Mass spectrometry samples protein precipitation

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

Quantification of TKIs in patients plasma samples is at present principally performed by liquid chromatography-mass spectrometry (LC-MS) after suitable plasma pretreatment, which implies most generally a protein precipitation with an organic... 

Hou W, Watters JW, McLeod HL (2004) Simple and rapid docetaxel assay in human plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B 804 263-267 Schuhmacher J, Zimmer D, Tesche F, Pickard V (2003) Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry practical approaches to their elimination. Rapid Communications in Mass Spectrometry 17 1950-1957 Shah PW (2001) Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration... 

High-performance liquid chromatography-tandem mass spectrometry method for the determination of gemifloxacin in human plasma was based on the protein precipitation of plasma samples with acetonitrile containing [ C Ha] gemifloxacin as an internal standard. The supernatant was injected onto a PLRP-S column without any further clean-up. The mass spectrometer was operated in positive ion mode, and the ions were detected in multiple reaction-monitoring (MRM) mode. The assay requires 50 gl of plasma and is precise and accurate within the range 10-5000 ng/ml [15]. 

Analyze samples by 15 % SDS-PAGE to check loading and cleavage efficiency. Combine protein thioester containing fractions and verify identity with mass spectrometry. Include always a sample of beads, because protein thioester might precipitate that will in turn reduce yield, if not realized. 

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