Analysis of Complexation Interactions between Metal Ions and Drugs under Pseudo-physiological pH Conditions by a High-throughput Screening Method Using a Solid-phase Extraction Cartridge

We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches.

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Gas Chromatographic/Mass Spectrometric Confirmation of Leucomalachite Green in Catfish (Ictalurus punctatus) Tissue

Journal of AOAC International ◽

10.1093/jaoac/78.4.971 ◽

1995 ◽

Vol 78 (4) ◽

pp. 971-977 ◽

Cited By ~ 27

Author(s):

Sherri B Turnipseed ◽

José E Roybal ◽

Jeffrey A Hurlbut ◽

Austin R Long

Keyword(s):

Solid Phase Extraction ◽

Ictalurus Punctatus ◽

Solid Phase ◽

Mass Spectrometric ◽

Phase Extraction ◽

Neutral Alumina ◽

Ms Analysis ◽

Solid Phase Extraction Cartridge ◽

Leucomalachite Green ◽

Chromatographic Mass

Abstract A gas chromatographic/mass spectrometric (GC/MS) method was developed to confirm the presence of leucomalachite green (LMG), a metabolite of the triphenylmethane dye malachite green (MG), in catfish tissue. Residues were isolated according to a previously described liquid chromatographic (LC)A/IS spectrometric analysis of MG and LMG in fish. In our isolation procedure, analytes are extracted from tissue with acetonitrile–buffer, partitioned into CH2CI2, and applied to neutral alumina and propylsulfonic acid solid-phase extraction cartridges. Before GC/MS analysis, extracts prepared for the LC determinative method are eluted from a cyano solid-phase extraction cartridge, extracted into organic solvent, and concentrated for GC/MS analysis. Selected ion monitoring was performed by using 5 diagnostic ions (m/z 330,329,253,210, and 165) of LMG. The method was validated by confirming LMG in tissue fortified with mixtures of MG and LMG (5 and 10 ng/g each) and in tissue from fish that had been exposed to low levels of MG.

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