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Rapid, Sensitive, and Quantitative LC/MS/MS Determination of Digitoxin and Digoxin in Plasma

Sigma-Aldrich Supelco

Using a Simple Procedure for Simultaneous Clean-up of Phospholipids and Proteins

18 Jan 2016

This application note describes the development of an LC/MS/MS method to measure digoxin and Digitoxin levels in the body, as these glycosides are used for treating heart conditions. Current methods using immunoassays are not selective between the two compounds due to the similarities between the molecules.

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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. 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Explore cardiovascular disease research and diagnostic tools in our peer-reviewed product directory; compare products, check reviews, and get pricing directly from manufacturers.HPLCHigh-performance liquid chromatography (HPLC) is a technique for separating, identifying, and quantifying components in mixtures. It is widely used in chemical, pharmaceutical, and environmental analysis to ensure purity and identify unknown compounds. Explore the best HPLC systems in our peer-reviewed product directory; compare products, read reviews, and get pricing directly from manufacturers.

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