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Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample. [ABSTRACT FROM AUTHOR]

Traditionally, curriculum change is a faculty responsibility. However, a first-year medical student, inspired by previous interactions with cancer patients and disillusioned with her education on the physician's role at the end of life, successfully initiated and sustained an end-of-life curriculum change. This article briefly describes the Preceptorship on End of Life Care and then shifts focus to five key dilemmas associated with student-led curriculum change. These dilemmas include articulating the benefits of student-initiated curriculum change, securing resources for curriculum change, the use of peer versus faculty facilitators, determining whether to create an elective or required curriculum, when to offer the course, and how to transition to new student leadership. Recommendations for students/residents seeking to initiate curriculum change are provided, highlighting the need for a collaborative approach of faculty, community affiliates, and students for sustained success. [ABSTRACT FROM AUTHOR]