A novel screening method for glucuronidated and non-glucuronidated drugs in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
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Abstract
Opioids are often prescribed to patients experiencing long-term, chronic pain. Although they have many benefits to the patient, opioids are highly addictive medications and have a high abuse potential. For this reason, pain management clinics regularly send patient urine samples to toxicology laboratories to ensure that the opioids they are prescribing are not being misused. Many labs operate under a strict budget, so being able to screen for the presence of opioids (in addition to a host of other drugs) quickly and economically is a top priority. Most labs screen bodily fluids using a form of immunoassay, but immunoassay techniques have a number of disadvantages, including their high cutoff values, low degree of specificity and selectivity, high expense, and lack of included drugs. Therefore, replacing the immunoassay technique with a more selective and sensitive technique like liquid chromatography-tandem mass spectrometry (LC-MS/MS) can prove to be a good alternative for labs looking to minimize expense.
Typical LC-MS/MS methods that analyze urine require samples to be prepared with a hydrolysis step. The hydrolysis reaction cleaves the glucuronide moiety that the liver attaches to certain drugs (like opioids) during the metabolism process. Although hydrolysis is effective in recovering the parent drug structure, it is expensive and time-intensive.
With the goal of a more efficient, sensitive, specific, and cost-effective analysis method, a novel screening method that replaces the immunoassay instrument and eliminates the hydrolysis reaction was created. The new method screened for the glucuronidated form of many drugs with an LC-MS/MS instrument. Drugs that are glucuronidated in the liver include morphine, hydromorphone, tapentadol, codeine, oxymorphone, oxazepam, and buprenorphine.
Creating the novel method required tuning the instrument for the glucuronidated drugs, adding these glucuronidated drugs to the lab’s existing LC-MS/MS method, and validating the new method. The method’s validation study evaluated several parameters, including linearity, precision and bias, interference, specificity, limit of detection (LOD), limit of quantitation (LOQ), client comparisons, and carryover, to ensure that the instrument could reliably detect every drug in the LC-MS/MS method accurately.
The method validation parameters were within acceptable limits, and the validation study was submitted to the New York State Department of Health for approval. However, there were two findings that are important to mention. Morphine glucuronide and hydromorphone glucuronide were not able to be individually resolved in the LC-MS/MS method. If one or two peaks are detected at morphine glucuronide/hydromorphone glucuronide’s retention time, the sample will carry a presumptive positive screen for both compounds to the confirmatory method. The confirmatory method will verify if the sample is positive for one, both, or neither drug. Second, it was not possible to detect alprazolam at its full, known concentration. Several possible explanations, including matrix effects, stability, and the possibility of glucuronidation, were evaluated with no increase in detection; therefore, alprazolam’s ion ratios require individual evaluation in order to identify a sample presumptively positive for alprazolam.
Although glucuronidated drugs have been detected in previous methods, this novel method is unique because it utilizes an LC-MS/MS to screen for six different glucuronidated drugs in addition to 21 non-glucuronidated drugs found in the urine of pain management patients. By replacing the immunoassay instrument and eliminating the hydrolysis step of the screening method, the laboratory not only saves time and expense but is able to more accurately screen for more drugs at lower concentrations.
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2024