Rapid Method Development

A stepwise procedure for rapid HPLC-MS/MS method development that was described recently by Xu et al. [10] is shown in Fig. 13.5. As shown in Fig. 13.5, there are three proposed checkpoints that are used to test the assay before it is used for the actual PK samples. If the assay (HPLC-MS/MS procedure) passes a 

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D. Wu, M. Berua, G. Maier and J. Johnson, An automated multidimensional sa eening approach for rapid method development in high-performance liquid cliromatography , 7. Pharm. Biomed. Anal. 16 57-68 (1997). 

Method development remains the most challenging aspect of chiral chromatographic analysis, and the need for rapid method development is particularly acute in the pharmaceutical industry. To complicate matters, even structurally similar compounds may not be resolved under the same chromatographic conditions, or even on the same CSP. Rapid column equilibration in SFC speeds the column screening process, and automated systems accommodating multiple CSPs and modifiers now permit unattended method optimization in SFC [36]. Because more compounds are likely to be resolved with a single set of parameters in SFC than in LC, the analyst stands a greater chance of success on the first try in SFC [37]. The increased resolution obtained in SFC may also reduce the number of columns that must be evaluated to achieve the desired separation. 

For discovery PK samples, rapid method development is required. For HPLC/MS/MS assays, method development can be achieved within 2 hr if no unusual problems are encountered. Xu et al.101 described a process for rapid method development as part of the discovery PK paradigm. As shown in Figure 7.4, the systematic process is based on using protein precipitation as the sample clean-up step and generic HPLC conditions for the HPLC/MS/MS assay. 

UHPLC (600 to 1000 bar) Significant runtime reduction for ultra-fast separation minimal solvent consumption Five-fold increase in speed for SIM Significantly higher efficiency for most complex separations Higher mass sensitivity Rapid method development... 

Zhao, Y., Woo, G, Thomas, S., Semin, D., Sandra, P. Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry. J. Chromatogr. A 2003, 1003, 157-166. 

Another potential application might be rapid method development screening—looking at four or eight columns under a certain set of conditions to investigate selectivity and retention. 

Several advantages offered by CE, such as a high efficiency, rapid method development, simple instrumentation, and low sample consumption, are the main reasons for its success in a variety of fields. UV-VIS spectrophotometry is probably the most widely used detection technique with CE because of the simplicity of the on-line configuration. However, its sensitivity, directly related to the optical path length afforded by the I.D. of capillaries, which is in the micrometer range, is low, and it remains the major bottleneck of this technique (see... 

Fig. 13.5 A schematic diagram showing the stepwise procedure for rapid method development of HPLC-MS/MS methods for discovery PK assays. Adapted from [10], with permission from the American Chemical Society.

The use of CE methods for routine quality control of synthetic or recombinant peptides-proteins necessitates optimization strategies for rapid method development. Ideally, the methods should be simple, fast, and robust. Because capillary electrophoresis in the zone format is the most simplistic, initial efforts should be directed toward the use of a simple buffer system [61]. The high efficiency and reproducibility in protein-pep-tide separations demands that interactions between the analyte and capillary wall be neglible. The use of low-pH buffers generally results in enhanced reproduciblity, and hence ruggedness, as slight variations in the capillary surface will have little impact on the already suppressed EOF. 

In summary, LC-MS offers excellent sensitivity for many classes of pharmaceutical compounds. Due to the fact that MS is becoming more routine (i.e., it is essentially another detector), LC-MS should be the first consideration for all cleaning verification assays that are less than 0.1 pg/swab. In the citations outlined above, LC-MS has been shown to offer excellent sensitivity and specificity for the analytes of interest. The mass spectrometric conditions can be optimized in a flow injection mode to allow for rapid method development. All LC-MS analytical methods were validated in a way consistent with the requirements outlined in Table 15.3. The applications cited utilized LC-MS because of the sensitivity requirements of the safety acceptance limits however, if the molecule of interest poses unique detection challenges such as a poor chromophote, LC-MS should be considered for assays at the level of 1.0 ig/unit area, or less. Above 1.0 xg/unit area there may be more attractive options for these swab determinations. 

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