Recent application of analytical methods to phase I and phase II drugs development: a review Marcello Locatelli*, Luciana Governatori, Giuseppe Carlucci, Salvatore Genovese, Adriano Mollica and Francesco Epifano ABSTRACT: Drug development is a time-consuming and costly process. It is usually divided into four phases, although it is not always possible to draw a sharp line between the various stages. In phase I and II there are many molecules investigate and it is necessary to analyze all of them in a short period of time, with lower costs, and with high-throughput assay. During phase I relevant chemical–physical parameters like the acid dissociation constant, lipophilicity, solubility and stability must be ana- lyzed. Classic techniques such as ‘shake-flask’ can be used, but instrumental analytical methods such as HPLC may be helpful to improve and enhance the productivity and reproducibility of the results. During phase II the activity of a drug and factors that may have an influence on it, like metabolic profile and transformations, impurities and plasma biding proteins, must be considered. In this field, recent hyphenated analytical methods, such as LC-MS/MS, GC-MS/MS or more complex couplings, can provide more complete information. The aim of this review is to report the processes required for the validation of drug efficacy with reference to the description of ‘classic’ and modern techniques used. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: analysis; analytical chemistry; high-throughput technologies; mass spectrometry; natural products; therapeutic drug monitoring; phase I and phase II drug development; analytical method; hyphenated techniques; legislation Introduction One goal of medicine is to find new drugs to replace ones with limited efficacy. The word ‘drug’ appears for the first time in Homer’ s Odyssey and later in Plato’ s Timaeus. More recently, the World Health Organization (WHO) provided a precise defini- tion for ‘drug’: ‘ all compounds that introduced in a living organ- ism may modify one or more functions’. The drug discovery process successful because of factors ranging from serendipity to a more scientific and rational approach. In the drug develop- ment process, several aspects must to be considered, as well reported and summarized into the empirical ‘4D’ rule (Fig. 1). The most efficient source of drugs is nature. Natural products, or natural-like compounds, coming from microbes, plants and animals (Balunas and Kinghorn, 2005; Tulp and Bohlin, 2004) can be identified by means of analytical techniques such as high-performance liquid chromatography coupled to mass spec- trometry (HPLC-MS), the role of the latter being to provide quan- titative and qualitative information about the analyte in mixtures that are analyzed by liquid chromatography (Korfmacher, 2005). Drug development can be divided into two main steps: (a) dis- covery, which includes target identification and lead optimiza- tion; and (b) development, which comprises pre-clinical and clinical studies. The development of a drug is a long and expen- sive process (Frank, 2003). For 50 molecules that reach phase II, only one will reach the market. Among the major limitations in the process of drug development is the need to select the best biological target and the best chemical entity (Joubert, 2001). The lack of adherence to the therapy by study participants is one of the major causes of unsatisfactory responses to treatment (Robiner, 2005). Adherence or ‘ compliance’ means the degree to which subjects comply with advice given to them by researchers or doctors who are supervising the study. In addition, greater participation of women in trials should be implemented to take into account any differences in efficacy and safety relating to gender (Pinnow et al., 2009; Berlin and Ellenberg, 2009). Pre-clinical studies Pre-clinical studies, ‘phase 0’ , are carried out to obtain prelimi- nary information about the mechanism of action and toxicity * Correspondence to: M. Locatelli, Dipartimento di Scienze del Farmaco, Uni- versità degli Studi “G. d’ Annunzio” Chieti-Pescara, Via dei Vestini 31, 66100 Chieti (CH), Italy. E-mail: m.locatelli@unich.it Dipartimento di Scienze del Farmaco, Università degli Studi ‘G. D’ Annunzio’ Chieti-Pescara, Via dei Vestini 31, 66100 Chieti (CH), Italy Abbreviations used: ACE, affinity capillary electrophoresis; AIDS, acquired immune deficiency syndrome; APCI, atmospheric pressure chemical ioniza- tion; CE, capillary electrophoresis; CI, chemical ionization; CMC, critical mi- cellar concentration; DMSO, dimethylsulfoxide; EI, electron impact; ELISA, enzyme-linked immunosorbent assay; EOF, electoosmotic flow; ESI, electro- spray ionization; FDA, Food and Drug Administration; FID, free induction decay; HPFA, high-performance frontal analysis; HPTLC, high-performance thin-layer chromatography; ICH, International Conference on Harmoniza- tion; IUPAC, International Union of Pure and Applied Chemistry; LADMET, liberation absorption distribution metabolism elimination toxicity; MAD, multiple ascending dose; MEKC, micellar electrokinetic chromatography; MEEKC, microemulsion electrokinetic chromatography; MTD, maximum tolerated dose; NCEs, new chemical entities; QqQ, triple quadrupole mass analyzer; Q, Trap quadrupole-ion trap mass analyzer; WHO, World Health Organization; SAD, single ascending dose; SPME, solid-phase micro- extraction; TOF, time of flight mass spectrometry; VACE, vacancy affinity capillary electrophoresis. Biomed. Chromatogr. 2012; 26: 283–300 Copyright © 2011 John Wiley & Sons, Ltd. Review Received: 4 May 2011, Revised: 13 June 2011, Accepted: 15 June 2011 Published online in Wiley Online Library: 19 August 2011 (wileyonlinelibrary.com) DOI 10.1002/bmc.1674 283