Sampling method development

As shown in Figure 7.2, most assays involve a common series of steps that must be completed in order to report results. These steps include sample receipt, method development, sample preparation, analysis, data processing, and data reporting. While most researchers focus on speeding the analysis step, any of these steps can become bottlenecks. Thus it is important to optimize the whole process. 

With decreasing LC run times, sample pretreatment, and the associated method development, sample analysis may be the rate-limiting step in bioanalytical assays. Both semi-automated and automated... 

A diode array detector is well suited to achieve these goals. Full range spectra should be collected for the method development samples and evaluated with two-dimensional or three-dimensional visualization to determine the best detection wavelength. 

Method development samples (appropriate DS batches, formulations (active and placebo), and stability samples) are available. 

Figure 12.4 Systematic methods development samples, (a) Heat plasma blank (b) plasma blank spiked with standards (c) 80% SFE window (d) heated plasma/standards (e) SFE win-dowed/plasma standards (f) SFE windowed patient sample.

Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difficult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. 

Isolated samples of these compounds are recommended for inclusion in the method development sample set, where applicable. 

Because of availability and time constraints, some of the peaks or samples listed may not be included in the method development sample set. However, during validation these peaks should be considered for the specificity assessments if available. 

It is common upon scale-up that recrystallization steps will be eliminated from the synthetic route therefore, it is prudent to include the mother liquor from the bond-forming step in the method development sample set even if there is evidence that the major synthetic by-products are being purged in the recrystallization steps. 

The selection of the set of samples that best covers the impurity and degradation profiles of the API is critical to successful method development. For a given API there are several ways to design the method development sample set some of which are more advantageous, but incur more risk in terms of the ability to track peaks from column to column and condition to condition. This section will address method development sample set design strategies, experimental concerns, and trade-offs. 

Impurity enhancement techniques such as fraction collection and phase equilibrium purification can be used to provide enriched samples for use in the method development process.23 When using the fraction collection approach, one or more cuts (fractions) of the chromatographic separation of a bulk lot or mother liquor are isolated. The excess solvent in these fractions is then evaporated to achieve the desired concentration enhancement. These fractions typically contain extraneous peaks because of the presence of salts in the mobile phase or sample degradation during the concentration step. The salts can be removed by extraction and/or a LC cleanup step. To insure that these extraneous peaks/artifacts are not identified as key peaks for separation, the original bulk lot or mother liquor should be included in the method development sample set. The same holds true for phase-equilibrium-purification supernatants. 

For a given API, there can be numerous ways to mix and match the available samples to construct the method development sample set. During early stages of development, only a few isolated intermediates and purposeful degradation samples may be available, making sample set selection relatively easy. Later in development, more isolated samples of the major degradants... 

Frequently, the objective of preparative HPLC as applied to natural products chemistry is to isolate a metabolite of unknown structure in sufficient quantity and purity to enable structure elucidation and biological assay. Alternatively, the objective might be to scale up the isolation of a known compound to provide material for further biological evaluation or for chemical studies. The purpose for which the compound is required will dictate the compound purity required, and this in turn may dictate the strategy adopted. Many factors may affect the success of a preparative HPLC-based natural product isolation. Some principal considerations—chromatographic method development, sample preparation, scale-up, and fractionation/fraction work-up—are now discussed. Examples are included wherever possible to illustrate the points in question and to emphasize the enormous versatility and resolving power of preparative HPLC. 

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