ABSTRACT:Bortezomib [N-(2,3-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid] is a potent first-in-class dipeptidyl boronic acid proteasome inhibitor that was approved in May 2003 in the United States for the treatment of patients with relapsed multiple myeloma where the disease is refractory to conventional lines of therapy. Bortezomib binds the proteasome via the boronic acid moiety, and therefore, the presence of this moiety is necessary to achieve proteasome inhibition. Metabolites in plasma obtained from patients receiving a single intravenous dose of bortezomib were identified and characterized by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Metabolite standards that were synthesized and characterized by LC/MS/MS and high field nuclear magnetic resonance spectroscopy (NMR) were used to confirm metabolite structures. The principal biotransformation pathway observed was oxidative deboronation, most notably to a pair of diastereomeric carbinolamide metabolites. Further metabolism of the leucine and phenylalanine moieties produced tertiary hydroxylated metabolites and a metabolite hydroxylated at the benzylic position, respectively. Conversion of the carbinolamides to the corresponding amide and carboxylic acid was also observed. Human liver microsomes adequately modeled the in vivo metabolism of bortezomib, as the principal circulating metabolites were observed in vitro. Using cDNA-expressed cytochrome P450 isoenzymes, it was determined that several isoforms contributed to the metabolism of bortezomib, including CYP3A4, CYP2C19, CYP1A2, CYP2D6, and CYP2C9. The development of bortezomib has provided an opportunity to describe the metabolism of a novel boronic acid pharmacophore.

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Novel opioid interferences were observed during the development of a high-resolution liquid chromatography-mass spectrometry urine drug testing method for 47 analytes from multiple drug classes. The interferences affected both analytes and internal standards and were only observed when the method was challenged with patient samples. Some interferences were attributable to isomeric opioid metabolites not previously reported while others were due to interference from in-source dissociations or 13C isotopic contributions from known opioid metabolites not typically monitored as analytes. Based on patient drug profiles, known and inferred metabolism, accurate mass, retention time and MS/MS spectrum, the putative identity of each interference was assigned and later confirmed, when possible, using an authentic standard. Opioids are some of the most frequently monitored analytes in urine drug testing laboratories. Because of the potential for co-purification, co-chromatography and spectral similarity, it is anticipated that the reported opioid metabolite interferences could be present with other method conditions and instrument platforms. The objectives of this work are to raise awareness of these interferences and emphasize the importance of evaluating patient samples for potential interferences during method development.

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