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2017 - 2018, The U.S. Food and Drug Administration defines as Precision or Personalized Medicine (PM) an innovative therapeutic approach that tailors therapy and prevention on patients based on inter-individual variabilities in molecular or environmental features and in lifestyles. The major goals of PM are to maximize treatment efficacy and to reduce cost, toxicities and therapy failure rates by early identification of patients who might benefit or not of a specific treatment. In this scenario, therapeutic drug monitoring (TDM) is an important laboratory tool for PM because of the possibility to measure several drugs and bioactive molecules in human biological matrices. TDM is based on the hypothesis that in the majority of drugs, there is a relationship between administered dose and circulating concentration of unbound fraction - and between this concentration and observed pharmacological effects. TDM is recommended for drugs with significant inter-individual pharmacokinetic variability and an established relationship between blood concentrations and clinical efficacy and/or toxicity. Moreover, TDM is also advisable in special populations such as pregnant women and children. To date, liquid chromatography and immunometric assay are still considered the standard for molecule measurement in biological fluids; however, in recent years, LC tandem mass spectrometry (LC-MS) is gaining popularity because of the possibility of in-depth and multiplexed analysis with high selectivity and specificity. During this Ph.D. program, we developed several high performance LC (HPLC)- and LC-MS/MS-based approaches for TDM of different drugs measured in various types of body fluids and validated according to EMA and FDA guidelines. In particular, we focused on: 1) TDM of hydroxychloroquine (HCQ) blood concentration, a drug with a wide therapeutic window. Our method was validated on a cohort of patients with Systemic Lupus Erythematosus treated with HCQ and blood concentrations were correlated to several clinical parameters, such quality of life. Moreover, TDM of HCQ was also used to monitor treatment adherence in those subjects. 2) TDM of a commonly used chemotherapeutic agent, the 5-fluorouracil (5-FU), which is known to have a narrow therapeutic window and a high toxicity 3) TDM of a new kinase inhibitor, Ruxolitinib, approved for the treatment of myeloproliferative hematologic disorders. 4) TDM of several drugs, such as caffeine and phenobarbital, in newborns who are at particular risk of uncorrected drug dosage. Due to the need to carry out analyses on very-small volume samples, we validated an analytical method using micro-sampling techniques such as the dried blood spot (DBS) sampling combined with LC-MS/MS analysis. [edited by author]

Hydroxychloroquine (HCQ) is an old antimalarial drug that has proven to be a safe and effective treatment for systemic lupus erythematosus (SLE) and other autoimmune diseases. Since hematic concentration of HCQ is closely related to the therapeutic response, monitoring the levels of the drug and its metabolites in the blood of HCQ-treated patients helps the clinician in the evaluation of partial or complete unresponsiveness to treatment. We developed and validated a novel ion-pairing HPLC-FL method for the simultaneous dosage of HCQ, and its major metabolites desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, after extraction from whole blood. This methodological approach was used for the analysis of real samples obtained from patients affected by SLE and undergoing HCQ treatment. The same samples were also analyzed using a previously validated LC/MS/MS method and data obtained with the two approaches were in substantial agreement with each other. Results presented in this work indicate that this approach can be successfully used to monitor the level of HCQ and its metabolites in the blood of various categories of patients (i.e. low and high responders, or those not adhering to the therapy). Comparison of HPLC-FL and LC/MS/MS data confirmed the efficacy of the proposed method for routine clinical analyses.