Why should drug testing labs consider performing preanalytical hydrolysis?

Drugs in the opioid and benzodiazepine classes undergo extensive metabolism through glucuronidation and/or sulfation, which produces conjugated metabolites that can be difficult to detect using immunoassay and mass spectrometry urine drug testing methods. In particular, studies have shown that immunoassay drug screens for benzodiazepines and opioids produce false negative results due to poor analyte cross-reactivity with various glucuronide metabolites. This can have serious consequences for pain management patients. An unexpected drug result that does not match a patient’s prescribed medication list could lead to accusations of noncompliance, an increase in the frequency of random drug testing, or dismissal from a pain management program altogether.

As a result, a common dilemma faced by drug testing laboratories is how to enhance analyte detection. One potential solution is to cleave conjugated metabolites prior to analysis using hydrolysis, which increases the parent drug concentration of therapeutics that are primarily metabolized to glucuronide or sulfate metabolites.

What limitations should labs be aware of when performing hydrolysis?

Hydrolysis can be mediated via an enzyme reaction with β-glucuronidase or chemically with hydrochloric acid or sodium hydroxide base. Both these methods have limitations related to sample processing.

Enzymatic hydrolysis procedures can be time-consuming, with hydrolysis incubation times ranging from 15 minutes to more than 24 hours. This increased sample preparation time inevitably leads to a longer overall turnaround time for laboratories. Moreover, prolonged incubation at temperatures greater than 50°C can lead to degradation and a loss of drug concentration in urine.

Although chemical hydrolysis is faster than enzymatic hydrolysis, acid can degrade benzodiazepine and opioid drugs, too, which may hinder accurate drug identification. Another limitation of acid hydrolysis is that the heroin metabolite 6-mono-acetylmorphine (6-AM) is de-acetylated and converted to morphine during the process, which impedes the ability to detect heroin use from the presence of 6-AM in urine.

Lastly, hydrolysis procedures may not produce 100% cleavage of the conjugated metabolites and the efficiency of various enzymatic and acid protocols varies between different drugs. Therefore, labs need to evaluate and select a hydrolysis method that is optimal for the analytes of interest.

Which other methods reduce inaccurate urine drug test results?

As an alternative to preanalytical hydrolysis, laboratories can select analytical methods with higher sensitivity to afford analyte detection at lower cutoffs and/or the ability to detect glucuronide metabolites. This enhances the detection of parent and conjugated metabolites in non-hydrolyzed samples.

When choosing a method, labs should assess whether hydrolysis increases detection of the parent drug concentration above the analytical cutoff and whether it improves analytical sensitivity in comparison with approaches that use non-hydrolyzed samples. Laboratories also should evaluate if their workflow and testing volume can accommodate this additional processing step.

Kamisha L. Johnson-Davis, PhD, DABCC (CC, TC), FAACC, is an associate professor in the department of pathology at the University of Utah Health Sciences Center in Salt Lake City, and is medical director of clinical toxicology at ARUP Laboratories. +EMAIL: kamisha.johnson-davis@aruplab.com