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

Abstract

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.

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the analysis of paroxetine in human plasma

“…In our laboratory, we have successfully used Quadra™ 96 for simultaneous protein precipitation , solid-phase (Shou et al, , 2002cChen et al, 2002;Eerkes et al, 2002a;Naidong et al, 2002a) and liquid-liquid (Eerkes and Naidong, 2003) extraction of 96 samples. Inadequate sensitivity was obtained when protein precipitation extraction was attempted for which the plasma size is typically limited to <0.10 mL.…”

Simultaneous determination of β‐blockers in human plasma using liquid chromatography–tandem mass spectrometry

“…Several gas chromatographic (Quaglio et al, 1993;Black et al, 1996;Hartonen and Riekkola, 1996;Amendola et al, 2000) and liquid chromatographic (Naidong et al, 2002;Maurer et al, 2004;do Carmo Borges et al, 2005;Josefsson and Sabanovic, 2006;Keski-Rahkonen et al, 2007;Li et al, 2007;Ding et al, 2007;Fanali et al, 2007) methods coupled to mass spectrometry, including tandem mass spectrometry (MS-MS), have been developed in order to determine the levels of acebutolol, labetalol, metoprolol and propranolol in biological samples. These methods utilized liquid-liquid extraction (LLE; Amendola et al, 2000;do Carmo Borges et al, 2005;KeskiRahkonen et al, 2007;Li et al, 2007) or off-line solidphase extraction (SPE; Quaglio et al, 1993;Black et al, 1996;Hartonen and Riekkola, 1996;Naidong et al, 2002;Maurer et al, 2004;Josefsson and Sabanovic, 2006) for specimen preparation and concentration prior to chromatography.…”

Pharmacokinetics of Ephedrine and Pseudoephedrine after Oral Administration of Kakkonto to Healthy Male Volunteers

“…Serum ephedrine and pseudoephedrine concentrations were measured using several methods with modifications [12][13][14][15][16][17][18][19] . Briefly, the d3 -form of each drug ／ （100 mL；1 ng／mL）was mixed with 1 mL serum in water, 3 mL 0.2 N HCl saturated with NaCl, and 5 mL n-hexane：ethyl acetate（9：1）for 5 min.…”