Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE.
Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States.
D000319:Adrenergic beta-Antagonists / Q000032:analysis / Q000097:blood; D001993:Bronchodilator Agents / Q000032:analysis / Q000097:blood; D002853:Chromatography, Liquid; D004809:Ephedrine / Q000032:analysis / Q000097:blood; D006634:Histamine H1 Antagonists / Q000032:analysis / Q000097:blood; D006801:Humans; D007917:Leprostatic Agents / Q000032:analysis / Q000097:blood; D008790:Metoprolol / Q000032:analysis / Q000097:blood; D004364:Pharmaceutical Preparations / Q000032:analysis*; D011786:Quality Control; D015203:Reproducibility of Results; D012293:Rifampin / Q000032:analysis / Q000097:blood; D012371:Robotics; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D016593:Terfenadine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood
D000319:Adrenergic beta-Antagonists; D001993:Bronchodilator Agents; D006634:Histamine H1 Antagonists; D007917:Leprostatic Agents; D004364:Pharmaceutical Preparations; D012997:Solvents; D016593:Terfenadine; C093230:fexofenadine; D008790:Metoprolol; D004809:Ephedrine; D012293:Rifampin