Opiate & Benzodiazepine Confirmations: To Hydrolyze or Not to Hydrolyze is the Question

Abstract

Background: Opiates/opioids and benzodiazepines are commonly prescribed drug therapies for acute and chronic pain. Urine drug testing is often employed to assess adherence to these mediations. Opioids and benzodiazepines are drug classes that undergo extensive metabolism through glucuronidation/sulfation. Conjugated glucuronide and sulfate drug metabolites can be difficult to detect by immunoassay and mass spectrometry methods. Consequently, false-negative or false-positive results can have a damaging impact on patient care. A common dilemma among drug-testing laboratories is whether to perform preanalytical hydrolysis to increase detection of drugs that are highly conjugated as metabolites. Methods: The purpose of hydrolysis is to cleave the glucuronide or sulfate compounds to enhance analyte detection by increasing the parent drug concentration of those drugs that are primarily metabolized by glucuronidation or sulfation. Hydrolysis procedures can be performed by acid, base, or enzyme sources (β-glucuronidase). Results: Preanalytical hydrolysis can improve the overall detection of most opioids and benzodiazepine drugs. However, the limitation of this procedure is that the process can be time-consuming and prolong the turnaround time to result. In addition, chemical hydrolysis has the potential to degrade opioid and benzodiazepine drugs, whereas incomplete hydrolysis and variable hydrolysis efficiencies can occur with an enzymatic approach. Conclusions: Preanalytical hydrolysis can improve the sensitivity of drug detection for drug classes such as opiates/opioids and benzodiazepines, which are highly metabolized by glucuronidation and sulfation and should be implemented in analytical procedures to convert conjugated metabolites into the free (unbound) form. IMPACT STATEMENT This article summarizes the advantages and limitations of performing preanalytical hydrolysis of urine samples in a pain management population. The purpose of hydrolysis is to increase the analytical sensitivity of drug detection. Hydrolysis method comparison studies have demonstrated an increase in analyte detection to nonhydrolyzed samples. These studies have shown that the sample preparation and analytical technique can have an impact on the accuracy of the results. Laboratories will need to develop drug assays to reduce the number of reported false-positive or false-negative results to support testing in a pain management population.

Exploring Enzymatic Hydrolysis of Urine Samples for Investigation of Drugs Associated with Drug-Facilitated Sexual Assault

“…The resulting conjugated metabolites usually have a longer half-life than the parent drug and thus pre-analytical deconjugation is typically implemented to enhance drug detection. This hydrolysis process cleaves glucuronide and/or sulfate conjugated metabolites and enables the detection of the parent drug [ 15 , 16 ]. Various sources of β-glucuronidases (mollusks, bovine, Escherichia coli ) are used but, recently, recombinant enzymes have shown promising results regarding efficient and fast hydrolysis at lower incubation temperatures compared to β-glucuronidases from other sources [ 17 , 18 ].…”

Optimisation of Benzodiazepine Immunoassay Using <i>β</i>-Glucuronidase Enzymatic Hydrolysis: A Comparison of Five Different <i>β</i>-Glucuronidase Enzymes

“…Using β-glucuronidase is a preferred method of hydrolysis over acid-catalysed hydrolysis, which is known to induce benzodiazepine degradation and transformation to increase cross-reactivity [1] [2] [3]. Metabolised forms of benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility [4] [5].…”