December 5, 2022

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HPLC-MS/MS Analysis Sample Preparation- Protein Precipitation To Solid Phase Extraction

HPLC-MS is a widely used analytical procedure to deal with the preparation of small sample volumes. The samples are prepared by solid-phase extraction and analyzed using HPLC analysis and mass spectrometry. A mass spectrometer is a tool that measures the mass-to-charge ratio of ions present in a given sample. Drug development and pharmacokinetics have seen the advanced applications of the HPLC method validation technique. The process is rapid and provides error-free results. The goal of the process is the isolation of a target analyte from the sample followed by the elimination of unwanted constituents from the mixture. The method provides an in-vivo sample extraction technique with minimal solvent use and reduced automation. 

HPLC-MS/MS Procedure 

Protein precipitation is a protocol used in the extraction of pharmaceuticals from samples, like plasma, serum, or blood. During the process of precipitation, solvents or heat denatures the protein, due to which it loses its binding ability.

In the technique, the mixture of any two solvents, for example, methanol, acetonitrile, and formic acid are used to denature the protein due to which it precipitates. The precipitated protein undergoes centrifugation, and the supernatant is extracted. 

Solid-phase extraction is another method used in sample preparation. The column is made of a particular material that has an affinity towards the sample. When the compound passes through the column, it adheres to the wall of the stationary phase. After the removal of impurities, another solvent having properties that will elude the sample compound from the column is used for the extraction. 

Following the extraction, the chromatography column is prepared for loading the sample. A solvent reservoir having a microfilter holds the mobile phase that flows through high pressure, and the high-pressure pump pushes the mobile phase through the column. The column is made of steel, glass, copper, and other materials. The length of the column is 20-25 cm and is packed with coarse packings. Separation takes place in the column, and the detector tests the sample components when they come out of the column. 

The detection system is usually a UV or visible spectrometer as it analyzes even a small quantity of sample. They detect specimens that lie in the range of 200-800nm, and hence minutest biological samples can be analyzed. 

After elution from the HPLC assay, the sample is directed to a mass spectrometer. In the mass spectrometer for LC MS analysis, the compound becomes charged due to ionization. These ionized particles move under high vacuum through mass analyzers. The mass analyzers, part of the mass spectrometer, is quadrupole, which means it consists of four cylindrical rods aligned parallel to each other. Its only function is to separate the charged ions based on their mass to charge ratios when the sample passes through each of these rods. 

The whole LC-MS method development is sensitive, and due to its high specificity wide variety of samples can be analyzed. The role of MS in the quantification of compounds and providing sensitive yet accurate results have increased its use in the process of biofluid extraction.


HPLC testing has excellent reproducibility and provides a good range of separation options for different compounds. The application and performance of the combined LS-MS method need to be recognized for laboratory use while updating existing methodologies for better, highly specific, and accurate results.