Sensitive determination of a pharmaceutical compound and its metabolites in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction.
This paper describes the determination of a drug candidate and two metabolites in human plasma by column-switching LC-MS/MS after protein precipitation. Starting from a standard method with a quantitation limit of 0.5 ng/mL, a highly sensitive assay was developed, employing UHPLC separation and detection on an API 5000 mass spectrometer. The injected plasma equivalent was increased from 6 to 20 μL; conventional column trapping for compound enrichment and removal of matrix constituents was combined with high-pressure analytical separation using small particle columns to improve resolution and signal-to-noise ratio. Quantitation limits were thus lowered to between 5 and 20 pg/mL, offering the possibility to provide bioanalytical support for microdosing studies in humans. Excellent assay quality and robustness were achieved by both methods.
Pharma Research, Non-clinical Safety, DMPK Development, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, Basel, Switzerland. katja.heinig@roche.com
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Heinig
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Wirz
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Bucheli
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Monin
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Gloge
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D057230:Limit of Detection; D008832:Microchemistry / Q000379:methods*; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry*; D010599:Pharmacokinetics; D015203:Reproducibility of Results; D052617:Solid Phase Microextraction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013678:Technology, Pharmaceutical
D004364:Pharmaceutical Preparations