Erin Strickland

Capillary electrophoresis-mass spectrometry determination of morphine and its isobaric glucuronide metabolites

The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide (M3G) and morphine-6-beta-d-glucuronide (M6G) is useful for therapeutic drug monitoring and forensic identification of drug use. In particular, capillary electrophoresis with mass spectrometry (CE-MS) represents an attractive tool for opioid analysis. Whereas volatile background electrolytes in CE often improve electrospray ionization for coupled MS detection, such electrolytes may reduce CE separation efficiency and resolution. To better understand the effects of background electrolyte (BGE) composition on separation efficiency and detection sensitivity, this work compares and contrasts method development for both volatile (ammonium formate and acetate) and nonvolatile (ammonium phosphate and borate) buffers. Peak efficiencies and migration times for morphine and morphine metabolites were optimal with a 25mM ammonium borate buffer (pH=9.5) although greater sensitivities were achieved in the ammonium formate buffer. Optimized CE methods allowed for the resolution of the isobaric morphine metabolites prior to high mass accuracy, electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS detection applicable to the analysis of urine samples in under seven minutes. Urine sample preparation required only a 10-fold dilution with BGE prior to analysis. Limits of detection (LOD) in normal human urine were found to be 1.0μg/mL for morphine and 2.5μg/mL for each of M3G and M6G by CE-ESI-QTOF-MS. These LODs were comparable to those for CE-UV analysis of opioid standards in buffer, whereas CE-ESI-QTOF-MS analysis of opioid standards in buffer yielded LODs an order of magnitude lower. Patient urine samples (N=12) were analyzed by this new CE-ESI-QTOF-MS method and no significant difference in total morphine content relative to prior liquid chromatography-mass spectrometry (LC-MS) results was found as per a paired-t test at the 99% confidence level. Whereas the LC-MS method applied to these samples determined only total morphine content, this new CE-ESI-QTOF-MS method allowed for species differentiation in addition to total morphine determination. By this method, it was found that M3G and M6G metabolites were present in a 5:1 concentration ratio, on average, in patient samples. Therefore, the CE-ESI-QTOF-MS method not only allows for total morphine concentration determination comparable to established LC-MS methods, but also allows for differentiation between morphine and its trace glucuronides, yielding additional biochemical information about drug metabolism.

Rapid Enzymatic Hydrolysis Using a Novel Recombinant β-Glucuronidase in Benzodiazepine Urinalysis

Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation.

Impact of β-Glucuronidase Mediated Hydrolysis on Haldol® Urinalysis

Reports have suggested that patients with mental health disorders including major depressive disorder and schizophrenia have dramatically low adherence levels to prescribed medications. Patients on haloperidol (Haldol®) therapy, regardless of their disease, were found to have higher adherence levels-though still strikingly low. This work shows that high levels of the glucuronidated form of haloperidol are present in patient urine samples. Time-of-Flight (TOF) mass spectrometry experiments are consistent with both the presence of haloperidol glucuronide and that hydrolysis of haloperidol patient urine samples leads to significantly increased concentrations of free haloperidol. Urine samples collected from patients prescribed haloperidol were tested with and without hydrolysis revealing a significant increase in the number of patients testing positive when the samples were hydrolyzed before analysis. These data demonstrate that hydrolysis greatly improves the sensitivity and consistency of results for patients on haloperidol therapy resulting in positivity data that strongly correlates with the dosage form administered.