Method validation and hyphenated techniques: recent trends and future perspectives

The recent applications of HPLC in bioanalytical chemistry provided the development of sensitive, selective and robust methods. All these procedures require validation to obtain reproducible analysis and analyze samples. The main international regulatory agencies, e.g., Food and Drug Administration (FDA), European Medicine Agency (EMA), and Agência Nacional de Vigilância Sanitária (ANVISA) edit three level of references for Guidelines setting up the basic parameters to validate bioanalytical assays. None of them covers the validation process successfully because the matrix effect (ME) is sometimes missed or partially included in the regulatory documents. In fact, the ME can affect the quality of results and still represents the “Achilles heel” in bioanalytical chemistry using the hyphenated techniques. Actually, several papers report the potential drawbacks of ME, and provide suitable strategies to suppress this phenomenon. Furthermore, different factors, e.g., the co-precipitation of biological samples, interfaces, chromatography and spectrometric conditions, can affect the bioanalytical assays and validation processes at different steps. This chapter discusses the potential strategies to suppress the ME and highlights the importance of validation process according to International Guidelines. In this attempt, the best solution should be the combination of on-line techniques miniaturized systems, new ionisation sources, chemometrics approach, high performance liquid chromatography based and hyphenated techniques.