Immunalysis Benzodiazepines Urine Enzyme Immunoassay, Immunalysis Multi-Drug Calibrators

K151771 · Immunalysis Corporation · JXM · Jan 5, 2016 · Clinical Toxicology

Device Facts

• Record ID: K151771
• Device Name: Immunalysis Benzodiazepines Urine Enzyme Immunoassay, Immunalysis Multi-Drug Calibrators
• Applicant: Immunalysis Corporation
• Product Code: JXM · Clinical Toxicology
• Decision Date: Jan 5, 2016
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3170
• Device Class: Class 2

Indications for Use

The Immunalysis Benzodiazepines Urine Enzyme Immunoassay is a homogeneous enzyme immunoassay with a cutoff of 200ng/mL. The assay is intended for use in laboratories for the qualitative and semi-quantitative analysis of Benzodiazepines in human urine with automated clinical chemistry analyzers. This assay is calibrated against Oxazepam. This in-vitro device is for prescription use only. The semi-quantitative mode is for purposes of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Gas Chromatography/ Mass Spectrometry (GC-MS) or permitting laboratories to establish quality control procedures. The Immunalysis Benzodiazepines Urine Enzyme Immunoassay Kit provides only a preliminary analytical test result. A more specific alternate chemical must be used in order to obtain a confirmed analytical result. GC-MS or Liquid Chromatography / Mass Spectrometry (LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used. The Immunalysis Multi-Drug Calibrators are intended for in vitro diagnostic use for the calibration of assays for the analytes currently listed in the package insert: Benzoylecgonine, Morphine and Oxazepam. The calibrators are designed for prescription use with immunoassays.

Device Story

The Immunalysis Benzodiazepines Urine Enzyme Immunoassay is a homogeneous enzyme immunoassay used in clinical laboratories to detect benzodiazepines in human urine. The device utilizes monoclonal antibodies to benzodiazepines, glucose-6-phosphate, and NAD in an antibody/substrate reagent, combined with a benzodiazepine derivative labeled with G6PDH enzyme. When benzodiazepines are present in the urine sample, they compete with the enzyme-labeled drug for antibody binding sites, resulting in a measurable change in enzyme activity. The assay is performed on automated clinical chemistry analyzers (e.g., Beckman Coulter AU 400e). Results are provided as qualitative (positive/negative relative to a 200ng/mL cutoff) or semi-quantitative. The output assists clinicians in identifying potential drug presence, which must be confirmed by GC-MS or LC/MS. The device aids in clinical decision-making regarding drug abuse testing and quality control procedures.

Clinical Evidence

Bench testing only. Precision/reproducibility evaluated over 20 days (N=80) at concentrations ±25%, ±50%, ±75%, and ±100% of the 200 ng/mL cutoff. Linearity confirmed from 100-1100 ng/mL. Method comparison with LC/MS using 86 clinical urine samples showed 98% positive agreement and 100% negative agreement. Interference testing performed for structurally related/unrelated compounds, endogenous substances, pH, and specific gravity.

Technological Characteristics

Homogeneous enzyme immunoassay; two-reagent liquid format (antibody/substrate and enzyme conjugate). Reagents contain monoclonal antibodies, G6PDH-labeled benzodiazepine derivative, G6P, NAD, Tris/HEPES buffers, and sodium azide preservative. Operates via spectrophotometric measurement at 340 nm. Designed for use on automated clinical chemistry analyzers. Storage at 2-8°C.

Indications for Use

Indicated for the qualitative and semi-quantitative analysis of benzodiazepines in human urine in laboratory settings using automated clinical chemistry analyzers. Intended for prescription use to provide preliminary analytical results requiring confirmation by GC-MS or LC/MS.

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® Benzodiazepines Assay (k930529)
• LZI Multiple Analyte Drugs of Abuse Calibrators and Controls (k051088)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k151771

B. Purpose for Submission:
New device

C. Measurand:
Benzodiazepines

D. Type of Test:
Qualitative and semi-quantitative homogeneous enzyme immunoassay

E. Applicant:
Immunalysis Corporation

F. Proprietary and Established Names:
Immunalysis Benzodiazepines Urine Enzyme Immunoassay
Immunalysis Multi-Drug Calibrators

G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| JXM – Benzodiazepine test system | Class II | 21 CFR §862.3170 | 91 – Toxicology |
| DKB – Clinical Toxicology Calibrator | Class II | 21 CFR §862.3200 | 91 – Toxicology |

H. Intended Use:
1. Intended use(s):
See Indication(s) for use below.
2. Indication(s) for use:

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Immunalysis Benzodiazepine Urine Enzyme Immunoassay

The Immunalysis Benzodiazepine Urine Enzyme Immunoassay is a homogeneous enzyme immunoassay with a cutoff of 200ng/mL. The assay is intended for use in laboratories for the qualitative and semi-quantitative analysis of Benzodiazepine in human urine with automated clinical chemistry analyzers. This assay is calibrated against Oxazepam. This in-vitro device is for prescription use only.

The semi-quantitative mode is for purposes of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Gas Chromatography/ Mass Spectrometry (GC-MS) or permitting laboratories to establish quality control procedures.

The Immunalysis Benzodiazepine Urine Enzyme Immunoassay Kit provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. GC-MS or Liquid Chromatography / Mass Spectrometry (LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

Immunalysis Multi-Drug Calibrators

The Immunalysis Multi-Drug Calibrators are intended for in vitro diagnostic use for the calibration of assays for the analytes currently listed in the package insert: Benzoylecgonine, Morphine and Oxazepam. The calibrators are designed for prescription use with immunoassays.

3. Special conditions for use statement(s):

For prescription use only.

4. Special instrument requirements:

Performance data was obtained using the Beckman AU400e clinical chemistry analyzer.

I. Device Description:

The Immunalysis Benzodiazepine Urine Enzyme Immunoassay Kit contains two reagents, which are provided as ready-to-use:

- Antibody/Substrate Reagent (RA) – This reagent contains monoclonal antibodies to Benzodiazepines, glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide (NAD) in Tris buffer with Sodium Azide as a preservative.
- Enzyme Conjugate Reagent (RE) – This reagent contains benzodiazepines derivative labeled with glucose-6-phosphate dehydrogenase (G6PDH) in HEPES buffer with Sodium Azide as a preservative.

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The Immunalysis Multi-Drug Calibrators set is liquid and ready-to-use. The negative calibrator is a processed, drug-free synthetic urine matrix with sodium azide as a preservative. Each calibrator level contains a known concentration of oxazepam spiked into the negative calibrator matrix.

J. Substantial Equivalence Information:

1. Predicate device name(s):

DRI® Benzodiazepines Assay

LZI Multiple Analyte Drugs of Abuse Calibrators and Controls

2. Predicate 510(k) number(s):

k930529

k051088

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Candidate Device | Predicate – k930529 |
| Intended Use | Same | For the qualitative and semiquantitative determination of the presence of Benzodiazepines in human urine at a cutoff of 200 ng/mL |
| Test Principle | Same | Homogeneous Enzyme Immunoassay |
| Cutoff | Same | 200 ng/mL |
| Reagent Form | Same | Liquid Ready-to-Use Two Reagent Assay |
| Reagent Storage | Same | 2 – 8°C |
| Differences | | |
| --- | --- | --- |
| Item | Candidate Device | Predicate – k930529 |
| Antibody | Monoclonal antibodies to Benzodiazepines | Sheep polyclonal antibody to Benzodiazepine |

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| Similarities | | |
| --- | --- | --- |
| Item | Candidate Device | Predicate – k051088 |
| Analyte | Benzoylecgonine, morphine, oxazepam | Benzoylecgonine, d-methamphetamine, methadone, morphine, oxazepam, secobarbital, phencyclidine and propoxyphene |
| Matrix | Same | Urine |
| Calibrator Levels | Same | 5 levels |
| Storage | Same | 2 – 8 °C |

K. Standard/Guidance Document Referenced (if applicable):

- CLSI EP5-A2, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—Second Edition
- CLSI EP7-A2, Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition

L. Test Principle:

The assay is based on the competition of Oxazepam labeled enzyme glucose-6-phosphate dehydrogenase (G6PDH) and the free drug in the urine sample for the fixed amount of antibody binding sites. In the absence of the free drug in the sample, the antibody binds the drug enzyme conjugate and enzyme activity is inhibited. This creates a dose response relationship between drug concentration in the urine sample and enzyme activity. The enzyme G6PDH activity is determined at 340 nm spectrophotometrically by the conversion of NAD to NADH.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

A precision/cutoff characterization study was performed for 20 days, 2 runs per day in duplicate (N=80) on drug-free negative urine samples spiked with oxazepam to concentrations of ±25%, ±50%, ±75%, and ±100% of each cutoff. The spiked concentrations were confirmed by mass spectrometry (MS). The results of the study are summarized below:

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| Qualitative Analysis | | | |
| --- | --- | --- | --- |
| Concentration (ng/mL) | % of cutoff | # of determinations | Result |
| 0 | -100% | 80 | 80 Neg / 0 Pos |
| 50 | -75% | 80 | 80 Neg / 0 Pos |
| 100 | -50% | 80 | 80 Neg / 0 Pos |
| 150 | -25% | 80 | 80 Neg / 0 Pos |
| 200 | Cutoff | 80 | 37 Neg / 43 Pos |
| 250 | +25% | 80 | 80 Pos / 0 Neg |
| 300 | +50% | 80 | 80 Pos / 0 Neg |
| 350 | +75% | 80 | 80 Pos / 0 Neg |
| 400 | +100% | 80 | 80 Pos / 0 Neg |
| Semi-quantitative Analysis | | | |
| --- | --- | --- | --- |
| Concentration (ng/mL) | % of cutoff | # of determinations | Result |
| 0 | -100% | 80 | 80 Neg / 0 Pos |
| 50 | -75% | 80 | 80 Neg / 0 Pos |
| 100 | -50% | 80 | 80 Neg / 0 Pos |
| 150 | -25% | 80 | 80 Neg / 0 Pos |
| 200 | Cutoff | 80 | 34 Neg / 46 Pos |
| 150 | +25% | 80 | 80 Pos / 0 Neg |
| 300 | +50% | 80 | 80 Pos / 0 Neg |
| 350 | +75% | 80 | 80 Pos / 0 Neg |
| 400 | +100% | 80 | 80 Pos / 0 Neg |

b. Linearity/assay reportable range:

A linearity study in the semi-quantitative mode was conducted by spiking a drug-free urine pool with a high concentration of oxazepam and generating serial dilutions to achieve concentrations ranging from 100 ng/mL to 1100 ng/mL. Each concentration was tested in triplicate and drug recovery calculated using the mean concentration of the replicates. The results are summarized below:

| Linearity/Recovery | | |
| --- | --- | --- |
| Expected Concentration (ng/mL) | Mean Concentration (ng/mL) | Recovery (%) |
| 100 | 94.2 | 94.2 |
| 200 | 213.7 | 106.8 |
| 300 | 313.9 | 104.6 |
| 400 | 389.7 | 97.4 |
| 500 | 511.6 | 102.3 |
| 600 | 637.2 | 106.2 |
| 700 | 693.2 | 99.0 |
| 800 | 820.7 | 102.6 |

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| 900 | 907.6 | 100.8 |
| --- | --- | --- |
| 1000 | 1025.3 | 102.5 |
| 1100 | 1061.1 | 96.5 |

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

## Traceability

The analyte in the calibrator is traceable to a commercially available oxazepam solution. This standard is diluted with synthetic negative urine to make the calibrators to the desired concentrations. The concentrations are confirmed by Gas Chromatography/Mass Spectrometry Analysis (GC/MS) and/or Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS).

## Value Assignment/Expected Values

The Negative Calibrator is a processed, drug free urine matrix. The standard is compared to a reference negative standard to ensure that it is free of analyte. A value is assigned when the test is within the acceptable range.

The non-zero calibrators are prepared by spiking known concentrations of oxazepam into the negative calibrator matrix. The concentrations are confirmed by GC/MS or LC/MS/MS. If any of the analytes are out of the acceptable range, then the calibrator is adjusted and re-tested. Values are assigned to the calibrator once the GC/MS or LC/MS/MS results are within the acceptable range.

## Calibrator Stability

Closed-vial accelerated stability and open vial stability studies were conducted for the calibrators. Real-time stability studies are ongoing. All stability protocols were reviewed and found to be acceptable. These studies support the closed vial stability claim of 12 months and the opened vial stability claim of 60 days when calibrators are stored at 2-8°C.

d. Detection limit:

Not applicable.

e. Analytical specificity:

## Structurally related compounds

The sponsor performed cross-reactivity studies in both qualitative and semi-quantitative modes by spiking various benzodiazepines or structurally related

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compounds into drug free urine at levels that will yield a result that is equivalent to the assay cutoff (200 ng/mL). The results were the same for the qualitative and semi-quantitative modes and are summarized below:

| Structurally Related Compounds Qualitative & Semi-quantitative | | |
| --- | --- | --- |
| Compound | Lowest Concentration Producing a Positive Result (ng/mL) | % Cross-reactivity |
| Oxazepam | 200 | 100.0 |
| Alpha-hydroxyalprazolam | 110 | 181.8 |
| Alprazolam | 120 | 166.7 |
| 7-Aminoclonazepam | 100,000 | < 0.002 |
| 7-Aminoflunitrazepam | 3,500 | 5.7 |
| 7-Aminonitrazepam | 35,000 | 0.6 |
| Bromazepam | 750 | 26.7 |
| Chlordiazepoxide | 1,600 | 12.5 |
| Clorazepate | 200 | 100.0 |
| Clobazam | 650 | 30.8 |
| Clonazepam | 180 | 111.1 |
| Demoxepam | 5,500 | 3.6 |
| Desalkyflurazepam | 75 | 266.7 |
| Diazepam | 100 | 200.0 |
| Estazolam | 225 | 88.9 |
| Flunitrazepam | 125 | 160.0 |
| Flurazepam | 110 | 181.8 |
| Lorazepam | 60 | 333.3 |
| Lorazepam glucuronide | 180 | 111.1 |
| Lormetazepam | 50 | 400.0 |
| Medazepam | 500 | 40.0 |
| Midazolam | 40 | 500.0 |
| Nitrazepam | 700 | 28.6 |
| Norchlordiazepoxide | 2,200 | 9.1 |
| Nordiazepam | 180 | 111.1 |
| Oxazepam glucuronide | 1,300 | 15.4 |
| Prazepam | 95 | 210.5 |
| Temazepam | 110 | 181.8 |
| Temazepam glucuronide | 700 | 28.6 |
| Triazolam | 50 | 400.0 |

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Non-structurally related compounds

Potential interference from non-structurally related drugs and metabolites was evaluated in the qualitative and semi-quantitative modes by spiking these compounds into drug free urine containing oxazepam at $\pm 25\%$ of the $200~\mathrm{ng / mL}$ cutoff (150 ng/mL and $250~\mathrm{ng / mL}$ respectively). The results were the same for the qualitative and semi-quantitative modes and are summarized below:

| Structurally Unrelated Compounds | | | |
| --- | --- | --- | --- |
| Compound | Concentration Tested (ng/mL) | -25% Cutoff (150 ng/mL) | +25% Cutoff (250 ng/mL) |
| 4-Bromo-2,5,Dimethoxyphenethylamine | 100,000 | Negative | Positive |
| 6-Acetylcodeine | 100,000 | Negative | Positive |
| 6-Acetylmorphine | 100,000 | Negative | Positive |
| Acetaminophen | 500,000 | Negative | Positive |
| Acetylsalicylic Acid | 500,000 | Negative | Positive |
| Amitriptyline | 100,000 | Negative | Positive |
| Amobarbital | 100,000 | Negative | Positive |
| S-(+)-Amphetamine | 100,000 | Negative | Positive |
| Benzoylecgonine | 500,000 | Negative | Positive |
| Benzylpiperazine | 100,000 | Negative | Positive |
| Buprenorphine | 100,000 | Negative | Positive |
| Bupropion | 100,000 | Negative | Positive |
| Butabarbital | 100,000 | Negative | Positive |
| Caffeine | 500,000 | Negative | Positive |
| Carbamazepine | 100,000 | Negative | Positive |
| Chlorpromazine | 100,000 | Negative | Positive |
| cis-Tramadol | 100,000 | Negative | Positive |
| Clomipramine | 100,000 | Negative | Positive |
| Cannabidiol | 100,000 | Negative | Positive |
| Cannabinol | 100,000 | Negative | Positive |
| Carisoprodol | 100,000 | Negative | Positive |
| Cocaine | 100,000 | Negative | Positive |
| Codeine | 100,000 | Negative | Positive |
| Cotinine | 100,000 | Negative | Positive |
| Cyclobenzaprine | 100,000 | Negative | Positive |
| Delta-9-THC | 100,000 | Negative | Positive |
| Desipramine | 100,000 | Negative | Positive |
| N-desmethyltapentadol | 100,000 | Negative | Positive |

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| Structurally Unrelated Compounds | | | |
| --- | --- | --- | --- |
| Compound | Concentration Tested (ng/mL ) | -25% Cutoff (150 ng/mL) | +25% Cutoff (250 ng/mL) |
| Dextromethorphan | 100,000 | Negative | Positive |
| Dihydrocodeine | 100,000 | Negative | Positive |
| Diphenhydramine | 500,000 | Negative | Positive |
| Doxepin | 100,000 | Negative | Positive |
| Ecgonine | 100,000 | Negative | Positive |
| Ecgonine methyl ester | 100,000 | Negative | Positive |
| EDDP | 100,000 | Negative | Positive |
| 1R,2S(-)-Ephedrine | 100,000 | Negative | Positive |
| 1S,2R(+)-Ephedrine | 100,000 | Negative | Positive |
| Ethyl β-D-glucuronide | 100,000 | Negative | Positive |
| Ethylmorphine | 100,000 | Negative | Positive |
| Fenfluramine | 100,000 | Negative | Positive |
| Fentanyl | 100,000 | Negative | Positive |
| Fluoxetine | 100,000 | Negative | Positive |
| Heroin | 100,000 | Negative | Positive |
| Hexobarbital | 100,000 | Negative | Positive |
| Hydrocodone | 100,000 | Negative | Positive |
| Hydromorphone | 100,000 | Negative | Positive |
| 11-hydroxy-delta-9-THC | 100,000 | Negative | Positive |
| Ibuprofen | 100,000 | Negative | Positive |
| Imipramine | 100,000 | Negative | Positive |
| Ketamine | 100,000 | Negative | Positive |
| Lamotrigine | 100,000 | Negative | Positive |
| Levorphanol Tartrate | 100,000 | Negative | Positive |
| Lidocaine | 100,000 | Negative | Positive |
| LSD | 100,000 | Negative | Positive |
| Maprotiline | 100,000 | Negative | Positive |
| (+)-MDA | 100,000 | Negative | Positive |
| MDEA | 100,000 | Negative | Positive |
| MDMA | 100,000 | Negative | Positive |
| Meperidine | 100,000 | Negative | Positive |
| Meprobamate | 100,000 | Negative | Positive |
| Methadone | 500,000 | Negative | Positive |
| S(+)-Methamphetamine | 100,000 | Negative | Positive |
| Methaquolone | 100,000 | Negative | Positive |
| Methylphenidate | 100,000 | Negative | Positive |

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| Structurally Unrelated Compounds | | | |
| --- | --- | --- | --- |
| Compound | Concentration Tested (ng/mL ) | -25% Cutoff (150 ng/mL) | +25% Cutoff (250 ng/mL) |
| Morphine | 100,000 | Negative | Positive |
| Morphine-3-glucuronide | 100,000 | Negative | Positive |
| Morphine-6-glucuronide | 100,000 | Negative | Positive |
| Nalorphine | 100,000 | Negative | Positive |
| Naloxone | 100,000 | Negative | Positive |
| Naltrexone | 100,000 | Negative | Positive |
| Norbuprenorphine | 100,000 | Negative | Positive |
| Norcodeine | 100,000 | Negative | Positive |
| Normorphine | 100,000 | Negative | Positive |
| Norpropoxyphene | 100,000 | Negative | Positive |
| Norpseudoephedrine | 100,000 | Negative | Positive |
| Nortriptyline | 100,000 | Negative | Positive |
| Oxycodone | 100,000 | Negative | Positive |
| Oxymorphone | 100,000 | Negative | Positive |
| PCP | 100,000 | Negative | Positive |
| Pentazocine | 100,000 | Negative | Positive |
| Pentobarbital | 100,000 | Negative | Positive |
| Phentermine | 100,000 | Negative | Positive |
| Phenobarbital | 100,000 | Negative | Positive |
| Phenylephedrine | 100,000 | Negative | Positive |
| Phenylpropanolamine | 100,000 | Negative | Positive |
| Phenytoin | 100,000 | Negative | Positive |
| PMA | 100,000 | Negative | Positive |
| Propoxyphene | 100,000 | Negative | Positive |
| Propranolol | 100,000 | Negative | Positive |
| Protriptyline | 100,000 | Negative | Positive |
| R,R(-)-Pseudoephedrine | 100,000 | Negative | Positive |
| S,S(+)-Pseudoephedrine | 100,000 | Negative | Positive |
| Ranitidine | 100,000 | Negative | Positive |
| Ritalinic Acid | 100,000 | Negative | Positive |
| Salicylic Acid | 100,000 | Negative | Positive |
| Secobarbital | 100,000 | Negative | Positive |
| Sertraline | 100,000 | Negative | Positive |
| Sufentanil Citrate | 100,000 | Negative | Positive |
| 11-nor-9carboxy THC | 100,000 | Negative | Positive |
| Theophylline | 100,000 | Negative | Positive |

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| Structurally Unrelated Compounds | | | |
| --- | --- | --- | --- |
| Compound | Concentration Tested (ng/mL) | -25% Cutoff (150 ng/mL) | +25% Cutoff (250 ng/mL) |
| Thioridazine | 100,000 | Negative | Positive |
| Trifluoromethylphenyl-piperazine | 100,000 | Negative | Positive |
| Trimipramine | 100,000 | Negative | Positive |
| Trazodone | 100,000 | Negative | Positive |
| Venlafaxine | 100,000 | Negative | Positive |
| Zolpidem Tartrate | 100,000 | Negative | Positive |

## Endogenous compounds

Potential interference from endogenous compounds was evaluated in the qualitative and semi-quantitative modes by spiking these compounds into drug free urine containing oxazepam at $\pm 25\%$ of the $200~\mathrm{ng/mL}$ cutoff (150 ng/mL and 250 ng/mL, respectively). The results were the same for the qualitative and semi-quantitative modes and are summarized below:

| Endogenous Compounds | | | |
| --- | --- | --- | --- |
| Compound | Concentration Tested (ng/mL) | -25% Cutoff (150 ng/mL) | +25% Cutoff (250ng/mL) |
| Acetone | 1.0 g/dL | Negative | Positive |
| Ascorbic Acid | 1.5 g/dL | Negative | Positive |
| Bilirubin | 0.002 g/dL | Negative | Positive |
| Creatinine | 0.5 g/dL | Negative | Positive |
| Ethanol | 1.0 g/dL | Negative | Positive |
| Galactose | 0.01 g/dL | Negative | Positive |
| γ-Globulin | 0.5 g/dL | Negative | Positive |
| Glucose | 2.0 g/dL | Negative | Positive |
| Hemoglobin | 0.115 g/dL | Negative | Positive |
| Human Serum Albumin | 0.5 g/dL | Negative | Positive |
| Oxalic Acid | 0.1 g/dL | Negative | Positive |
| Riboflavin | 0.0075 g/dL | Negative | Positive |
| Sodium Azide | 1% w/v | Negative | Positive |
| Sodium Chloride | 6.0 g/dL | Negative | Positive |
| Sodium Fluoride | 1% w/v | Negative | Positive |
| Urea | 6.0 g/dL | Negative | Positive |

Boric Acid. Boric Acid was also evaluated. Boric Acid at a concentration of $1\%$ w/v was found to cause false negative results at $+25\%$ and $+50\%$ (250 ng/mL and 300 ng/mL, respectively) at the 200 ng/mL cutoff in both the qualitative and semiquantitative modes and the following statement is provided in the limitations

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section of the labeling: "Boric Acid at 1% w/v may cause false negative results. Boric Acid is not recommended as a preservative for urine".

## pH and Specific Gravity

To evaluate potential interference from the pH of urine, device performance in the qualitative and semi-quantitative modes was tested using a range of urine pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0). All test samples were prepared in drug free urine containing oxazepam at ±25% of the 200 ng/mL cutoff (150 ng/mL and 250 ng/mL, respectively). No positive or negative interference was observed at urine pH values ranging from 3.0 to 11.0 for each test mode.

To evaluate potential interference from the specific gravity of urine, device performance in the qualitative and semi-quantitative modes was tested using a range of urine specific gravity values (1.000, 1.002, 1.005, 1.010, 1.015, 1.020, 1.025 and 1.030). All test samples were prepared in drug free urine containing oxazepam at ±25% of the 200 ng/mL cutoff (150 ng/mL and 250 ng/mL, respectively). No positive or negative interference was observed at urine specific gravity values ranging from 1.000 to 1.030 for each test mode.

## f. Assay cut-off:

Characterization of how the device performs analytically around the claimed cutoff concentrations of 200 ng/mL is described in the precision section, M.1.a. above.

## 2. Comparison studies:

### a. Method comparison with predicate device:

Eighty-six unaltered urine samples from clinical testing laboratories were analyzed for Benzodiazepine by the candidate device on the Beckman Coulter AU400e clinical chemistry analyzer and with LC/MS. The results were identical in both qualitative and semi-quantitative modes and are summarized below:

| Candidate Device Results | <50% cutoff (< 100 ng/mL) | -50% to cutoff (100 ~ 199 ng/mL) | cutoff to +50% (200 ~ 300 ng/mL) | >50% cutoff (> 300 ng/mL) |
| --- | --- | --- | --- | --- |
| Positive | 0 | 0 | 4 | 38 |
| Negative | 36 | 7 | 1* | 0 |

*sample contained 187 ng/mL Nordiazepam, which is equivalent to 207.8 ng/mL of Oxazepam (the calibrator drug).

% agreement among positives is 98%

% agreement among negatives is 100%

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b. Matrix comparison:
Not applicable.

3. Clinical studies:
a. Clinical Sensitivity:
Not applicable.
b. Clinical specificity:
Not applicable.
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable.

4. Clinical cut-off:
Not applicable.

5. Expected values/Reference range:
Not applicable.

N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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