title: A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma.
abstract: A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model. This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.
authors.affiliation: Preclinical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb, New Brunswick, NJ 08903, USA. yongjun.xue@bms.com
authors.forename: Y-J
authors.initials: YJ
authors.lastname: Xue
authors.forename: J Billy
authors.initials: JB
authors.lastname: Akinsanya
authors.forename: Jane
authors.initials: J
authors.lastname: Liu
authors.forename: Steve E
authors.initials: SE
authors.lastname: Unger
mesh_terms: D001798:Blood Proteins / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D005240:Feasibility Studies; D006801:Humans; D004364:Pharmaceutical Preparations / Q000097:blood*; D011522:Protons; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D001798:Blood Proteins; D002412:Cations; D004364:Pharmaceutical Preparations; D011522:Protons