DRI Benzodiazepine Assay

K173963 · Microgenics Corporation · JXM · Feb 21, 2018 · Clinical Toxicology

Device Facts

• Record ID: K173963
• Device Name: DRI Benzodiazepine Assay
• Applicant: Microgenics Corporation
• Product Code: JXM · Clinical Toxicology
• Decision Date: Feb 21, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3170
• Device Class: Class 2

Intended Use

The DRI Benzodiazepine Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semi-quantitative determination of the presence of benzodiazepines and their metabolites in human urine at a cutoff concentration of 200 ng/mL. The assay is intended to be used in laboratories and provides a simple and rapid analytical screening procedure to detect benzodiazepines in human urine. The assay is designed for use with a number of clinical chemistry analyzers. This assay is calibrated against Oxazepam. This product is intended to be used by trained professionals only. The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/tandem mass spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures. The assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Gas chromatography/ mass spectrometry (GC/MS) or Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is the preferred confirmatory method. Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary results are used. For In Vitro Diagnostic Use Only.

Device Story

Homogeneous enzyme immunoassay for benzodiazepine screening in human urine; utilizes competition between drug in sample and G6PDH-labeled drug for specific antibody binding sites. Enzyme activity, measured spectrophotometrically at 340 nm, is inversely proportional to drug concentration. Used in clinical laboratories by trained professionals on chemistry analyzers (e.g., Beckman AU680). Provides preliminary results; requires confirmation via GC/MS or LC-MS/MS. Enables semi-quantitative estimation for dilution guidance or QC. Benefits include rapid screening to support clinical decision-making regarding drug presence.

Clinical Evidence

No clinical trials performed. Evidence consists of analytical bench testing including precision (n=80 per condition), linearity (100-1000 ng/mL), cross-reactivity with structurally related/unrelated compounds, and interference testing (endogenous substances, pH, specific gravity). Method comparison study (n=106) against LC-MS/MS showed concordance, with discordant samples attributed to cross-reactivity with specific metabolites (e.g., 7-amino-clonazepam, alpha-hydroxy-alprazolam).

Technological Characteristics

Homogeneous enzyme immunoassay; reagents contain sheep polyclonal anti-benzodiazepine antibodies and G6PDH-labeled benzodiazepine derivative. Tris buffer with sodium azide preservative. Spectrophotometric detection at 340 nm. Calibrated against Oxazepam. Designed for automated clinical chemistry analyzers. Standards referenced: CLSI EP05-A3, EP07-A2, EP09-A3, EP06-A, EP25-A.

Indications for Use

Indicated for the qualitative and/or semi-quantitative detection of benzodiazepines and their metabolites in human urine at a 200 ng/mL cutoff. Intended for use by trained professionals in laboratory settings as a preliminary screening procedure.

Regulatory Classification

Identification

A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and urine. The benzodiazepine compounds include chlordiazepoxide, diazepam, oxazepam, chlorzepate, flurazepam, and nitrazepam. Measurements obtained by this device are used in the diagnosis and treatment of benzodiazepine use or overdose and in monitoring levels of benzodiazepines to ensure appropriate therapy.

Special Controls

*Classification.* Class II (special controls). A benzodiazepine test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (*e.g.,* programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).

Predicate Devices

• DRI Benzodiazepine Assay (k930529)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

February 21, 2018

MICROGENICS CORPORATION MINOTI PATEL MANAGER, REGULATORY AFFAIRS 46500 KATO ROAD FREMONT, CA 94538

Re: K173963

Trade/Device Name: DRI Benzodiazepine Assay Regulation Number: 21 CFR 862.3170 Regulation Name: Benzodiazepine test system Regulatory Class: Class II Product Code: JXM Dated: December 27, 2017 Received: December 28, 2017

Dear Minoti Patel:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR

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Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

# Kellie B. Kelm -S

for Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) k173963

Device Name DRI Benzodiazepine Assay

#### Indications for Use (Describe)

The DRI Benzodiazepine Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semiquantitative determination of the presence of benzodiazepines and their metabolites in human urine at a cutoff concentration of 200 ng/mL. The assay is intended to be used in laboratories a simple and rapid analytical screening procedure to detect benzodiazepines in human urine. The assay is designed for use with a number of clinical chemistry analyzers. This assay is calibrated against Oxazepam. This product is intended to be used by trained professionals only.

The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/tandem mass spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical test result. A more specific alternative chemical must be used to obtain a confirmed analytical result. Gas chromatography/ mass spectrometry (GC/MS) or Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is the preferred confirmatory method.

Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary results are used. For In Vitro Diagnostic Use Only.

Type of Use (Select one or both, as applicable)

| <span style="font-family: DejaVu Sans, sans-serif">☑</span> Prescription Use (Part 21 CFR 801 Subpart D) | ☐ Over-The-Counter Use (21 CFR 801 Subpart C) |
|----------------------------------------------------------------------------------------------------------|-----------------------------------------------|
|----------------------------------------------------------------------------------------------------------|-----------------------------------------------|

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#### k173963

# 510(k) Summary

This 510(k) Summary of Safety and Effectiveness is being submitted in accordance with the requirements of Safe Medical Device Act of 1990 and 21 CFR 807.92.

#### A. Device Information

| Category | Comments |
|-------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------|
| Sponsor: | Microgenics Corporation<br>Thermo Fisher Scientific<br>46500 Kato Road<br>Fremont, CA 94538<br>Phone: [phone]<br>FAX: [phone] |
| Correspondent Contact<br>Information: | Minoti Patel, RAC<br>Manager, Regulatory Affairs<br>Email: [e-mail]<br>Phone: [phone]<br>FAX: [phone] |
| Device Common Name: | Benzodiazepine Enzyme Immunoassay |
| Trade or Proprietary Name | DRI Benzodiazepine Assay |
| Candidate Device Product<br>Code, Classification,<br>Classification Name &<br>Panel | JXM, Class II, 21 CFR 862.3170 – Benzodiazepine test<br>system, 91 - Toxicology |

#### Predicate Device Information:

| Predicate Device: | Benzodiazepine Enzyme Immunoassay |
|-----------------------------------------------|-----------------------------------|
| Predicate Device<br>Manufacturer: | Diagnostic Reagents, Inc. |
| Predicate Device Premarket<br>Notification #: | K930529 |

# B. Date Summary Prepared

February 20, 2018

#### C. Description of Device

The DRI Benzodiazepine Assay is a homogeneous enzyme immunoassay with liquid ready-to-use reagents. The assay uses a specific antibody which can detect most benzodiazepines and their metabolites in urine. The assay is based on the competition of an enzyme glucose- 6-phosphate dehydrogenase (G6PDH) labeled drug and the drug from the urine sample for a fixed amount of specific antibody binding sites. In the absence of free drug from the sample, the enzyme-labeled drug is bound by the specific antibody and the enzyme activity is inhibited. This phenomenon

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creates a relationship between drug concentration in urine and the enzyme activity. The enzyme G6PDH activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.

The assay consists of reagents A and E.

Reagent A: Contains sheep polyclonal anti-benzodiazepine antibodies, glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide (NAD) in Tris buffer with sodium azide as a preservative.

Reagent E: Contains benzodiazepine derivative labeled with glucose-6-phosphate dehydrogenase (G6PDH) in Tris buffer with sodium azide as a preservative.

# D. Intended Use

# DRI Benzodiazepine Assay:

The DRI Benzodiazepine Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semi-quantitative determination of the presence of benzodiazepines and their metabolites in human urine at a cutoff concentration of 200 ng/mL. The assay is intended to be used in laboratories and provides a rapid analytical screening procedure to detect benzodiazepines in human urine. The assay is designed for use with a number of clinical chemistry analyzers. This assay is calibrated against Oxazepam. This product is intended to be used by trained professionals only.

The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/tandem mass spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Gas chromatography/ mass spectrometry (GC/MS) or Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is the preferred confirmatory method.1,2

Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary results are used. For In Vitro Diagnostic Use Only.

| Characteristic | Candidate Device:<br>DRI Benzodiazepine Assay | Predicate Device:<br>DRI Benzodiazepine Assay<br>(K930529). |
|----------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| Intended Use | The DRI Benzodiazepine<br>Assay is a homogeneous<br>enzyme immunoassay<br>intended for the qualitative<br>and/or semi-quantitative<br>determination of the presence | Same |

# E. Comparison to Predicate Device

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| Characteristic | Candidate Device:<br>DRI Benzodiazepine Assay<br>of benzodiazepines and their<br>metabolites in human urine at<br>a cutoff concentration of 200<br>ng/mL. | Predicate Device:<br>DRI Benzodiazepine Assay<br>(K930529). |
|----------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| Operating<br>Principle<br>(Technology) | DRI | Same |
| Measured<br>Analyte | Benzodiazepine and its<br>metabolites | Same |
| Test Matrix | Urine | Same |
| Cutoff Levels | 200 ng/mL | Same |
| Methodology | Homogeneous Enzyme<br>Immunoassay | Same |
| Reagents Form | Liquid ready-to-use. | Same |
| Antibody | Sheep Polyclonal antibody | Goat Polyclonal antibody |
| Storage | 2-8°C until expiration date. | Same |
| Principal<br>Operator | Trained professionals | Same |

#### F. Test Principle

The DRI Benzodiazepine Assay is a homogeneous enzyme immunoassay with liquid readyto-use reagents. The assay uses a specific antibody which can detect most benzodiazepines and their metabolites in urine. The assay is based on the competition of an enzyme glucose-6-phosphate dehydrogenase (G6PDH) labeled drug and the drug from the urine sample for a fixed amount of specific antibody binding sites. In the absence of free drug from the sample, the enzyme-labeled drug is bound by the specific antibody and the enzyme activity is inhibited. This phenomenon creates a relationship between drug concentration in urine and the enzyme activity. The enzyme G6PDH activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.

# G. Summary of Supporting Data

#### 1. Analytical Performance:

Performance is evaluated at the manufacturer's site on the AU680 clinical analyzer.

#### a) Precision

Precision studies were performed in accordance with CLSI Guideline EP05-A3. Samples were prepared by spiking Oxazepam into drug free urine at the cutoff, 25%, 50%. 75% and 100% above and below the cutoff and tested in both qualitative and semiquantitative modes. Results presented below were generated by testing all samples in

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replicates of 2, twice per day for 20 days, total n=80. The results are summarized in the table below.

| Spiked<br>Concentration<br>(ng/mL) | % of Cutoff<br>(200 ng/mL) | LC-MS/MS<br>(ng/mL) | Total Precision (n=80) | |
|------------------------------------|----------------------------|---------------------|------------------------|-----------------------------------------------|
| | | | # of<br>Determinants | Immunoassay<br>Results<br>(Negative/Positive) |
| 0 | -100% | N/A | 80 | 80/0 |
| 50 | -75% | 56.0 | 80 | 80/0 |
| 100 | -50% | 102.0 | 80 | 80/0 |
| 150 | -25% | 161.5 | 80 | 80/0 |
| 200 | 100% | 214.0 | 80 | 16/64 |
| 250 | +25% | 255.5 | 80 | 0/80 |
| 300 | +50% | 299.0 | 80 | 0/80 |
| 350 | +75% | 348.0 | 80 | 0/80 |
| 400 | +100% | 403.0 | 80 | 0/80 |

# Quantitative Study Analysis

#### Semi-Quantitative Study Analysis

| Spiked<br>Concentration<br>(ng/mL) | % of Cutoff<br>(200 ng/mL) | LC-MS/MS<br>(ng/mL) | # of<br>Determinants | Total Precision (n=80)<br>Immunoassay<br>Results<br>(Negative/Positive) |
|------------------------------------|----------------------------|---------------------|----------------------|-------------------------------------------------------------------------|
| 0 | -100% | N/A | 80 | 80/0 |
| 50 | -75% | 56.0 | 80 | 80/0 |
| 100 | -50% | 102.0 | 80 | 80/0 |
| 150 | -25% | 161.5 | 80 | 80/0 |
| 200 | 100% | 214.0 | 80 | 27/53 |
| 250 | +25% | 255.5 | 80 | 0/80 |
| 300 | +50% | 299.0 | 80 | 0/80 |
| 350 | +75% | 348.0 | 80 | 0/80 |
| 400 | +100% | 403.0 | 80 | 0/80 |

# b) Spike Recovery

The study was performed for 20 replicates. This study was carried out by testing spiked samples containing Oxazepam at the cutoff calibrator and control levels. The spiked samples were prepared by spiking Oxazepam into drug free urine. Samples were tested in both Qualitative and Semi-Quantitative mode. The qualitative results are summarized in the table below.

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| | 150 ng/mL | 250 ng/mL |
|-----------------|------------------|------------------|
| Replicates | (n=20) | (n=20) |
| 1 | Negative | Positive |
| 2 | Negative | Positive |
| 3 | Negative | Positive |
| 4 | Negative | Positive |
| 5 | Negative | Positive |
| 6 | Negative | Positive |
| 7 | Negative | Positive |
| 8 | Negative | Positive |
| 9 | Negative | Positive |
| 10 | Negative | Positive |
| 11 | Negative | Positive |
| 12 | Negative | Positive |
| 13 | Negative | Positive |
| 14 | Negative | Positive |
| 15 | Negative | Positive |
| 16 | Negative | Positive |
| 17 | Negative | Positive |
| 18 | Negative | Positive |
| 19 | Negative | Positive |
| 20 | Negative | Positive |
| Overlap | No | No |
| Relative to C/O | All 20 below C/O | All 20 above C/O |

# Qualitative Data

# c) Analytical Recovery and Linearity

Linearity studies were performed in accordance with CLSI Guideline EP06-A. To demonstrate the dilution linearity for purposes of sample dilution and quality control of the entire assay range, drug free urine was spiked to the high level calibrator using Oxazepam (1000 ng/mL) and diluted with drug free urine to generate 10 intermediate levels.

Each sample was run in replicates of five in semi-quantitative mode and the average was used to determine percent recovery compared to the expected target value. The percent recovery is summarized in the table below.

| Level | Expected<br>Concentration<br>(ng/mL) | Observed<br>Concentration<br>(ng/mL) | Recovery (%) |
|-------|--------------------------------------|--------------------------------------|--------------|
| 1 | 0 | -1.0 | N/A |
| 2 | 100 | 104.5 | 104.5 |
| 3 | 200 | 196.2 | 98.1 |

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| Level | Expected<br>Concentration<br>(ng/mL) | Observed<br>Concentration<br>(ng/mL) | Recovery (%) |
|-------|--------------------------------------|--------------------------------------|--------------|
| 4 | 300 | 314.7 | 104.9 |
| 5 | 400 | 455.3 | 113.8 |
| 6 | 500 | 565.2 | 113.0 |
| 7 | 600 | 661.0 | 110.2 |
| 8 | 700 | 764.7 | 109.2 |
| 9 | 800 | 872.1 | 109.0 |
| 10 | 900 | 937.3 | 104.1 |
| 11 | 1000 | 1024.9 | 102.5 |

# d) Method Comparison and Accuracy

The method comparison study was performed in accordance with CLSI Guideline EP09-A3. One hundred and six patient samples were analyzed by the DRI Benzodiazepine Assay in both qualitative and semi-quantitative modes and the results were compared to LC-MS/MS. The overall concordance between LC-MS/MS and DRI Benzodiazepine Assay is 96.2%. The qualitative and semi-quantitative results are summarized in the tables below.

| Candidate<br>Device<br>Results | Negative<br>by LC-MS/MS | < 50% of<br>Cutoff<br>concentration<br>by LC-MS/MS<br>(< 100ng/mL) | Near Cutoff<br>Negative<br>(Between 50%<br>below the<br>cutoff and the<br>cutoff<br>concentration<br>as determined<br>by LC-<br>MS/MS) (100<br>- 199 ng/mL) | Near Cutoff<br>Positive<br>(Between the<br>cutoff and 50%<br>above the<br>cutoff<br>concentration<br>as determined<br>by LC-MS/MS)<br>(200 – 300<br>ng/mL) | High Positives<br>(Greater than<br>50% above<br>cutoff<br>concentration<br>(> 300 ng/mL) |
|--------------------------------|-------------------------|--------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|
| Positive | 0 | 1* | 3* | 5 | 45 |
| Negative | 48 | 2 | 2 | 0 | 0 |

# Qualitative Accuracy study with LC-MS/MS as reference method

Negative agreement is 52/56 * 100% = 92.9% Positive agreement is 50/50 * 100% = 100%

Overall agreement is 102/106 * 100% = 96.2%

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#### Semi-Quantitative Mode Accuracy study with LC-MS/MS as reference method

| Candidate<br>Device<br>Results | Negative<br>by LC-MS/MS | < 50% of<br>Cutoff<br>concentration<br>by LC-MS/MS<br>(< 100ng/mL) | Near Cutoff<br>Negative<br>(Between 50%<br>below the<br>cutoff and the<br>cutoff<br>concentration<br>as determined<br>by LC-<br>MS/MS) (100<br>- 199 ng/mL) | Near Cutoff<br>Positive<br>(Between the<br>cutoff and 50%<br>above the<br>cutoff<br>concentration<br>as determined<br>by LC-MS/MS)<br>(200 – 300<br>ng/mL) | High Positives<br>(Greater than<br>50% above<br>cutoff<br>concentration<br>(> 300 ng/mL) |
|--------------------------------|-------------------------|--------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|
| Positive | 0 | 1* | 3* | 5 | 45 |
| Negative | 48 | 2 | 2 | 0 | 0 |

Negative agreement is 52/56 * 100% = 92.9% Positive agreement is 50/50 * 100% = 100% Overall agreement is 102/106 * 100% = 96.2%

#### *Discordant samples

| Sample ID | Qualitative | Semi-<br>Quantitative | LC-MS/MS |
|--------------|-----------------------|-----------------------|---------------------------------------------|
| | Negative/<br>Positive | Negative/<br>Positive | Total Benzodiazepine<br>Parent Only (ng/mL) |
| CA160606-045 | Positive | Positive | 86.20 |
| CA160926-057 | Positive | Positive | 175.08 |
| CA170605-001 | Positive | Positive | 151.52 |
| CA160908-003 | Positive | Positive | 192.87 |

These four samples are discordant because of the presence of Benzodiazepine metabolites. Sample CA160606-045 contains 3154.59 ng/mL 7-Aminoclonazepam .

Sample CA160926-057 contains 13.46 ng/mL a-Hydroxyalprazolam and 410.69 ng/mL 7-Aminoclonazepam.

Sample CA170605-001 contains 1.43 ng/mL a-Hydroxyalprazolam and 560.37 ng/mL 7-Aminoclonazepam.

Sample CA160908-003 contains 96.27 ng/mL α-Hydroxyalprazolam.

# e) Specificity

The cross-reactivity of benzodiazepine compounds and their metabolites were evaluated by adding known amounts of each compound to drug-free negative urine. The results are summarized in the tables below.

Cross reactivity of benzodiazepine compounds and structurally unrelated compound*

| Structurally related and | Tested Concentration | Cross-reactivity |
|--------------------------|----------------------|------------------|
| unrelated compounds | (ng/mL) | (%) |
| α-Hydroxyalprazolam | 110 | 182 |
| α-Hydroxytriazolam | 140 | 143 |
| Alprazolam | 110 | 182 |

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| Structurally related and | Tested Concentration | Cross-reactivity |
|--------------------------|----------------------|------------------|
| unrelated compounds | (ng/mL) | (%) |
| 7-Aminoclonazepam | 2,500 | 8 |
| 7-Aminoflunitrazepam | 300 | 67 |
| 7-Aminonitrazepam | 300 | 67 |
| Bromazepam | 170 | 118 |
| Chlordiazepoxide | 700 | 29 |
| Clobazam | 150 | 133 |
| Clonazepam | 210 | 95 |
| Clorazepate | 135 | 148 |
| Delorazepam | 150 | 133 |
| Demoxepam | 220 | 91 |
| Desalkylflurazepam | 130 | 154 |
| Diazepam | 110 | 182 |
| Estazolam | 100 | 200 |
| Flunitrazepam | 120 | 167 |
| Flurazepam | 150 | 133 |
| 2-Hydroxyethylflurazepam | 120 | 167 |
| Lorazepam | 700 | 29 |
| Lorazepam glucuronide | 50,000 | <0.4 |
| Lormetazepam | 275 | 73 |
| Medazepam | 325 | 62 |
| Midazolam | 180 | 111 |
| Nitrazepam | 130 | 154 |
| Norchlordiazepoxide | 800 | 25 |
| Nordiazepam | 110 | 182 |
| *Oxaprozin | 125,000 | 0.16 |
| Oxazepam | 200 | 100 |
| Oxazepam glucuronide | 50,000 | 0.4 |
| Prazepam | 200 | 100 |
| Temazepam | 160 | 125 |
| Temazepam glucuronide | 50,000 | <0.4 |
| Triazolam | 130 | 154 |

Structurally unrelated compounds were evaluated by adding each substance to Oxazepam spiked at low control, 150 ng/mL (-25% of the cutoff concentration) and the high control, 250 ng/mL (+25% of the cutoff concentration), at the concentrations indicated. As shown in the table below, the Controls were detected accurately, Low Control as Negative and the High Control as Positive, indicating that all the compounds evaluated exhibited no significant cross-reactivity at the concentrations tested.

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Structurally unrelated compounds spiked at the concentration listed below into Low and High control urine

| Structurally Unrelated Compounds | Tested Concentration (ng/mL) | Spiked Oxazepam Level | | |
|-------------------------------------|-------------------------------------|--------------------------------------|---------------------------------------|---------------------------------------|
| | | Low Control Positive/Negative | High Control Positive/Negative | |
| 6-Acetyl morphine | 100,000 | Negative | Positive | |
| 10,11 Dihydrocarbamazepine | 100,000 | Negative | Positive | |
| 11-nor-Δ9-THC-COOH | 100,000 | Negative | Positive | |
| Acetaminophen | 1,000,000 | Negative | Positive | |
| Acetylsalicylic acid | 1,000,000 | Negative | Positive | |
| Amitriptyline | 100,000 | Negative | Positive | |
| Amoxicillin | 100,000 | Negative | Positive | |
| Amphetamine | 100,000 | Negative | Positive | |
| Amisulpride | 100,000 | Negative | Positive | |
| Benzotropine Mesylate | 100,000 | Negative | Positive | |
| Benzoylecgonine | 100,000 | Negative | Positive | |
| Brompheniramine | 100,000 | Negative | Positive | |
| Buprenorphine | 100,000 | Negative | Positive | |
| Caffeine | 100,000 | Negative | Positive | |
| Captopril | 100,000 | Negative | Positive | |
| Chlorpromazine | 100,000 | Negative | Positive | |
| Chloroquine | 100,000 | Negative | Positive | |
| Cimetidine | 100,000 | Negative | Positive | |
| Clomipramine | 100,000 | Negative | Positive | |
| Codeine | 100,000 | Negative | Positive | |
| Desipramine | 100,000 | Negative | Positive | |
| Dextromethorphan | 100,000 | Negative | Positive | |
| Digoxin | 100,000 | Negative | Positive | |
| Dihydrocodeine | 100,000 | Negative | Positive | |
| Diphenhydramine | 500,000 | Negative | Positive | |
| Doxepine HCl | 100,000 | Negative | Positive | |
| EDDP | 100,000 | Negative | Positive | |
| EMDP | 25,000 | Negative | Positive | |
| Enalapril | 100,000 | Negative | Positive | |
| Fentanyl | 100,000 | Negative | Positive | |
| Fluoxetine | 500,000 | Negative | Positive | |
| Fluophenazine | 100,000 | Negative | Positive | |
| Haloperidol | 100,000 | Negative | Positive | |
| Heroin | 100,000 | Negative | Positive | |
| Hydrocodone | 100,000 | Negative | Positive | |
| Hydromorphone | 100,000 | Negative | Positive | |
| Hydroxychloroquine | 100,000 | Negative | Positive | |
| Hydroxyzine | 100,000 | Negative | Positive | |
| Structurally Unrelated<br>Compounds | Tested<br>Concentration<br>(ng/mL) | Spiked Oxazepam Level | | |
| | | Low Control<br>Positive/<br>Negative | High Control<br>Positive/<br>Negative | |
| Ibuprofen | 100,000 | Negative | Positive | |
| Imipramine | 100,000 | Negative | Positive | |
| LAAM | 100,000 | Negative | Positive | |
| Levorphanol | 100,000 | Negative | Positive | |
| Levothyroxine | 100,000 | Negative | Positive | |
| Maprotiline | 100,000 | Negative | Positive | |
| Meperidine | 100,000 | Negative | Positive | |
| Methadone | 100,000 | Negative | Positive | |
| Methamphentamine | 100,000 | Negative | Positive | |
| Morphine | 100,000 | Negative | Positive | |
| Morphine-3β-D-glucuronide | 100,000 | Negative | Positive | |
| Morphine-6β-D-glucuronide | 100,000 | Negative | Positive | |
| Nalbuphine | 100,000 | Negative | Positive | |
| Nalorphine | 100,000 | Negative | Positive | |
| Naloxone | 100,000 | Negative | Positive | |
| Naltrexone | 100,000 | Negative | Positive | |
| Naproxen | 100,000 | Negative | Positive | |
| Nifedipine | 100,000 | Negative | Positive | |
| Norcodeine | 100,000 | Negative | Positive | |
| Norhydrocodone | 100,000 | Negative | Positive | |
| Norfluoxetine | 500,000 | Negative | Positive | |
| Noroxycodone | 100,000 | Negative | Positive | |
| Noroxymorphone | 100,000 | Negative | Positive | |
| Norpropoxyphene | 100,000 | Negative | Positive | |
| Norsertraline | 62,500 | Negative | Positive | |
| Nortryptiline | 100,000 | Negative | Positive | |
| Oxycodone | 100,000 | Negative | Positive | |
| Oxymorphone | 100,000 | Negative | Positive | |
| Paroxetine | 100,000 | Negative | Positive | |
| Perphenazine | 100,000 | Negative | Positive | |
| Phencyclidine | 100,000 | Negative | Positive | |
| Phenobarbital | 100,000 | Negative | Positive | |
| Procyclidine | 100,000 | Negative | Positive | |
| Propoxyphene | 100,000 | Negative | Positive | |
| Protriptyline | 100,000 | Negative | Positive | |
| Ranitidine | 100,000 | Negative | Positive | |
| Secobarbital | 100,000 | Negative | Positive | |
| Sertraline | 62,500 | Negative | Positive | |
| Sulpiride | 100,000 | Negative | Positive | |
| | Structurally Unrelated<br>Compounds | Tested<br>Concentration<br>(ng/mL) | Spiked Oxazepam Level | |
| | | | Low Control<br>Positive/<br>Negative | High Control<br>Positive/<br>Negative |
| | Tapentadol | 100,000 | Negative | Positive |
| | Thioridazine | 100,000 | Negative | Positive |
| | Tramadol | 100,000 | Negative | Positive |
| | Triprolidine | 100,000 | Negative | Positive |
| | Verapamil | 100,000 | Negative | Positive |
| | Zaleplon | 100,000 | Negative | Positive |
| | Zolpidem | 100,000 | Negative | Positive |
| | Zopiclone | 100,000 | Negative | Positive |

{12}------------------------------------------------

{13}------------------------------------------------

#### f) Interference

The interference studies were performed in accordance with CLSI Guideline EP07-A2, using both Qualitative and Semi-quantitative modes. The potential interference of pH and endogenous physiologic substances on recovery of Oxazepam using DRI Benzodiazepine Assay was assessed by spiking known compounds of potentially interfering substances into the Low Control, 150 ng/mL (-25% of the cutoff concentration) and High Control, 250 ng/mL (+25% of the cutoff concentration). In the presence of the compounds listed below, the controls were detected accurately, indicating that these compounds did not show interference in the assay.

# Interference substances

| Compound | Tested Concentration<br>(mg/dL) | Spiked Oxazepam Level | |
|---------------------|---------------------------------|----------------------------------------------|-----------------------------------------------|
| | | Low Control<br>-25% of cutoff<br>(150 ng/mL) | High Control<br>+25% of cutoff<br>(250 ng/mL) |
| Acetone | 500 | Negative | Positive |
| Ascorbic acid | 150 | Negative | Positive |
| Creatinine | 400 | Negative | Positive |
| Ethanol | 1000 | Negative | Positive |
| Galactose | 5 | Negative | Positive |
| Glucose | 1000 | Negative | Positive |
| Hemoglobin | 150 | Negative | Positive |
| Human serum albumin | 200 | Negative | Positive |
| Oxalic acid | 50 | Negative | Positive |
| Riboflavin | 3 | Negative | Positive |
| Sodium Chloride | 1000 | Negative | Positive |
| Urea | 1000 | Negative | Positive |

{14}------------------------------------------------

| Compound | Tested Concentration<br>(mg/dL) | Spiked Oxazepam Level | |
|----------|---------------------------------|----------------------------------------------|-----------------------------------------------|
| | | Low Control<br>-25% of cutoff<br>(150 ng/mL) | High Control<br>+25% of cutoff<br>(250 ng/mL) |
| pH | pH | | |
| pH | 3.0 | Negative | Positive |
| pH | 4.0 | Negative | Positive |
| pH | 5.0 | Negative | Positive |
| pH | 6.0 | Negative | Positive |
| pH | 7.0 | Negative | Positive |
| pH | 8.0 | Negative | Positive |
| pH | 9.0 | Negative | Positive |
| pH | 10.0 | Negative | Positive |
| pH | 11.0 | Negative | Positive |

# g) Specific Gravity

Drug free urine samples with specific gravity ranging in value within 1.000 to 1.030 were split and spiked with Oxazepam to a final concentration of either 150 ng/mL or 250ng/mL (the Low Control and High Concentrations, respectively). These samples were then evaluated in both qualitative and semi-quantitative modes. The Controls were detected accurately, indicating that no interference was observed.

| Specific Gravity | Spiked Oxazepam Level<br>Low Control | High Control |
|------------------|--------------------------------------|--------------|
| 1.004 | Negative | Positive |
| 1.005 | Negative | Positive |
| 1.007 | Negative | Positive |
| 1.010 | Negative | Positive |
| 1.011 | Negative | Positive |
| 1.013 | Negative | Positive |
| 1.019 | Negative | Positive |
| 1.023 | Negative | Positive |
| 1.025 | Negative | Positive |
| 1.029 | Negative | Positive |

# H. Conclusion

The information supports a determination of substantial equivalence between DRI Benzodiazepine Assay and the predicate device Benzodiazepine Enzyme Immunoassay (K930529)