Paul Jannetto, Ph.D.
Associate Professor of Laboratory Medicine and Pathology
Division of Clinical Biochemistry
Mayo Clinic, Rochester, Minnesota
Hello, my name is Dr. Paul Jannetto, and I am the director of the Clinical and Forensic Toxicology Laboratory, the Clinical Mass Spectrometry Laboratory, and the Metals Laboratory here at the Mayo Clinic.
I have nothing to disclose.
The objectives of my talk are to describe the clinical utility and limitations of traditional laboratory or point-of-care-based immunoassays and mass spectrometry-based screening assays used to monitor benzodiazepines. Secondly, to define the metabolic profiles of benzodiazepines commonly prescribed and discuss how to interpret screening and definitive test results.
Benzodiazepines are a large class of central nervous system (CNS) depressant medications with useful sedative, hypnotic, anxiolytic, and anticonvulsant properties. There are short-, intermediate-, and long-acting benzodiazepines, which are commonly prescribed for generalized anxiety disorders, panic disorders, social phobias, insomnia, seizures, or they can be used for premedication for anesthetic procedures.
Benzodiazepines work by raising the levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain. Some commonly prescribed benzodiazepines include diazepam, alprazolam, clonazepam, and lorazepam.
Benzodiazepines are listed by the U.S. Drug Enforcement Administration (DEA) as schedule IV-controlled substances, which means they have the potential for abuse, addiction, and diversion. As a result, urine drug tests are recommended by numerous clinical practice guidelines to monitor compliance/adherence to these medications. Unfortunately, with the opioid epidemic in America, benzodiazepines are also found in greater than 30% of opioid overdoses, which further drives the need to perform urine drug testing for these medications.
So, what types of urine tests are being used by physicians to determine compliance to benzodiazepines? Physicians may use screening assays or definitive assays. However, physicians are primarily using screening assays with or without confirmatory tests to verify adherence of benzodiazepines.
There are two types of screening assays. Traditional screening assays that use antibodies directed against a drug or drug metabolite. These immunoassays may be in a point-of-care format so the test can be done right in the physician’s office or clinic, or they can be commercially-based immunoassays run in CLIA-certified laboratories. Alternatively, targeted laboratory-developed screening assays using mass spectrometry have also started to emerge as a screening tool, like the benzodiazepine assay I am talking to you about today. Each of these types of assays has advantages and disadvantages.
Point-of-care tests have the advantage of having the fastest turnaround time, so the physicians can get an immediate result, which is good for patients who have a high risk for abuse or reside far from care. Laboratory-based immunoassays typically are more economical. However, all immunoassays suffer from higher cutoffs, limited sensitivity, and specificity. On the other hand, targeted screens have better sensitivity and specificity, but aren’t widely available at all local laboratories.
Cross-reactivity with immunoassays is a big issue that needs to be considered when interpreting screening results. For example, in the urine benzodiazepine immunoassay, the antibody used in most manufacturer kits is directed against oxazepam or nordiazepam. It has limited-to-no cross-reactivity with all of the other commonly prescribed benzodiazepines. As you can see, a standard urine benzodiazepine immunoassay has a pretty good cross-reactivity with the metabolite of alprazolam (a-hydroxyalprazolam). However, a patient’s urine sample would need to have a much higher concentration (greater than 20,000 ng/mL) of temazepam glucuronide (the metabolite of temazepam) to get a positive result using this immunoassay with a 100 ng/mL cutoff. It should be noted that most benzodiazepines actually appear in the urine largely as the glucuronidated conjugate. Only a very small or negligible amount of the parent drug is excreted unchanged in the urine.
Now, let’s look at one clinical case study which uses a traditional immunoassay approach with reflexing to confirmatory testing when positive.
In this case, we have a 55-year-old female with an anxiety disorder who is being prescribed lorazepam (a benzodiazepine). The patient’s anxiety is well-controlled, but per institutional guidelines, the physician wants to order a urine drug test that uses several different immunoassays to screen for the prescribed benzodiazepine as well as commonly abused drugs. The purpose of the urine drug tests is to check for adherence to the prescribed medication and make sure the patient isn’t abusing other medications. However, you can see the immunoassay screening results all came back negative, including the benzodiazepine immunoassay.
As a result, how should the physician interpret these results? Is the patient compliant? The patient is prescribed lorazepam and admits taking this medication as prescribed daily. The last dose was taken in the morning prior to collection of the urine sample. However, the benzodiazepine immunoassay test result was negative. The physician must remember and understand the limitations of the immunoassay. The doctor should contact the lab to determine the cross-reactivity of the immunoassay used with the prescribed benzodiazepine. The clinician must also remember that most benzodiazepines appear in the urine as the glucuronidated form, and this assay has a very poor cross-reactivity to lorazepam glucuronide. Therefore, this result could be a false negative result since the concentration of lorazepam glucuronide would have to be greater than 19,615 ng/mL to give a positive result.
This slide shows a simplified metabolic pathway for benzodiazepines. Each blue box contains a separate prescribable benzodiazepine (e.g., parent drug). The arrows then indicate the metabolites formed in the body when the drug is broken down.
As I stated earlier, the antibody in most commercial benzodiazepine immunoassays is targeted against oxazepam. Therefore, these immunoassays then tend to be good at detecting benzodiazepines in this common pathway outlined by the red box. However, our patient was prescribed lorazepam, which is metabolized to lorazepam glucuronide and then eliminated in the urine. As you may recall, the cross-reactivity of the benzodiazepine immunoassay is relatively poor to lorazepam glucuronide, so even when it’s present, you are more likely to get a false negative result. As a result, the physician using this benzodiazepine immunoassay needs to order additional confirmatory testing.
In this case, the physician then called the laboratory back to add on a confirmatory benzodiazepine test that uses liquid chromatography-tandem mass spectrometry to specifically look for lorazepam. The confirmatory test also uses a hydrolysis reaction to cleave off the glucuronide to enable better detection of the parent form of the drug, if present. In this case, the results show lorazepam was present at a concentration of 8,250 ng/mL, which also explains why the immunoassay was negative, since the concentration of lorazepam glucuronide would have had to have been greater than 19,615 ng/mL to trip this immunoassay positive. In the end, the patient is compliant and taking lorazepam despite the initial false negative immunoassay result.
Now, let’s look at Mayo Clinic’s high-resolution targeted benzodiazepine screen (Mayo ID: TBSU).
The use of mass spectrometry-based technologies to screen for benzodiazepines is consistent with the evidence-based laboratory medicine practice guideline entitled, “Using Clinical Laboratory Tests to Monitor Drug Therapy in Pain-Management Patients,” that was published in January 2018 in the Journal of Applied Laboratory Medicine.
One of the recommendations was that qualitative definitive tests should be used instead of immunoassays since they are more effective at identifying prescribed medications and illicit substances in pain management patients.
The features and benefits of the high-resolution targeted benzodiazepine screen, TBSU, include:
Here is the complete list of the 27 benzodiazepines and metabolites along with the cutoffs or concentrations required to give a positive or present result. This one test will enable clinicians so that they don’t have to remember which benzodiazepine does or does not cross-react with the urine benzodiazepine immunoassay or have to order additional costly confirmation tests to look for all of the different benzodiazepines.
The high-resolution targeted benzodiazepine screen will also improve test utilization without compromising turnaround times. It will reduce the need for confirmatory testing that is required with traditional immunoassays since they don’t specifically identify which benzodiazepine is present when the assay is positive. Because it detects both parent and metabolite of a benzodiazepine, we can identify spiked samples where patients crush up pills and then add them into the urine instead of ingesting them. Lastly, this test comes with an enhanced PDF report that has interpretative comments to aid the clinician with the interpretation of the test results.
Here is an example of an enhanced report. If anything was positive, those results would have been pulled up into the top section of the report so the clinician can immediately see what was present or detected. It also provides some additional information regarding brand names or if something is a metabolite of another drug. The next section is the interpretation section where a comment is given providing guidance regarding the test results. The last section shows all the analytes tested for that were negative or not detected.
In addition to being a separate orderable test, the high-resolution targeted benzodiazepine screen (TBSU) will also be available as part of the controlled substance monitoring panel (CSMP). The controlled substance monitoring panel starts with adulterant testing looking at the creatinine, specific gravity, pH, and oxidants. It includes both the high-resolution targeted opioid and benzodiazepine assays along with traditional immunoassays for common drugs of abuse.
This slide shows the algorithm for the CSMP where adulteration or specimen validity testing is performed first. If the sample is adulterated, testing stops, and no charge is issued to the client/patient. If the sample is not adulterated, testing then resumes with the targeted screens for opioids and benzodiazepines along with the traditional immunoassays.
In summary, objective measures like laboratory tests are needed to identify and evaluate adherence or misuse, or abuse of controlled substances.
The high-resolution targeted benzodiazepine assay can identify 27 different benzodiazepines and/or metabolites where immunoassays are not adequate (improved specificity); has lower detection limits (improved sensitivity); reduces the need for confirmatory testing required with traditional immunoassay screens; improves test utilization; and is available with enhanced reports and interpretative comments.
In the end, all urine drug test results must be interpreted in the context of the test, drugs prescribed, specimen type, time since last dose and sample collection, specimen validity test results, and the patient. Unexpected or unexplained results should be discussed with the patient and laboratory, and additional testing should be performed if needed.
Thank you for your attention. I hope you found this presentation useful. If you have any questions, please contact Mayo Clinic Laboratories.
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