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Choosing the Right Benzodiazepine Assay: Impact on Clinical Decision Making

Deborah French PhD, Judy A. Stone PhD, Judy S. Chang MSc, Alan H.B. Wu PhD
DOI: http://dx.doi.org/10.1309/LMDNLG1ZVOS2R8TV 196-200 First published online: 1 April 2010
  • benzodiazepines
  • immunoassay
  • toxicology

Clinical History


A 51-year-old, non-ambulatory, homeless African-American woman was admitted to a long-term care facility in September 2008 and used the San Francisco General Hospital Emergency Department as her primary care facility. She was a known intravenous drug abuser and was currently on several prescribed medications including Vicodin (hydrocodone and acetaminophen), Catapres (clonidine), and methadone. Her stay at the long-term care facility was dependent upon her refraining from additional drug abuse.

Chief Complaint:


History of Present Illness:

The patient admitted taking a non-prescribed clonazepam tablet obtained from outside the care facility, prompting her clinician to order a urine drug of abuse screen (DAU) using a commercial CEDIA HS Benzodiazepine immunoassay. It reported positive for benzodiazepines. The clinician proceeded to order DAUs on this patient almost daily to determine reuse of non-prescribed drugs.

Principal Laboratory Findings:

This patient came to the laboratory’s attention when the clinician called approximately 2 weeks after the first positive DAU. The DAU had been positive for a prolonged period and then negative in 1 sample. However, in the next sample, it was positive again. The clinician wanted to know if this positive sample indicated reuse of non-prescribed benzodiazepines by this patient. The clinical laboratory first normalized the semi-quantitative benzodiazepine results to urine creatinine in order to help determine if this was reuse. Seven-aminoclonazepam spiking studies were undertaken using the CEDIA HS immunoassay to try and replicate the high semi-quantitative values seen with this patient. Additionally, the laboratory compared the results from a targeted liquid chromatography tandem mass spectrometry method (LC-MS/MS) to 5 other benzodiazepine assays carried out on the same 9 urine samples from this patient (Table 2, Table 3, and Table 4) in order to further investigate the clinician’s question.


  1. If the semi-quantitative urine benzodiazepine results are normalized to urine creatinine, can reuse of non-prescribed benzodiazepines be ruled out?

  2. What is the degree of cross reactivity of the benzodiazepine assay for 7-aminoclonazepam, the major excreted metabolite of clonazepam; the drug the patient self-reported to taking?

  3. By comparing the results of 5 assays including 4 immunoassays and a GC-MS assay to a targeted LC-MS/MS assay, is the same conclusion regarding patient reuse reached?

  4. Is there utility in using targeted LC-MS/MS assays for the detection of benzodiazepines?

  5. Is a laboratory clinical consultation service for interpretation of toxicology results warranted?

Possible Answers

1. The physician’s question was investigated and meanwhile, she continued to order DAUs on this patient on an almost daily basis. The laboratory suggested she also order a urine creatinine on each sample since the drug:creatinine ratio proved useful in differentiating new drug use from residual excretion when considering 11-nor-9-carboxy-Δ9-tetrahydrocannabinol1,2 and diazepam metabolites.3 All available urine samples from this patient that had been previously drug screened were retrieved, and urine creatinine testing was performed.

The numerical results (ie, the raw data instrument printouts) from the semi-quantitative benzodiazepine assay for all urine samples obtained from the patient after single self-reported clonazepam use were analyzed (Figure 1A). The highest numerical value (653 ng/mL) was found in sample A, the first sample obtained after the admitted drug use. The subsequent samples (B–H) trended toward a decrease in concentration over time until there was a negative sample (<200 ng/mL; sample I; day 15 after admitted clonazepam use). The next urine sample was positive (≥200 ng/mL; sample J; day 18 after admitted clonazepam use), and this result was therefore questioned by the clinician (sample J indicated by arrow in Figure 1A). When these semi-quantitative numerical benzodiazepine assay results were normalized to urine creatinine concentration (Figure 1B), an increase in benzodiazepine concentration was apparent in samples D and E (obtained on days 5 and 7 after the self-reported drug use); but this was not the sample the clinician was concerned about (sample J indicated by the arrow in Figure 1B). Subsequently, samples F–V (obtained between days 8 and 46 after self-reported drug use) were all decreasing in benzodiazepine concentration normalized to urine creatinine.

Figure 1

(A) Serial urine benzodiazepine levels (ng/mL) from the patient. Arrow indicates the sample questioned by the physician (sample J). The 9 urine samples studied in this case are labeled A and K–R. A positive result is ≥200 ng/mL (dotted horizontal line). (B) Serial urine benzodiazepine levels (ng/mL) in our patient normalized to urine creatinine (mg/dL). Eight of the 9 urine samples studied in this case are labeled K–R. For sample A, the urine creatinine was not available. Arrow indicates the sample questioned by the physician (sample J).

Clonazepam, the drug the patient had reported taking, undergoes extensive biotransformation with only approximately 0.5% of parent drug excreted in the urine. The major metabolite is 7-aminoclonazepam, formed in the liver and/or gut by reduction of the 7-nitro group via CYP3A4.4 Polymorphisms in this enzyme are very common in the general population and may account for the variation in both concentrations of clonazepam metabolites and the length of time these metabolites are still found in the urine. However, none of the reported polymorphisms have been shown to have any effect on the expression of CYP3A4, and to date, no clinically meaningful data have been published.5 Additionally, concurrent use of CYP3A4 inducers or inhibitors may also have an effect on the metabolism of this drug.68 The patient in this case was currently prescribed methadone when the self-reported clonazepam use occurred. Methadone has a very long half-life of approximately 15–55 hours, and 1 of the major metabolic pathways is N-demethylation via CYP3A4.910 Therefore, in the patient presented here, the clonazepam and methadone may be competing for the same enzyme for metabolism, accounting for the observed positive benzodiazepine results for an extended period of time. However, a recent in vitro study showed that methadone is also an inducer of CYP3A4, which may enhance the clearance of drugs metabolized via this enzyme, including clonazepam, thereby decreasing the length of time metabolites would be found in the patient’s urine.11

There are relatively few studies on the elimination of 7-aminoclonazepam in urine, but 1 laboratory gave a 3 mg dose of clonazepam to 10 volunteers and measured the urine concentration of both parent drug and this metabolite at 6 hours and at 1, 3, 5, 8, 10, 14, 21, and 28 days after dose administration by negative ion chemical ionization gas chromatography.12 They found that in all 10 volunteers, there were no detectable concentrations of the parent drug at any time point, and there were still detectable amounts of 7-aminoclonazepam in the urine 14 days after administration of the dose. In 8 of the 10 volunteers, there were detectable levels of 7-aminoclonazepam after 21 days, and in 1 of the 10 volunteers, this metabolite was still detectable after 28 days (Table 1). Additionally, the urine concentrations of 7-aminoclonazepam in the 10 volunteers over the study period ranged from 0.073–183.2 ng/mL, with peak concentrations being observed at day 1 (6 volunteers), day 3 (1 volunteer), day 5 (2 volunteers), and day 8 (1 volunteer) after administration of the drug.

This information prompted the laboratory to inform the clinician that the conclusion was that the results were consistent with elimination following cessation of chronic dosing rather than reuse. The limitations of this information were also discussed including the immunoassay being a crude measure of drug level in the urine because of precision, specificity, and linearity limitations, and that the assay may not be sensitive enough to detect all instances of single reuse by this patient due to these limitations.

View this table:
Table 1

Elimination of 7-aminoclonazepam in Urine After a Single Dose*

SubjectUrine Concentration at 21 Days (ng/mL)Urine Concentration at 28 Days (ng/mL)
  • ND, not detected.

  • * data extracted from reference 12

2. In the laboratory at San Francisco General Hospital, the qualitative CEDIA HS Benzodiazepine homogeneous enzyme immunoassay with β-glucuronidase from Microgenics (Thermo Fisher Scientific, Waltham, MA) was used. Although a minor metabolic pathway for clonazepam, this hydrolysis pre-step enables detection of other benzodiazepines that are largely excreted as the glucuronide, for example, lorazepam. A positive benzodiazepine result is classified as ≥200 ng/mL. Since this patient only self-reported taking 1 clonazepam tablet, studies were undertaken in the laboratory in which concentrations of 7-aminoclonazepam (the major metabolite of clonazepam) ranging from 0–1,000 ng/mL were spiked into drug free urine and measured using the CEDIA HS immunoassay. The aim of these studies was to see if the 653 ng/mL semi-quantitative benzodiazepine concentration reported for sample A, obtained from the patient after the admitted ingestion, could be replicated. We could not replicate this value indicating there may have been more than 1 type of benzodiazepine present in the patient urine, since the cross-reactivity of the immunoassay with 7-aminoclonazepam is 96% according to the manufacturer.

3. No, by using different assays (immunoassays, GC-MS, and LC-MS/MS), the same conclusion regarding patient reuse was not reached.

There has been a recent explosion of studies in the literature looking at new, more sensitive, and more specific methods for measuring both urine and serum benzodiazepines, indicating the clinical world is well aware of the limitations in the current methods.1318 The new methodologies include LC-MS/MS, GC-MS/MS, and LC-MS-TOF (time of flight), and they are rapidly becoming regarded as gold standard methods in clinical laboratory toxicology testing.

The laboratory asked colleagues in the local area to run 9 available urine samples from this patient on the benzodiazepine assays used in their laboratories (labeled A and K–R in chronological order). Using a targeted LC-MS/MS assay (Table 2), urine samples A and B were positive for 7-aminoclonazepam, the major metabolite of clonazepam. Since all samples were positive for oxazepam (samples A and K–R), 4 were positive for temazepam (samples A, K–M), and 1 was positive for nordiazepam (sample A), this suggested that the patient may have also taken diazepam since these are all metabolites of this drug. However, the concentrations were decreasing over time indicating no reuse. The single low positive lorazepam (sample P) could indicate reuse by this patient as it is not a metabolite of any of the other drugs found. Alternatively, it could represent an artifact of the assay, but this is unlikely given the specificity of LC-MS/MS.

View this table:
Table 2

LC-MS/MS Assay Results

SampleDays After Ingestion7-Aminoclonazepam (ng/mL; LOD=5)Clonazepam (ng/mL; LOD=5)Oxazepam (ng/mL; LOD=5)Nordiazepam (ng/mL; LOD=2.5)Temazepam (ng/mL; LOD=2.5)Lorazepam (ng/mL; LOD=5)
  • * LOD, limit of detection.

The CEDIA HS assay identified samples A, K, and L positive and the rest negative. Using the Abbott AxSYM assay (Abbott Park, IL), all samples were negative. The Syva EMIT II Plus (Siemens Healthcare Diagnostics, Deerfield, IL) and Biosite Triage (Biosite, San Diego, CA) assays identified sample A positive and the rest negative (Table 3).

The GC-MS assay determined sample A to be positive for 7-aminoclonazepam with oxazepam around the limit of detection of the assay. The 8 remaining samples were determined to be negative using this assay (Table 4).

The CEDIA HS, Biosite Triage, and Syva EMIT assays used herein have similar cross-reactivity for clonazepam (71%, 86%, and 78% respectively). The CEDIA HS assay has greater cross-reactivity than the other assays for 7-aminoclonazepam, oxazepam, lorazepam, and nordiazepam as indicated in the product inserts for these assays, which may explain the higher semi-quantitative values obtained in almost all the patient samples with this assay. This assay also used β-glucuronidase, unlike the other immunoassays used in this study, allowing the glucuronidated benzodiazepine metabolites to also be quantified, therefore accounting for the higher values.

The Syva EMIT assay has greater cross-reactivity with temazepam than the other assays. This may explain why sample A has such a high semi-quantitative value using this assay.

View this table:
Table 3

Immunoassay Results

SampleDays After IngestionCEDIA HSAbbott AxSYMSyva EMIT II PlusBiosite Triage
ng/mL; (cut-off = 200)ng/mL; (cut-off = 200)ng/mL; (cut-off = 100)ng/mL; (cut-off = 300)
  • * cut-off - test is reported as positive above this concentration.

The Abbott AxSYM assay has no published percentage cross reactivity for any benzodiazepines. The only available information is that at a concentration of 200 ng/mL, clonazepam, oxazepam, temazepam, and lorazepam cross react. The manufacturer did not report any cross reactivity of this kind with 7-aminoclonazepam, which may explain the negative result for all 9 urine samples using this assay.

View this table:
Table 4

GC-MS Assay Results (LOD 100ng/mL)

SampleDays After Ingestion7-Aminoclonazepam (ng/mL)Oxazepam (ng/mL)Nordiazepam (ng/mL)Temazepam (ng/mL)Lorazepam (ng/mL)
K21not testedundetectedundetectedundetectedundetected
L22not testedundetectedundetectedundetectedundetected
M23not testedundetectedundetectedundetectedundetected
N24not testedundetectedundetectedundetectedundetected
O25not testedundetectedundetectedundetectedundetected
P26not testedundetectedundetectedundetectedundetected
Q30not testedundetectedundetectedundetectedundetected
R31not testedundetectedundetectedundetectedundetected
  • LOD, limit of detection.

The structural similarities, extensive conjugation of the metabolites, short elimination half-lives, and low daily doses of benzodiazepines make them notoriously difficult to detect. They are 1 of the most common drugs used in drug-facilitated crimes making this an extremely important issue to address. A recent study compared the ability of 4 commercially available immunoassays (Roche Benzodiazepine Plus KIMS assay, Microgenics CEDIA Benzodiazepine assay, Microgenics CEDIA HS Benzodiazepine assay, and Microgenics DRI reagent ready Benzodiazepine assay) to detect urine benzodiazepines in more 10,000 samples with positive samples confirmed by GC-MS.19 They found all 4 assays demonstrated acceptable precision and linearity, and the sensitivity was good with structurally unrelated compounds. Of 71 samples screened positive by at least 1 kit, 61 were confirmed positive by GC-MS. Of the 61 confirmed positive samples, the DRI, Roche, standard CEDIA, and CEDIA HS assays detected 67.2%, 34.4%, 59%, and 98.4% respectively. The Microgenics CEDIA HS Benzodiazepine assay (used in the San Francisco General Hospital laboratory) showed the highest positive screening rate and highest confirmation rate, due to the use of β-glucuronidase in this assay, allowing for approximately 40% improved detection of these drugs compared to the standard CEDIA assay.

4. The results presented herein indicate the use of a targeted LC-MS/MS assay is more sensitive than the other assays at detecting benzodiazepines and their metabolites, and immunoassays using different detection mechanisms have varying cross-reactivity with benzodiazepines and their metabolites. Additionally, the targeted LC-MS/MS assay was more sensitive in detecting benzodiazepines than the GC-MS. Specifically concerning the case illustrated here, the LC-MS/MS results indicated the patient may have reused benzodiazepines since sample P was positive for lorazepam, and the remaining 8 samples included in this study were negative for this drug. There may also have been illicit use of oxazepam since the concentrations of this drug are decreasing in samples A–Q, but in sample R, there is an increase in the concentration of this drug. However, if only an immunoassay was used, this could not be conclusively determined due to variations in the cross-reactivity for the different benzodiazepines that may have been taken by the patient, and due to the inability of some immunoassays to detect the glucuronidated metabolites. Additionally, if only GC–MS was used, reuse by the patient could not be conclusively determined due to the high limit of detection of the assay.

5. This case came to the attention of the laboratory due to the clinician calling the toxicologist for help in interpreting the benzodiazepine assay results. The possible impact of the results in this case was high, since the patient would be removed from a long-term care facility if drug abuse was proven. This case highlights the utility of a counseling service for clinicians to discuss and receive help in the interpretation of often complicated toxicology results. However, in providing a laboratory counseling service, it would be necessary for the clinical laboratory to have access to mass spectrometry capabilities so individual drugs, their metabolites, and congeners can be detected.


We would like to thank Dr. Richard Gilbert at Santa Clara Valley Medical Center for performing the GC–MS assay and Joanne Wong at UCSF Clinical Laboratories for performing the Abbott AxSYM immunoassay on the patient samples. We would also like to thank Dr. Firoozeh Parsa Nezhad for consulting with us regarding the interpretation of the toxicology results, Susan Gross for insightful discussions, and members of the Chemistry Laboratory at San Francisco General Hospital.


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