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HelpMayo Test ID BNZX Benzodiazepines Confirmation, Chain of Custody, Random, Urine
Specimen Required
Supplies: Chain of Custody Kit (T282)
Container/Tube: Chain of custody kit containing the specimen containers, seals, and documentation required
Specimen Volume: 5 mL
Collection Instructions: Collect specimen in the container provided, seal, and submit with the associated documentation to satisfy the legal requirements for chain-of-custody testing.
Additional Information: Submitting less than 5 mL will compromise the ability to perform all necessary testing.
Useful For
Detecting drug use involving benzodiazepines such as alprazolam, chlordiazepoxide, clonazepam, diazepam, midazolam, oxazepam, temazepam, clobazam, flunitrazepam, flurazepam, lorazepam, prazepam, triazolam, and zolpidem, in urine specimens handled through the chain-of-custody process
Providing chain of custody for when the results of testing could be used in a court of law. Its purpose is to protect the rights of the individual contributing the specimen by demonstrating that it was always under the control of personnel involved with testing the specimen; this control implies that the opportunity for specimen tampering would be limited.
Additional Tests
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| COCH | Chain of Custody Processing | No | Yes |
| ADLTX | Adulterants Survey, CoC, U | Yes | Yes |
Testing Algorithm
Testing for adulterants will be performed on all chain-of-custody urine samples per regulatory requirements.
Method Name
Immunoassay/Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)
Reporting Name
Benzodiazepines Conf, CoC, USpecimen Type
UrineSpecimen Minimum Volume
1 mL
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Urine | Refrigerated (preferred) | 7 days | |
| Frozen | 14 days |
Reject Due To
| Gross hemolysis | Reject |
| Gross icterus | Reject |
Clinical Information
Benzodiazepines are any of a group of compounds having a common molecular structure and acting similarly as depressants of the central nervous system. As a class of drugs, benzodiazepines are among the most prescribed drugs in the western hemisphere because of their efficacy, safety, low addiction potential, minimal side effects, and high public demand for sedative and anxiolytic agents.
Chain of custody is a record of the disposition of a specimen to document each individual who collected, handled, and performed the analysis. When a specimen is submitted in this manner, analysis will be performed in such a way that it will withstand regular court scrutiny.
Reference Values
Negative
Immunoassay Screen: <100 ng/mL
Cutoff concentrations by liquid chromatography tandem mass spectroscopy:
Alprazolam: 10 ng/mL
Alpha-hydroxyalprazolam: 10 ng/mL
Chlordiazepoxide: 10 ng/mL
Clonazepam: 10 ng/mL
7-Aminoclonazepam: 10 ng/mL
Diazepam: 10 ng/mL
Nordiazepam: 10 ng/mL
Midazolam: 10 ng/mL
Alpha-hydroxy midazolam: 10 ng/mL
Oxazepam: 10 ng/mL
Temazepam: 10 ng/mL
Clobazam: 10 ng/mL
N-Desmethylclobazam by LC-MS/MS: 10 ng/mL
Flunitrazepam: 10 ng/mL
7-Aminoflunitrazepam: 10 ng/mL
Flurazepam: 10 ng/mL
2-Hydroxy ethyl flurazepam: 10 ng/mL
Lorazepam: 10 ng/mL
Prazepam: 10 ng/mL
Triazolam: 10 ng/mL
Alpha-hydroxy triazolam: 10 ng/mL
Zolpidem: 10 ng/mL
Zolpidem phenyl-4-carboxylic acid: 10 ng/mL
Interpretation
Benzodiazepines are extensively metabolized, and the parent compounds are not detected in urine. This test screens for (and confirms) the presence of:
-Alprazolam
-Alpha-hydroxyalprazolam (metabolite of alprazolam)
-Chlordiazepoxide
-Clonazepam
-7-Aminoclonazepam (metabolite of clonazepam)
-Diazepam (separate prescribable drug and metabolite of medzazepam)
-Nordiazepam (metabolite of clorazepate, halazepam, prazepam, diazepam and medazepam)
-Midazolam
-Alpha-hydroxy midazolam (metabolite of midazolam)
-Oxazepam (separate prescribable drug and metabolite of clorazepate, halazepam, prazepam, medazepam, temazepam, and diazepam)
-Temazepam (separate prescribable drug and metabolite of medazepam and diazepam)
-Clobazam
-N-Desmethylclobazam (metabolite of clobazam)
-Flunitrazepam
-7-Aminoflunitrazepam (metabolite of flunitrazepam)
-Flurazepam
-2-Hydroxy ethyl flurazepam (metabolite of flurazepam)
-Lorazepam
-Prazepam
-Triazolam
-Alpha-hydroxy triazolam (metabolite of triazolam)
-Zolpidem
-Zolpidem phenyl-4-carboxylic acid (metabolite of zolpidem)
The clearance half-life of long-acting benzodiazepines is more than 24 hours. It takes 5 to 7 half-lives to clear 98% of a drug dose. Therefore, the presence of a long-acting benzodiazepine greater than the limit of quantification indicates exposure within a 5 to 20-day interval preceding specimen collection. Following a dose of diazepam, the drug and its metabolites appear in the urine within 30 minutes. Peak urine output is reached between 1 and 8 hours. For additional information including metabolism, clearance (half-life), and approximate detection times, see Optimize Urine Drug Monitoring for CNS Depressants.
Cautions
No significant cautionary statements
Clinical Reference
1. Gudin JA, Mogali S, Jones JD, Comer SD. Risks, management, and monitoring of combination opioid, benzodiazepines, and/or alcohol use. Postgrad Med. 2013;125(4):115-130. doi:10.3810/pgm.2013.07.2684
2. Dowell D, Ragan KR, Jones CM, Baldwin GT, Chou R. CDC Clinical Practice Guideline for Prescribing Opioids for Pain - United States, 2022. MMWR Recomm Rep. 2022;71(3):1-95
3. Baselt RC. Disposition of Toxic Drugs and Chemical in Man. 12th ed. Biomedical Publications; 2020
4. Langman LJ, Bechtel LK, Holstege C. Clinical toxicology. In: Rifai N, Chiu RWK, Burnham CD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023:chap 43
Method Description
Preliminary screen is performed by immunoassay.
The benzodiazepine assay is based on the kinetic interaction of microparticles in a solution as measured by changes in light transmission. In the absence of sample drug, soluble drug conjugates bind to antibody-bound microparticles causing the formation of particle aggregates. As the aggregation reaction proceeds in the absence of sample drug, the absorbance increases. When a urine sample contains the drug in question, this drug competes with the drug derivative conjugate for microparticle-bound antibody. Antibody bound to sample drug is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The presence of sample drug diminishes the increasing absorbance in proportion to the concentration of drug in the sample. Sample drug content is determined relative to the value obtained for a known cutoff concentration of drug.(Package insert: BNZ2. Roche Diagnostics; V 2.0, 04/2024)
Benzodiazepines are extensively metabolized by the liver and subsequently exist in the urine primarily as conjugated esters (-glucuronides). The conjugated metabolites are cleaved during a mild hydrolysis utilizing the enzyme glucuronidase. Stable isotope forms of the compounds are added as internal standards to account for extraction losses. An aliquot of the hydrolyzed sample is diluted and the analytes are separated by liquid chromatography tandem mass spectroscopy and analyzed by multiple reaction monitoring.(Unpublished Mayo method)
Specimen Retention Time
14 daysPerforming Laboratory
Mayo Clinic Laboratories in Rochester
Test Classification
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.CPT Code Information
80347
G0480 (if appropriate)
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| BNZX | Benzodiazepines Conf, CoC, U | 90890-5 |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| 608452 | Benzodiazepines Immunoassay Screen | 14316-4 |
| 608280 | Alprazolam by LC-MS/MS | 59615-5 |
| 608281 | Alpha-Hydroxyalprazolam by LC-MS/MS | 16348-5 |
| 608282 | Chlordiazepoxide by LC-MS/MS | 20522-9 |
| 608283 | Clonazepam by LC-MS/MS | 16229-7 |
| 608284 | 7-aminoclonazepam by LC-MS/MS | 51776-3 |
| 608285 | Diazepam by LC-MS/MS | 16227-1 |
| 608286 | Nordiazepam by LC-MS/MS | 16228-9 |
| 608287 | Midazolam by LC-MS/MS | 16233-9 |
| 608288 | Alpha-Hydroxy Midazolam by LC-MS/MS | 59590-0 |
| 608289 | Oxazepam by LC-MS/MS | 16201-6 |
| 608290 | Temazepam by LC-MS/MS | 20559-1 |
| 608291 | Clobazam by LC-MS/MS | 59635-3 |
| 608292 | N-Desmethylclobazam by LC-MS/MS | 97160-6 |
| 608293 | Flunitrazepam by LC-MS/MS | 20528-6 |
| 608294 | 7-aminoflunitrazepam by LC-MS/MS | 51777-1 |
| 608295 | Flurazepam by LC-MS/MS | 16231-3 |
| 608296 | 2-Hydroxy Ethyl Flurazepam by LC-MS/MS | 97159-8 |
| 608297 | Lorazepam by LC-MS/MS | 17088-6 |
| 608298 | Prazepam by LC-MS/MS | 17479-7 |
| 608299 | Triazolam by LC-MS/MS | 16232-1 |
| 608300 | Alpha-Hydroxy Triazolam by LC-MS/MS | 49876-6 |
| 608301 | Zolpidem by LC-MS/MS | 72770-1 |
| 608302 | Zolpidem Phenyl-4-Carboxylic acid by LC-MS/MS | 72768-5 |
| 608449 | Benzodiazepines Interpretation | 69050-3 |
| 608450 | Chain of Custody | 77202-0 |
Day(s) Performed
Monday through Friday