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Method Optimization for LC-MS Analysis of Vitamin D Metabolite Critical Pairs in Serum

Sigma-Aldrich Supelco
14 Jul 2014

Analysis of vitamin D metabolites has continued to be a topic of interest in recent publications, primarily as biomarkers for possible disease states and vitamin deficiency. There is also an interest in analytical means to differentiate the D2 and D3 forms from the D2 and D3 epimers because of their different degrees of bioactivity. Ascentis® Express F5 gives a fast and efficient analytical method for 25-hydroxyvitamin D and related forms from serum samples.

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Ascentis® Express F5 HPLC Columns

Sigma-Aldrich Supelco

The pentafluorophenylpropyl (PFP) stationary phase of Ascentis® Express F5 provides a stable, reversed-phase packing with electron-deficient phenyl rings due to the presence of electronegative fluorines. In addition to forming p-p and mildly steric interactions, F5 phases also retain compounds by polar interactions. As a result of having both polar and non-polar character, F5 phases can show dual-mode retention behavior, sometimes producing a “U-shaped” retention as a function of acetonitrile content of the mobile phase, with retention increasing at both low and high concentrations of ACN (reversed-phase and HILIC retention modes). Ascentis Express F5 can be used for basic, acidic, or neutral compounds with alternate selectivity from C18. Column Details: Chemistry: Reversed Phase, Hydrophilic Interaction Particle Size: 2.7, 5.0 μm Pore Size: 90 Å Length: 20, 30, 50, 75, 100, 150, 250 mm ID: 2.1, 3.0, 4.6 mm

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HybridSPE®-Precipitation, 96-well Plate, pk of 1

Sigma-Aldrich Supelco

Patent pending HybridSPE – Precipitation (HybridSPE-PPT) technology is a simple and generic sample prep platform designed for the gross level removal of endogenous protein and phospholipid interferences from biological plasma and serum prior to LC-MS or LC-MS/MS analysis. Biological plasma or serum is first subjected to protein precipitation via the addition and mixing of acidified acetonitrile. Precipitated proteins are then removed by centrifugation and the resulting supernatant is loaded on the HybridSPE-PPT 96-well plate or cartridge which acts as a chemical filter that specifically targets the removal of endogenous sample phospholipids. The phospholipid retention mechanism is based on a highly selective Lewis acid-base interaction between the proprietary zirconia ions functionally bonded to the HybridSPE-PPT stationary phase and the phosphate moiety consistent with all phospholipids. The resulting eluent is ready for immediate LC-MS or LC-MS-MS analysis. An alternative “In-well Precipitation” method is available for the HybridSPE-PPT 96-well version in which biological plasma/serum is first added to the 96-well plate followed by acidified acetonitrile (precipitation agent). After a brief mixing/vortexing step, vacuum is applied to the 96-well plate. Because the 96-well version contains a series of low porosity hydrophobic filters/frits, the packed-bed filter/frit assembly acts as a depth filter facilitating the concurrent removal of both phospholipids and precipitated proteins during the extraction process.

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UHPLC and HPLCHigh performance liquid chromatography (HPLC) and ultra high performance liquid chromatography (UHPLC), also known as UPLC, are analytical techniques used to separate, identify and quantitate components of complex mixtures including biological samples such as proteins and lipids as well as chemical mixtures of pesticides, drugs and oils. Both techniques are liquid chromatographic methods but differ by operating pressures (HPLC < 6000 psi < UHPLC ). Components of HPLC and UHPLC systems include columns, detectors, pumps, autosamplers and column heaters. Explore a range of UHPLC and HPLC columns for your specific sample needs including reverse phase, normal phase, ion exchange, HILIC, ion exclusion and size exclusion columns. For more specialized HPLC, explore FPLC, countercurrent LC and simulated moving bed systems. Find the best UHPLC and HPLC equipment in our peer reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.LC-MSLC-MS (liquid chromatography-mass spectrometry) systems and equipment are used for separation and quantitative analysis of complex mixtures, combining liquid chromatography and mass spectrometry. Quantify proteins, contaminants, pesticides or screen for drug metabolites with a high level of sensitivity. LC-MS systems and equipment include reverse phase, normal phase and specialized columns integrated with various MS detectors such as time-of-flight (TOF), quadrupole, orbitrap or ion trap mass analyzers. LC-MS/MS instruments equipped with a qTOF or triple quadrupole analyzer give greater sensitivity and resolving power to your analysis. Find the best LC-MS equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Mass SpectrometryMass spectrometry (MS) is a powerful analytical technique used to identify and quantify molecules based on the mass-to-charge ratio of gas-phase ions. It provides detailed information about the structure, composition, and properties of compounds and is widely used across fields such as environmental monitoring, materials science, drug discovery and development, food and beverage testing, and wider chemical research. Key MS techniques include tandem mass spectrometry (MS/MS), liquid chromatography–mass spectrometry (LS-MS) and inductively coupled plasma (ICP-MS). Choosing from these wide range of techniques and technologies can be a daunting task, so keep up to date with scientific applications, performance expectations, and customer reviews here all in one place. Visit our product directory to receive quotes direct from the manufacturer. BiomarkersBiomarkers are biological markers which can be measured and evaluated to indicate a biological state. The use of biomarkers in research and diagnosis can indicate a normal or disease state or drug response of cells / tissues. Biomarkers include genetic markers, cell surface markers such as antigens, antibodies or receptors and secreted molecules such as cytokines. An assay system is required for identification of biomarkers. :Clinical ChemistryBiochemistry (or clinical chemistry) involves the analysis of bodily fluids using chemical tests. Techniques used include HPLC, chromatography, spectroscopy, mass spectrometry, immunochemical, electrophoresis, turbidometric / spectrophotometric assay, MRI and ISE analysis. Tests are often carried out on plasma or serum but urine (urinalysis) and fecal specimens are also processed.MetabolomicsMetabolomics is the study of small metabolites (the intermediates and products of metabolism). It involves the identification and quantification of cellular metabolites using analytical technologies such as GC, HPLC, NMR, and LC/MS.World CupVitamin D

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Method Optimization for LC-MS Analysis of Vitamin D Metabolite Critical Pairs in Serum