PO0026 Utilizing Purifiedβ-Glucuronidase and Arylsulfatase to Accurately Quantitate Metabolites in Human Urine

Several metabolites are known to be glucuronidated and sulfated in the human body. Monitoring the aglycones with the gluruco-nidated and sulfated metabolites creates six additional masses to monitor on tandem mass spectrometry, which can limit the total number of metabolites being monitored. In addition, the conjugated standards are very expensive and often not available. Herein,we report the use of purifiedβ-glucuronidase (IMCSzyme®) and recombinant arylsulfatase that effectively cleave glucuronidesand sulfates at neutral pH with no detectable CYP activity or esterase activity. The initial studies explored urinary corticosteroidmetabolites on tandem mass spectrometry before and after cleaving glucuronides and sulfates with the purified enzymes. Among corticosteroids, glucuronidated allo-tetrahydrocortisol (aTHF) has been reported to be recalcitrant substrate for enzymatic decon-jugation. We demonstrate rapid hydrolysis within 10 minutes, while the reported 4 hour incubation using crude snail enzyme is in-adequate for recovering the higher levels of a THF glucuronide (1,2).

PO0016 Rapid Hydrolysis of Glucuronidated Opiates and Opioids in Urine Using IMCSzyme®

• Recreational opiate and opioid use has become an increasing cause of deaths due to drug over dose.
• Codeine-6-β-D-glucuronide is one of the more challenging drug metabolites for β-glucuronidases to hydrolyze.
• >80% hydrolysis of 30,000 ng/mL codeine-6-β-D-glucuronide was achieved in 15 minutes using only 3000 units of IMCSzyme (60µL per 30µL urine).

PO0015 The Importance of pH in β-Glucuronidase Hydrolysis Efficiency

•Different enzymes have different optimal hydrolysis pH
•pH optima are substrate dependent
•Urine samples have a range of pH 4.0-9.0 and require buffers to adjust pH for the most efficient hydrolysis
•Comparison of twoβ-glucuronidase hydrolysis efficiencies at their optimal pH in both fortified synthetic urine and authentic patient urine samples

PO0014 Rapid Enzyme Hydrolysis Using a Novel Recombinant β-glucuronidasein β-agonists Meat Analysis

PO0013 Enzyme Activities (Fishman units) Correlate Poorly with Hydrolysis Efficiencies

•Activity of five differentβ-glucuronidases was tested via a phenolphthalein-glucuronide chromogenic assay.
•Hydrolysis efficiency of ten drugs was monitored for all fiveβ-glucuronidases at fivetime points.
•Activity level measured with phenolphthalein-glucuronide did not correlate well withdrug hydrolysis efficiency.

JA0006 Variation in Enzymatic Hydrolysis Efficiencies for Amitriptyline and Clyclobenzaprine in Urine

A collaborative study was conducted to investigate discrepancies in recoveries of two commonly prescribed compounds, amitriptyline and cyclobenzaprine, in patient urine samples when hydrolyzed with different enzymes from different sources. A 2- to 10-fold increase in analyte recoveries was seen for patient samples hydrolyzed using a recombinant β-glucuronidase (IMCSzyme™) over samples hydrolyzed with β-glucuronidase from Haliotis rufescens. We report outcomes from four commercially available β-glucuronidase enzymes (IMCSzyme™, Patella vulgata, Helix pomatia and H. rufescens) on patient samples that tested positive for amitriptyline and cyclobenzaprine. Our results confirm reduced hydrolysis of glucuronides by β-glucuronidases isolated from mollusks, but near complete conversion when using the recombinant enzyme. Our premise is that systematic differences in hydrolysis efficiencies due to varying substrate affinity among enzyme subtypes potentially impacts accuracy and reliability of measurements.

JA0007 Internal Hydrolysis Indicator for Sample Specific Monitoring of Beta-Glucuronidase Activity

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.

JA0008 Comparison of Purified β-glucuronidases in Patient Urine Samples Indicates a Lack of Correlation Between Enzyme Activity and Drugs of Abuse Metabolite Hydrolysis Efficiencies Leading to Potential False Negatives

Pain management laboratories analyze biological fluids (urine, saliva or blood) from patients treated for chronic pain to ensure compliance and to detect undisclosed drug use. The quantitation of multi-panel drugs in urine and tissues utilizes β-glucuronidase to cleave the glucuronic acid and liberate the parent drug for mass spectrometry analysis. This work focuses on the comparison of three different, purified and commercially available β-glucuronidases across 83 patient urine samples. One enzyme is genetically modified, expressed in bacteria and the other two enzymes are purified from abalone. The results indicate that the source of β-glucuronidase plays an important role in substrate specificity which in turn dictates hydrolysis efficiency. Contaminants in the enzyme solutions also interfere with analyte detection. Altogether, these factors impact precision and accuracy of data interpretation, leading up to 13% positive/negative disagreement.

AN0033 胎粪中药物的9秒快速筛查分析

胎粪通常被用来筛查新生儿体内的药物残留。药物通过胎儿排泄进胆汁及羊水后聚集在胎粪中，通常胎粪中药物的检测方法是免疫检测。为了能够提高样本的回收率同时减少胎粪的使用量，研究人员建立了一种激光二极管热脱附质谱（Laser Diode Thermal Desorption Mass Spectrometry，LDTD®-MS/MS）的方法，并对胎粪中26种滥用药物进行检测。LDTD离子源的激光二极管可对96孔板中的样品蒸发干燥，随后样品通过气流进入电晕放电区，可产生高效质子化作用和强烈的抗离子抑制反应。这个过程只需9秒。

AN0030 尿液中阿密曲替林和环苯扎林代谢物的酶水解效率评估

阿密曲替林是一种广谱的三环抗抑郁药物，环苯扎林是一种三环骨骼肌松弛剂，其在代谢中均形成季铵葡萄糖苷酸复合物。为了能够精确的定量这些代谢物，需对复合物进行水解。本研究比较了四种β-葡萄糖醛酸酶对这两种复合物的水解效率。