High-throughput quantitative bioanalysis by LC/MS/MS

Abstract

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.

Determination of Serum Cortisol by Isotope-Dilution Liquid-Chromatography Electrospray Ionization Tandem Mass Spectrometry with On-line Extraction

“…In the future, LC-tandem MS systems will probably be more widely used clinically for therapeutic drug monitoring and metabolic profiling (6). Our results demonstrate that this technology -if available -may also be useful for highly specific serum cortisol measurement.…”

“…The use of stable isotope internal standards is applicable to the emerging technique of liquid chromatography-mass spectrometry as well (6). The interfacing of HPLC to mass spectrometers by use of atmospheric pressure ionization is a major milestone for the application of chromatographic techniques in the clinical laboratory.…”

Quantification des opiacés, cocaïniques et amphétaminiques par chromatographie liquide haute performance / spectrométrie de masse en tandem aprés préparation en ligne de l'échantillon

“…Ces dernières années, de nombreux travaux ont conjugué l'utilisation de grands débits sur des colonnes préparatives de grosse granulométrie (25-50 μm) et de « colonnes à accès restreints » [11,12]. Ces grosses particules autorisent de hauts débits de 2 à 4 mL/min (vitesse linéaire 8-10 cm/seconde) qui transforment le flux laminaire en flux turbulent (d'où la notion de turbulent flow chromatography, TFC) sans générer de pressions trop éle-vées : ce courant tourbillonnant facilite les transferts de masses entre les phases [13,14]. Les techniques par TFC sont généralement conduites sur des phases polymériques mélangées hydrophile-hydrophobe qui supportent l'injection de gros volumes de plasma.…”

“…Les protéines ne pénètrent pas dans les pores et sont éluées pendant que les petites molécules y sont retenues. L'élution se fait généralement à contre-courant (back flush) vers la source du détecteur de masse ou vers une colonne analytique si une chromatographie préalable est nécessaire [11][12][13][14][15].…”

Liquid Chromatography-Tandem Mass Spectrometry – Application in the Clinical Laboratory

“…Typical applications of high-performance liquid chromatography (HPLC) in clinical chemistry are, for example, the quantification of urinary catecholamines and metabolites (metanephrines, vanillin mandelic acid, homovanillinic acid), of urinary 5-hydroxyindole acetic acid and serotonin, porphyrins, the measurement of serum vitamin A, E, B 1 , B 6 concentrations, and the monitoring of some drugs (e.g., amiodarone and itraconazole). Other HPLC methods compete with commercially available automated immunoassays (e.g., 25-OH-vitamin D; homocysteine, desoxypyridinolin).…”