Throughput, analytical method development

Salm et al.44 developed a high-throughput analytical method to measure cyclosporine in whole blood. They used a simple SPE procedure, followed by HPLC-MS/MS. An Agilent 1100 liquid chromatograph was coupled with an Agilent Zorbax Bonus C18 reversed-phase column (50 x 2.1 mm, 5 jt/rn particle size). The column temperature was maintained at 70°C in a column oven. The mobile phase consisted of 80% methanol and 20% 40mM ammonium acetate buffer (pH 5.1) delivered isocratically at a flow of 0.4 mL/min. D12 cyclosporine was the IS. 

Dr. Perry G. Wang is currently a principal scientist at Teleflex Medical. His interests include analytical method development and validation, medicated device products, and environmental engineering. His expertise focuses on high-throughput analysis of drugs and their metabolites in biological matrices with LC/MS/MS. 

The methods chemist must be able to make the method work when operating conditions of time, instrument limitations, and other techniques that could baby the method are not used. Normal operating conditions in quality control laboratories require a robust method that can be run routinely by different chemists on different instruments in a high throughput mode. In some cases, the method should be automated to take advantage of greater laboratory efficiency. It is the responsibility of the analytical methods development chemist to build these important elements into the methods. 

In general, however, detailed protocols for the direct analysis of stereochemical features by mass spectrometric means have not been achieved so far. Instead, other analytical methods, such as X-ray analysis or nuclear magnetic resonance (NMR), elegantly address these issues [3]. However, with respect to the analysis of trace compounds and in the context of high-throughput analytical methods, it would be beneficial to develop tools for rapid stereochemical assays by mass spectrometric means or hyphenated techniques [4,5]. 

An overview of several recent applications of UHPLC-MS(/MS) methods for the multi-residue analysis of pesticides in food products has been presented. In order to cope with the necessity of high throughput and fast analysis of pesticide residues in food, all aspects of analytical method development, that is, sample extraction and clean-up, chromatographic analysis, and quantitation and confirmation aspects, must be taken into account. 

In the development of analytical methods one has to consider also cases where a fast response is required, e.g. clinical and forensic chemists or toxicologists need methods which yield results in a few minutes or hours to allow a fast response in cases of poisoning. In this event, accurate quantitative results may be of less importance, but the time from sampling to result may be lifesaving, whereas the throughput (i.e. number of analyses per day) is not so much of concern. 

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

Tandem mass spectrometric methods have demonstrated superb specificity because of their ability to isolate analytes selectively in the presence of endogenous interferences. Attempts to further increase sample throughput led to the idea of using LC/MS/MS without the LC. Traditional chromatographic separations were replaced with flow injection analysis (FLA) or nanoelectrospray infusion techniques. The MS-based columnless methods attracted a lot of attention because of their inherent fast cycle times and no need for LC method development. 

In this chapter we report recent advantages in analytical method hyphenation and chemometric approach applied to drug development, especially related to last trends in pharmaceutical field and to advantages in terms of high-throughputs procedure and instrumentations. 

When developing a CE method for routine analysis of samples in our laboratory, the first three parameters to be checked are reproducibility, sensitivity, and throughput. There is little use for any analytical method that does not meet these three requirements. 

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