HPLC method development trends

HPLC Method Development Trends in Pharmaceutical Analysis... 

Impurity testing of pharmaceuticals is one of the most difficult HPLC method development tasks because of their requirements for both high-resolution and trace analysis in additional to stringent regulatory and reporting guidelines.20,21 There are several recent trends for these methods 22,23... 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

Rao RN, Nagaraju V. An overview of the recent trends in development of HPLC methods for determination of impurities in drugs. Journal of Pharmaceutical and Biomedical Analysis 33, 335-377, 2003. 

Difficulties in using HPLC methods frequently arise when a method is developed in one laboratory and is then transferred for use in another laboratory. To overcome this problem, there is a current trend towards developing method transfer protocols to facilitate a smoother transition. These protocols not only involve additional testing but also may even involve visits (for observation and supervised introduction to carrying out the method) of staff between sites. This seems over elaborate and it remains to be seen whether this trend will continue given that it should not be necessary if a method has been rigorously documented and validated. 

Another current trend that is well underway is the use of more specific analytical instrumentation that allows less extensive sample preparation. The development of mass spectrometric techniques, particularly tandem MS linked to a HPLC or flow injection system, has allowed the specific and sensitive analysis of simple extracts of biological samples (68,70-72). A similar HPLC with UV detection would require significantly more extensive sample preparation effort and, importantly, more method development time. Currently, the bulk of the HPLC-MS efforts have been applied to the analysis of drugs and metabolites in biological samples. Kristiansen et al. (73) have also applied flow-injection tandem mass spectrometry to measure sulfonamide antibiotics in meat and blood using a very simple ethyl acetate extraction step. This important technique will surely find many more applications in the future. 

Kirkland, J.J. Trends in HPLC column design for improved method development. Am.Lab., 1994,26(9), 28K-28R [simultaneous desipramine, doxepin, trimipramine]... 

From today s point of view, the following trends in HPLC in the context of method development/optimization can be discerned ... 

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

Current trends in HPLC favor the development of faster methods that exhibit high resolution and use less mobile phase. 

Different developed analytical method are discussed in this chapter related to the determination of illicit substances in blood (either whole blood, plasma, or serum), OF, urine, and hair. These methods take into consideration the particular chemical and physical composition of the matrix and applies each time a suitable pretreatment to remove interfering and matrix effect, to maximize recoveries and to achieve a suitable enrichment if necessary. For liquid matrices the applications of the most common techniques are considered from simple PPT to SPE and LLE the results of recent works from literature are reported and new trends as online SPE, pSPE, automated LLE (SLE) or MAE are examined. Several stationary phases have been shown to be suitable for determination of illicit drugs Cl8, pentafluorophenyl, strong cation-exchange, and HILIC columns. The trend toward fast chromatography is investigated, both UHPLC and HPLC with appropriate arrangements moreover, results obtained with different ion sources, ESI, A PCI, and APPI are compared. 

Newer approaches to the development of ion exchange separation methods in biochemistry are represented by two general trends (a) medium and high pressure liquid chromatography (MPLC, HPLC), (b) chromatofocusing both trends are briefly discussed here. 

The HPLC-MS/MS and UPLC-MS/MS bioanalytical methods described in this chapter have been utilized for determination of PK parameters for support of drug discovery, preclinical development, and clinical development programs. The trend has been to evaluate PK parameters much earlier in the drug discovery process and the bioanalytical methods have been validated following optimization by DoE. Development and optimization of the assays is performed more efficiently by DoE and require only a fraction of the experiments if each factor were changed separately. The main emphasis has been to develop bioanalytical methods with high selectivity and sensitivity, since sample throughput has never been the bottleneck. The main focus today is on automatization of the whole workflow for a PK evaluation in different species and matrices. 

Ultra high performance liquid chromatography (UHPLC) or ultra performance liquid chromatography (UPLC) is a quite novel technique, but it is one of the most popular nowadays, due to the fact that it is much more rapid than conventional LC. UHPLC systems can work at very high pressures and are compatible with stationary phases with particle sizes of around two micrometers or even smaller. Thus, the development of UHPLC methods [69,77, 80] or the conversion of HPLC to UHPLC methods is a current trend, due mainly to the speed of analysis and low solvent use. 

Multiresidue methods are the most powerful procedures for the analysis of pesticides in environmental, food, and/or feed samples. The maximum residue limits of the pesticides prescribed by health authorities include not only the residues of the parent compounds but their toxic metabolites as well. There is also a trend toward inclusion of residues of highly polar and conjugated metabolites (126). The ever-increasing demands on the quality of food, feed, and environmental samples, and the lowering of the maximum residue limits, require the development of new and more sensitive methods. To distinguish the pesticide signals from the interfering coextractives of samples demands more selective detection. TLC methods alone are not sufficient and should be combined with GLC, HPLC, mass spectrometry, etc. 

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