extraction and LC-MS/MS method capable of independent identification and quantitation of native sequence insulins (endogenous, Insulin Regular, Insulin NPH), and analogues Glargine, Lispro, Detemir, and Aspart with an analytical sensitivity for endogenous insulin of between 1 and 2 μU/mL in patient serum samples. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry
We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directl...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Direct Measurement of Free Estradiol in Human Serum and Plasma by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry
We describe a direct method of measurement of free estradiol using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum aliquots and internal standards are extracted by liquid-liquid extraction using methyl-tert-butyl ether (MTBE) followed by derivatization with dansyl chloride. An API 5500 mass spectrometer operated in positive electrospray mode is used for detection. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Simultaneous Quantitation of Estradiol and Estrone in Serum Using Liquid Chromatography Mass Spectrometry
Accurate measurement of the endogenous estrogens, estrone (E1) and estradiol (E2), is important in the clinical diagnosis and monitoring of multiple disorders. Typically, given the efficacy and low cost, radioimmunoassays (RIA) and enzyme-linked immunoassays (EIA) are used to quantify these hormones in biological samples. Unfortunately, at low levels these assays lack the necessary sensitivity and specificity for diagnosis of certain disorders in adult and pediatric endocrinology and oncology. In response to this need, we developed a fast and sensitive high performance liquid chromatography negative electrospray ionization...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 56™ Mass Spectrometer
Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple ...
Source: Springer protocols feed by Imaging/Radiology - November 27, 2015 Category: Radiology Source Type: news

siRNA Nanoparticles for Ultra-Long Gene Silencing In Vivo
Small interfering RNA (siRNA)-mediated gene silencing has shown prominent therapeutic effects in treating various diseases. However, adequate delivery and persistent gene silencing remain challenging. A nanoparticle-based delivery system which assembled by layering siRNAs between protease degradable polypeptides to show ultra-long gene silencing effect in vivo is developed. Gold nanoparticle is used as a scaffold for its unique properties including uniform size, biocompatibility, ready synthesis, and easy functionalization. A simple layer-by-layer fabrication approach, based on the electrostatic interaction between positiv...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news

Whole-Body Scanning PCR, a Tool for the Visualization of the In Vivo Biodistribution Pattern of Endogenous and Exogenous Oligonucleotides in Rodents
Characterizing the in vivo biodistribution pattern and relative expression levels of oligonucleotide-based molecules such as mRNA, miRNA, siRNA, and anti-miRNAs in animal models, could be a helpful first-step in the successful development of therapeutic oligonucleotides. Here we describe a simple procedure called “Whole-Body Scanning PCR” (WBS-PCR), which combines the power of PCR with that of imaging. WBS-PCR relies on 384 well-defined extractions across a mouse whole-body section followed by a single dilution step which renders the lysates compatible with various qPCR-based assays. The in vivo biodistribution...
Source: Springer protocols feed by Imaging/Radiology - October 11, 2015 Category: Radiology Source Type: news