"Investigation of the pre-analytical treatment of biological fluids for the determination of biomarkers by LC-MS/MS spectrometry"

Abstract

Biomarkers are molecules whose determination in clinical laboratories has great diagnostic and clinical value. The determination of biomarkers can be performed in various biological fluids, with blood and urine being the most widely used. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) is one of the most widely applied analytical techniques in biomarker analysis, due to its advantages over others, such as high specificity and sensitivity, speed, small sample volume, simultaneous determination of multiple analytes, determination of thermally unstable and non-volatile compounds, the avoidance of derivatization of the analytes, etc.

The pre-analytical processing of biological samples is a mandatory step for the determination of various biomarkers by liquid chromatography-mass spectrometry. Its importance is decisive, and ensures reliable results for the correct diagnosis, prediction, monitoring of diseases and determination of drug levels.

The purpose of this work is to review and present selected sample preparation techniques of biological fluids suitable for the analysis of biomarkers, applying the LC-MS/MS methodology.

Protein precipitation, solid phase extraction and liquid-liquid extraction are the mainly used pretreatment techniques. Moreover, new techniques or evolution of classic techniques are used today, allowing the analysis of smaller volumes of biological fluids by using smaller volumes of elution solvents. These include solid phase microextraction, salting-out assisted liquid–liquid extraction, supported liquid extraction, fabric phase sorptive extraction, stir bar sorptive extraction, microextraction by packed sorbent and monolithic spin column extraction. Additional technologies, such as phospholipid removal plates, magnetic beads, turbulent flow extraction, as well as, the use of newer materials, such as carbon nanotubes, restricted access materials, immunosorbents, molecularly imprinted polymers and aptamers, enable greater recovery of analytes and removal of interfering substances, and resulting to more specific and sensitive biomarkers identification and quantification.

In conclusion, the selection of the most suitable sample preparation technique for the determination of specific biomarker, should be based on the evaluation of the following parameters: the chemical properties of the biomarker, the cost and the time required to perform the specific analysis as well as the available laboratory equipment. Furthermore, the performance offered by each technique in terms of sensitivity and specificity is probably the most important factor that will affect the choice of the applied technique for routine laboratory analyses.

PAPACHARISIS Vasilios