Sensitive and reliable analysis of endogenous compounds is critically important for many physiological and pathological studies. Methods based on LC–MS have progressed to become the method of choice for analyzing endogenous compounds. However, the analysis can be challenging due to various factors, including inherent low concentrations in biological samples, low ionization efficiency, undesirable chromatographic behavior and interferences of complex biological. The integration of chemical derivatization with LC–MS could enhance its capabilities in sensitivity and selectivity, and extend its application to a wider range of analytes. In this article, we will review the derivatization strategies in the LC–MS analysis of various endogenous compounds, and provide applications highlighting the impact of these important techniques in the evaluation of pathological events.

Background: The replacement of hydrogen with deuterium invokes a kinetic isotope effect. Thus, this method is an attractive way to slow down the metabolic rate and modulate pharmacokinetics. Purpose: Enzalutamide (ENT) acts as a competitive inhibitor of the androgen receptor and has been approved for the treatment of metastatic castration-resistant prostate cancer by the US Food and Drug Administration in 2012. To attenuate the N-demethylation pathway, hydrogen atoms of the N–CH₃ moiety were replaced by the relatively stable isotope deuterium, which showed similar pharmacological activities but exhibited favorable pharmacokinetic properties. Methods: We estimated in vitro and in vivo pharmacokinetic parameters for ENT and its deuterated analog (d₃-ENT). For in vitro studies, intrinsic primary isotope effects (K_H/K_D) were determined by the ratio of intrinsic clearance (CL_int) obtained for ENT and d₃-ENT. The CL_int values were obtained by the substrate depletion method. For in vivo studies, ENT and d₃-ENT were orally given to male Sprague Dawley rats separately and simultaneously to assess the disposition and metabolism of them. We also investigated the main metabolic pathway of ENT by comparing the rate of oxidation and hydrolysis in vitro. Results: The in vitro CL_int (maximum velocity/Michaelis constant [V_max/K_m]) of d₃-ENT in rat and human liver microsomes were 49.7% and 72.9% lower than those of the non-deuterated compound, corresponding to the K_H/K_D value of ~2. The maximum observed plasma concentration, C_max, and area under the plasma concentration -time curve from time zero to the last measurable sampling time point (AUC_0–t) were 35% and 102% higher than those of ENT when orally administered to rats (10 mg/kg). The exposure of the N-demethyl metabolite M2 was eightfold lower, whereas that of the amide hydrolysis metabolite M1 and other minor metabolites was unchanged. The observed hydrolysis rate of M2 was at least ten times higher than that of ENT and d₃-ENT in rat plasma. Conclusion: ENT was mainly metabolized through the “parent→M2→M1” pathway based on in vitro and in vivo elimination behavior. The observed in vitro deuterium isotope effect translated into increased exposure of the deuterated analog in rats. Once the carbon–hydrogen was replaced with carbon–deuterium (C–D) bonds, the major metabolic pathway was retarded because of the relatively stable C–D bonds. The systemic exposure to d₃-ENT can increase in humans, so the dose requirements can be reduced appropriately.

AST1306 is an orally active irreversible small molecule inhibitor of EGFR (erbB1), HER2 (erbB2) and HER4 (erbB4) signaling. This is a phase I, open-label, dose-escalation study to evaluate the safety and tolerability, pharmacokinetics (PK), and preliminary anti-tumor effects of oral AST1306. In addition the effects of food on PK was tested.