PubMed · 11729570
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P Troiano. Automating the medication distribution process.. https://pubmed.ncbi.nlm.nih.gov/11729570/
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Rapid identification of metabolites of compound X using data dependent scan function of a quadrupole ion trap mass spectrometer and semi-automated metabolite identification software is described. Compound X is metabolized via monooxygenation and desmethylation. LC-ESI-MS spectra obtained, following incubations of Compound X with microsomes in the presence and absence of chemical inhibitors specific for CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2E1, were processed using semi-automated metabolite identification software to extract information and to identify the cytochrome P450 enzymes responsible for metabolite formation. Chemical inhibition data suggests that the primary cytochrome P450 (CYP450) isozyme responsible for the metabolism of compound X is CYP3A4 with a minor contribution from both CYP2D6 and CYP2E1. Additionally, neither CYP2C9 nor CYP1A2 appears to contribute to the metabolism of compound X.
Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X-ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X-ray analysis and bioinformatics stages once diffraction-quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large-scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high-throughput techniques.
A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.