LZI Methadone II Enzyme Immunoassay

K192433 · Lin-Zhi International, Inc. · DJR · Oct 4, 2019 · Clinical Toxicology

Device Facts

• Record ID: K192433
• Device Name: LZI Methadone II Enzyme Immunoassay
• Applicant: Lin-Zhi International, Inc.
• Product Code: DJR · Clinical Toxicology
• Decision Date: Oct 4, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3620
• Device Class: Class 2

Intended Use

The LZI Methadone II Enzyme Immunoassay is an in vitro diagnostic test intended for the qualitative and semi-quantitative determination of methadone in human urine. The cutoff for both the qualitative and semi-quantitative modes of the assay is 300 ng/mL for methadone. The assay is designed for prescription use on automated clinical chemistry analyzers. The semi-quantitative mode is for purposes of (1) enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas or liquid chromatography/mass spectrometry (GC/MS or LC/MS) or (2) permitting laboratories to establish quality control procedures. The assay provides only a preliminary analytical result. A more specific alternative analytical chemistry method must be used in order to obtain a confirmed analytical result. Gas or liquid chromatography/mass spectrometry (GC/MS or LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be exercised with any drug of abuse test result, particularly when the preliminary test result is positive.

Device Story

LZI Methadone II Enzyme Immunoassay is a homogeneous enzyme immunoassay for urine methadone detection. Input: human urine sample. Principle: competitive binding between sample methadone and methadone-labeled G6PDH enzyme for fixed antibody; free methadone displaces labeled enzyme, increasing enzyme activity. Output: absorbance change measured spectrophotometrically at 340 nm, correlating to methadone concentration. Used in clinical laboratories on automated chemistry analyzers by trained personnel. Provides preliminary screening results; requires confirmatory testing via GC/MS or LC/MS for clinical decision-making. Assists in identifying methadone presence for clinical or laboratory quality control purposes.

Clinical Evidence

Bench testing included precision (NCCLS-EP5, 88 runs), linearity (100-1000 ng/mL), cross-reactivity, and interference studies. Clinical accuracy evaluated using 94 unaltered clinical samples compared against LC/MS. Semi-quantitative accuracy showed 97.8% positive agreement and 97.9% negative agreement. Qualitative accuracy showed 97.8% positive agreement and 97.9% negative agreement.

Technological Characteristics

Homogeneous enzyme immunoassay. Reagents: R1 (mouse monoclonal anti-methadone antibody, G6P, NAD, stabilizers, 0.09% sodium azide); R2 (methadone-labeled G6PDH, buffer, 0.09% sodium azide). Sensing: spectrophotometric absorbance at 340 nm. Form factor: liquid reagents for automated clinical chemistry analyzers. Storage: 2-8°C.

Indications for Use

Indicated for qualitative and semi-quantitative determination of methadone in human urine at a 300 ng/mL cutoff. Intended for prescription use on automated clinical chemistry analyzers to provide preliminary analytical results. Requires confirmation by GC/MS or LC/MS.

Regulatory Classification

Identification

A methadone test system is a device intended to measure methadone, an addictive narcotic pain-relieving drug, in serum and urine. Measurements obtained by this device are used in the diagnosis and treatment of methadone use or overdose and to determine compliance with regulations in methadone maintenance treatment.

Special Controls

*Classification.* Class II (special controls). A methadone 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).

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health and Human Services logo on the left and the FDA logo on the right. The FDA logo is a blue square with the letters "FDA" in white, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue.

October 4, 2019

Lin-Zhi International, Inc Bernice Lin VP Operations 2945 Oakmead Village Court Santa Clara, CA 95051

Re: K192433

Trade/Device Name: LZI Methadone II Enzyme Immunoassay Regulation Number: 21 CFR 862.3620 Regulation Name: Methadone test system Regulatory Class: Class II Product Code: DJR Dated: August 30, 2019 Received: September 5, 2019

Dear Bernice Lin:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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

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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) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Kellie B. Kelm, Ph.D. Acting Director Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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### Indications for Use

510(k) Number (if known) k192433

Device Name LZI Methadone II Enzyme Immunoassay

#### Indications for Use (Describe)

The LZI Methadone II Enzyme Immunoassay is an in vitro diagnostic test intended for the qualitative and semi-quantitative determination of methadone in human urine. The cutoff for both the qualitative and semi-quantitative modes of the assay is 300 ng/mL for methadone. The assay is designed for prescription use on automated clinical chemistry analyzers.

The semi-quantitative mode is for purposes of (1) enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas or liquid chromatography/mass spectrometry (GCMS or LC/MS) or (2) permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical result. A more specific alternative analytical chemistry method must be used in order to obtain a confirmed analytical result. Gas or liquid chromatography/mass spectrometry (GC/MS or LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be exercised with any drug of abuse test result, particularly when the preliminary test result is positive.

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

| Prescription Use (Part 21 CFR 801 Subpart D) | <span></span> |
|----------------------------------------------|---------------|
| Over-The-Counter Use (21 CFR 801 Subpart C) | <span></span> |

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# 510(k) Summary of Safety and Effectiveness

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

## Submitted On

August 30, 2019

## Last Updated On

October 3, 2019

### Introduction

According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.

### Submitter Name, Address, and Contact:

Lin-Zhi International, Inc. 2945 Oakmead Village Court Santa Clara, CA 95051 Phone: [phone] Fax: [phone] e-mail: [e-mail]

| Contact: | Bernice Lin, Ph.D. |
|----------|--------------------|
| | VP Operations |

### Device Name and Classification

| Classification Name: | Enzyme Immunoassay, Methadone<br>Class II, DJR (91 Toxicology),<br>21 CFR 862.3620 |
|----------------------|------------------------------------------------------------------------------------|
| Common Name: | Homogeneous Methadone Enzyme Immunoassay |
| Proprietary Name: | LZI Methadone II Enzyme Immunoassay |

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# Legally Marketed Predicate Device(s)

The LZI Methadone II Enzyme Immunoassay (EIA) is substantially equivalent to the Methadone Enzyme Immunoassay (k023317) manufactured by Lin-Zhi International, Inc. The LZI Methadone II Enzyme Immunoassay is identical or similar to its predicate in terms of intended use, method principle, device components, and clinical performance.

# Device Description

The LZI Methadone II Enzyme Immunoassay is a homogeneous enzyme immunoassay with ready-to-use liquid reagents. The assay is based on competition between methadone in the sample and methadone labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for a fixed amount of antibody in the reagent. Enzyme activity decreases upon binding to the antibody, and the methadone concentration in the sample is measured in terms of enzyme activity. In the absence of methadone in the sample, methadone-labeled G6PDH conjugate is bound to antibody, and the enzyme activity is inhibited. On the other hand, when free methadone is present in the sample, antibody would bind to free methadone; the unbound methadone-labeled G6PDH then exhibits its maximal enzyme activity. Active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH, resulting in an absorbance change that can be measured spectrophotometrically at 340 nm.

The LZI Methadone II Enzyme Immunoassay is a kit comprised of two reagents, an R1 and R2 which are bottled separately but sold together within the kit.

The Ri solution contains mouse monoclonal anti-methadone antibody, glucose-6-phosphate (G6P) nicotinamide adenine dinucleotide (NAD), stabilizers, and sodium azide (0.09 %) as a preservative. The R2 solution contains glucose-6-phosphate dehydrogenase (G6PDH) labeled with methadone in buffer with sodium azide (0.09 %) as a preservative.

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# Intended Use

The LZI Methadone II Enzyme Immunoassay is an in vitro diagnostic test intended for the qualitative and semi-quantitative determination of methadone in human urine. The cutoff for both the qualitative and semi-quantitative modes of the assay is 300 ng/mL for methadone. The assay is designed for prescription use on automated clinical chemistry analyzers.

The semi-quantitative mode is for purposes of (1) enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas or liquid chromatography/mass spectrometry (GC/MS or LC/MS) or (2) permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical result. A more specific alternative analytical chemistry method must be used in order to obtain a confirmed analytical result. Gas or liguid chromatography/mass spectrometry (GC/MS or LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be exercised with any drug of abuse test result, particularly when the preliminary test result is positive.

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# Comparison to Predicate Device

The LZI Methadone II Enzyme Immunoassay is substantially equivalent to the Lin-Zhi International, Inc. Methadone Enzyme Immunoassay, Calibrators, and Controls for Hitachi systems cleared by the FDA under the premarket notification k023317 for their stated intended use.

The following table compares the LZI Methadone II Enzyme Immunoassay with the predicate device.

| Device<br>Characteristics | Subject Device | Predicate Device (k023317) |
|---------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The LZI Methadone II Enzyme Immunoassay<br>The LZI Methadone II Enzyme<br>Immunoassay is an in vitro diagnostic test<br>intended for the qualitative and<br>semi-quantitative determination of<br>methadone in human urine. The cutoff for<br>the qualitative and semi-quantitative<br>modes of the assay is 300 ng/mL for<br>methadone. The assay is designed for<br>prescription use on automated clinical<br>chemistry analyzers.<br>The semi-quantitative mode is for<br>purposes of (1) enabling laboratories to<br>determine an appropriate dilution of the<br>specimen for confirmation by a<br>confirmatory method such as gas or liquid<br>chromatography/mass spectrometry<br>(GC/MS or LC/MS) or (2) permitting<br>laboratories to establish quality control<br>procedures.<br>The assay provides only a preliminary analytical<br>result. A more specific alternative analytical<br>chemistry method must be used in order to obtain a<br>confirmed analytical result. Gas or liquid<br>chromatography/mass spectrometry (GC/MS or<br>LC/MS) is the preferred confirmatory method.<br>Clinical consideration and professional judgment<br>should be exercised with any drug of abuse test<br>result, particularly when the preliminary test result<br>is positive. | Methadone Enzyme Immunoassay<br>The Lin-Zhi International, Inc. (LZI)<br>Methadone Enzyme Immunoassay is<br>intended for the qualitative and<br>semi-quantitative determination of<br>methadone in human urine at a cutoff value<br>of 300 ng/mL. The assay is designed for<br>professional use with a number of<br>automated clinical chemistry analyzers.<br>This assay provides a rapid screening procedure<br>for determining the presence of methadone<br>metabolite in urine. The assay provides only a<br>preliminary analytical result. A more specific<br>alternative chemical method must be used in order<br>to obtain a confirmed analytical result. Gas or<br>liquid chromatography/mass spectrometry (GC/MS<br>or LC/MS) is the preferred confirmatory method.<br>Clinical consideration and professional judgment<br>should be exercised with any drug of abuse test<br>result, particularly when the preliminary test result<br>is positive. |
| Analyte | methadone | methadone |
| Cutoff | 300 ng/mL | 300 ng/mL |
| Matrix | urine | urine |
| Calibrators Level | 0, 150, 300, 600, and 1000 ng/mL | 0, 150, 300, 600, and 1000 ng/mL |
| Controls Level | 225 ng/mL and 375 ng/mL | 225 ng/mL and 375 ng/mL |
| Storage | 2-8°C until expiration date | 2-8°C until expiration date |

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# Performance Characteristics Summary: Beckman Coulter® AU680 Analyzer

### Precision

The assay tested in qualitative (△OD, mAU) and semi-quantitative (ng/mL) mode using a modified NCCLS-EP5 protocol. Methadone sample concentrations were prepared by spiking a methadone standard into a pool of negative human urine at concentrations ±25%, ±50%, ±75%, and ±100% of cutoff concentration.

Results shown below were obtained by testing all samples in replicate of two, two runs a day (one in the morning and one in the afternoon) for 22 days on one AU680 automatic clinical analyzer for a total of 88 runs. Samples were evaluated against the assay's calibration curve in both qualitative and semi-quantitative modes. One single lot of reagents, calibrators, and controls were used and stored at 2-8℃ when not in use.

| Methadone<br>Concentration | Within Run (N=22)<br>Qualitative<br>Response | Total Precision (N=88)<br>Qualitative Response |
|----------------------------|----------------------------------------------|------------------------------------------------|
| 0 ng/mL | - | - |
| 75 ng/mL | - | - |
| 150 ng/mL | - | - |
| 225 ng/mL | - | - |
| 300 ng/mL | - | - |
| 375 ng/mL | + | + |
| 450 ng/mL | + | + |
| 525 ng/mL | + | + |
| 600 ng/mL | + | + |

#### Semi-Quantitative Precision Analysis Summary: Qualitative Results

| Semi-Quantitative Positive/Negative Results: | | |
|----------------------------------------------|--|--|
| | | |

| 300 ng/mL Cutoff Result: | | Within Run (N=22) | | Total Precision (N=88) | |
|----------------------------|-------------|-------------------|--------------|------------------------|---------------|
| Methadone<br>Concentration | % of Cutoff | # of Samples | EIA Result | # of Samples | EIA Result |
| 0 ng/mL | 0.0% | 22 | 22 Negative | 88 | 88 Negative |
| 75 ng/mL | 25.0% | 22 | 22 Negative | 88 | 88 Negative |
| 150 ng/mL | 50.0% | 22 | 22 Negative | 88 | 88 Negative |
| 225 ng/mL | 75.0% | 22 | 22 Negative | 88 | 88 Negative |
| 300 ng/mL | 100.0% | 22 | 18 Neg/4 Pos | 88 | 66 Neg/22 Pos |
| 375 ng/mL | 125.0% | 22 | 22 Positive | 88 | 88 Positive |
| 450 ng/mL | 150.0% | 22 | 22 Positive | 88 | 88 Positive |
| 525 ng/mL | 175.0% | 22 | 22 Positive | 88 | 88 Positive |
| 600 ng/mL | 200.0% | 22 | 22 Positive | 88 | 88 Positive |

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# Performance Characteristics Summary, continued:

Beckman Coulter® AU680 Analyzer

| 300 ng/mL Cutoff Result: | | Within Run (N=22) | | Total Precision (N=88) | |
|----------------------------|-------------|-------------------|---------------|------------------------|---------------|
| Methadone<br>Concentration | % of Cutoff | # of Samples | EIA Result | # of Samples | EIA Result |
| 0 ng/mL | 0.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 75 ng/mL | 25.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 150 ng/mL | 50.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 225 ng/mL | 75.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 300 ng/mL | 100.0 % | 22 | 17 Neg/ 5 Pos | 88 | 59 Neg/29 Pos |
| 375 ng/mL | 125.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 450 ng/mL | 150.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 525 ng/mL | 175.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 600 ng/mL | 200.0 % | 22 | 22 Positive | 88 | 88 Positive |

#### Qualitative Positive/Negative Results:

### Linearity

To demonstrate linearity of the entire assay range, a drug free-urine pool spiked with methadone at 1000 ng/mL was serially diluted. Each sample was run in 10 replicates and the average was used to determine percent recovery compared to the expected target value.

Observed values were obtained and acceptable if measurements were ±15% of the expected values. Recovery values (Expected Value divided by the Observed Value) were considered acceptable between 85 - 115%.

Samples from the linear range of the assay (100 ng/mL to 1000 ng/mL) were tested with recovery ranging from 93.7% to 104.9%.

| Expected Value<br>(ng/mL) | Observed Value<br>(ng/mL) | % Recovery |
|---------------------------|---------------------------|------------|
| 1000 | 950.5 | 95.0% |
| 900 | 848.6 | 94.3% |
| 800 | 767.9 | 96.0% |
| 700 | 668.0 | 95.4% |
| 600 | 572.2 | 95.4% |
| 500 | 463.0 | 92.6% |
| 400 | 393.4 | 98.4% |
| 300 | 287.8 | 95.9% |
| 200 | 187.4 | 93.7% |
| 100 | 104.9 | 104.9% |
| 20 | 15.8 | 78.9% |
| 0 | -7.1 | N/A |

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## Method Comparison - Clinical Samples

A total of ninety-four (94) unaltered clinical samples were tested with the LZI Methadone II Enzyme Immunoassay on the Beckman Coulter® AU680 automated clinical analyzer. Samples were evaluated against the assay's calibration curve in both qualitative and semi-quantitative modes. All samples were tested in singlet.

All samples were confirmed with LC/MS for both methadone and methadone metabolite concentrations. Samples were collected by Lin-Zhi International, Inc. (LZI) and from the University of California, San Francisco (UCSF).

### Semi-Quantitative Accuracy Study

Discrepant samples determined as compared to methadone concentration from LC/MS.

| Candidate<br>Device<br>Results | Negative | <50 %<br>of<br>Cutoff | Near Cutoff<br>Negative<br>(between -50<br>% of cutoff<br>to the cutoff) | Near Cutoff<br>Positive<br>(between cutoff<br>and +50 % of<br>cutoff) | High<br>Positive<br>(>50 %<br>above<br>cutoff) | %<br>Agreement |
|--------------------------------|----------|-----------------------|--------------------------------------------------------------------------|-----------------------------------------------------------------------|------------------------------------------------|----------------|
| Positive | 0 | 0 | 1* | 4 | 41 | 97.8 % |
| Negative | 20 | 23 | 4 | 1** | 0 | 97.9 % |

| Sample<br># | LC/MS<br>Methadone<br>(ng/mL) | Pos/Neg<br>Result | AU680 EIA<br>Pos/Neg<br>Result |
|-------------|-------------------------------|-------------------|--------------------------------|
| 48* | 274 | - | + |
| 50** | 317 | + | - |

* Discrepant between 50% below cutoff and cutoff concentration (150 – 299,9 ng/mL)

** Discrepant between cutoff and 50% above cutoff concentration (300 - 449.9 ng/mL)

### Qualitative Accuracy Study

| Candidate<br>Device<br>Results | Negative | <50 %<br>of<br>Cutoff | Near Cutoff<br>Negative<br>(between -50<br>% of cutoff<br>to the cutoff) | Near Cutoff<br>Positive<br>(between cutoff<br>and +50 % of<br>cutoff) | High<br>Positive<br>(>50 %<br>above<br>cutoff) | %<br>Agreement |
|--------------------------------|----------|-----------------------|--------------------------------------------------------------------------|-----------------------------------------------------------------------|------------------------------------------------|----------------|
| Positive | 0 | 0 | 1* | 4 | 44 | 97.8 % |
| Negative | 20 | 23 | 4 | 1** | 0 | 97.9 % |

| Sample<br># | LC/MS<br>Methadone<br>(ng/mL) | Pos/Neg<br>Result | AU680 EIA<br>Qualitative<br>Result (mAU) | Pos/<br>Neg<br>Result | Qualitative<br>Cutoff Rate<br>(mAU) |
|-------------|-------------------------------|-------------------|------------------------------------------|-----------------------|-------------------------------------|
| 48* | 274 | - | 176.5 | + | 133.5 |
| 50** | 317 | + | 95.4 | - | 165.0 |

* Discrepant between 50% below cutoff and cutoff concentration (150 - 299.9 ng/mL)

** Discrepant between cutoff and 50% above cutoff concentration (300 - 449.9 ng/mL)

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### Cross-reactivity

The Cross-reactivity of various potentially interfering drugs were tested by spiking various concentrations of each substance into a pool of negative human urine and then evaluated against the assay's calibration curve in both qualitative and semi-quantitative modes. All samples were tested in replicates.

The table below lists the concentration of each test compound that gave a response approximately equivalent to that of the cutoff calibrator (as positive) or the maximal concentration of the compound tested that gave a response below the response of the cutoff calibrator (as negative). Compounds tested at high concentration with results below the cutoff value were listed as Not Detected (ND).

| Compound | Target<br>Concentration<br>(ng/mL) | % Cross-<br>reactivity |
|-------------------------------------------|------------------------------------|------------------------|
| Methadone | 300 | 100.00% |
| EDDP | 100,000 | <0.30% |
| EMDP | 100,000 | <0.30% |
| (-)-α-Noracetylmethadol<br>(Nor-LAAM) HCl | 75,000 | 0.40% |
| LAAM HCl | 25,000 | 1.20% |
| (±)-α-Methadol | 26,500 | 1.13% |
| (-)-Isomethadone HCl | 35,000 | 0.86% |

### Methadone and Structurally Related Compounds:

Structurally unrelated compounds were additionally spiked into pooled negative human urine to desired concentrations (as described above). These solutions were then split into three portions; one without methadone, and the remaining two that were further spiked with methadone standards to a final methadone concentration of 225 ng/mL (as negative or positive controls, ±25% cutoff concentration, respectively). Samples were then evaluated against the assay's calibration curve in both qualitative and semi-quantitative modes. All samples were tested in replicates.

### Structurally Unrelated Pharmacological Compounds:

| | Spiked [ ]<br>(ng/mL) | Spiked Methadone Concentration | | |
|--------------------------------------------------------|-----------------------|--------------------------------|----------------------|-------------------|
| Cross-reactant | 0 ng/mL | 225 ng/mL<br>Control | 375 ng/mL<br>Control | |
| Acetaminophen | 100,000 | ND | Neg | Pos |
| 6-Acetylmorphine | 100,000 | ND | Neg | Pos |
| Acetylsalicylic Acid | 100,000 | ND | Neg | Pos |
| Amitriptyline | 100,000 | ND | Neg | Pos |
| Amlodipine Besylate | 100,000 | ND | Neg | Pos |
| Amoxicillin | 100,000 | ND | Neg | Pos |
| d-Amphetamine | 100,000 | ND | Neg | Pos |
| Atorvastatin | 100,000 | ND | Neg | Pos |
| Acetaminophen | 100,000 | ND | Neg | Pos |
| 6-Acetylmorphine | 100,000 | ND | Neg | Pos |
| Cross-reactant | Spiked [] (ng/mL) | 0 ng/mL | 225 ng/mL Control | 375 ng/mL Control |
| Acetylsalicylic Acid | 100,000 | ND | Neg | Pos |
| Amitriptyline | 100,000 | ND | Neg | Pos |
| Amlodipine Besylate | 100,000 | ND | Neg | Pos |
| Amoxicillin | 100,000 | ND | Neg | Pos |
| d-Amphetamine | 100,000 | ND | Neg | Pos |
| Atorvastatin | 100,000 | ND | Neg | Pos |
| Benzoylecgonine | 100,000 | ND | Neg | Pos |
| Buprenorphine | 100,000 | ND | Neg | Pos |
| Bupropion | 100,000 | ND | Neg | Pos |
| Caffeine | 100,000 | ND | Neg | Pos |
| Carbamazepine | 100,000 | ND | Neg | Pos |
| Cetirizine | 100,000 | ND | Neg | Pos |
| Chlorpheniramine | 100,000 | ND | Neg | Pos |
| Chlorpromazine | 100,000 | ND | Neg | Pos |
| Clomipramine | 100,000 | ND | Neg | Pos |
| Codeine | 100,000 | ND | Neg | Pos |
| Desipramine | 100,000 | ND | Neg | Pos |
| Diphenhydramine | 100,000 | ND | Neg | Pos |
| Duloxetine | 50,000 | ND | Neg | Pos |
| Fentanyl | 100,000 | ND | Neg | Pos |
| Fluoxetine | 100,000 | ND | Neg | Pos |
| Fluphenazine | 100,000 | ND | Neg | Pos |
| Gabapentin | 100,000 | ND | Neg | Pos |
| Hydrocodone | 100,000 | ND | Neg | Pos |
| Hydromorphone | 100,000 | ND | Neg | Pos |
| Ibuprofen | 100,000 | ND | Neg | Pos |
| Imipramine | 100,000 | ND | Neg | Pos |
| Lisinopril | 100,000 | ND | Neg | Pos |
| Losartan | 100,000 | ND | Neg | Pos |
| Loratidine | 100,000 | ND | Neg | Pos |
| MDA (3,4-methylenedioxy-amphetamine) | 100,000 | ND | Neg | Pos |
| MDEA | 100,000 | ND | Neg | Pos |
| MDMA (3,4-methylenedioxy-methamphetamine) | 100,000 | ND | Neg | Pos |
| Meperidine | 100,000 | ND | Neg | Pos |
| Metformin | 100,000 | ND | Neg | Pos |
| Metoprolol | 100,000 | ND | Neg | Pos |
| d-Methamphetamine | 100,000 | ND | Neg | Pos |
| Morphine | 100,000 | ND | Neg | Pos |
| Nicotine | 100,000 | ND | Neg | Pos |
| Nortriptyline | 100,000 | ND | Neg | Pos |
| Omeprazole | 100,000 | ND | Neg | Pos |
| Cross-reactant | Spiked [ ] (ng/mL) | Spiked Methadone Concentration | | |
| | | 0 ng/mL | 225 ng/mL Control | 375 ng/mL Control |
| Oxazepam | 100,000 | ND | Neg | Pos |
| Oxycodone | 100,000 | ND | Neg | Pos |
| Oxymorphone | 100,000 | ND | Neg | Pos |
| Phenobarbital | 100,000 | ND | Neg | Pos |
| (1S,2S)-(+)Pseudoephedrine | 100,000 | ND | Neg | Pos |
| Quetiapine | 100,000 | ND | Neg | Pos |
| Ranitidine | 100,000 | ND | Neg | Pos |
| Salbutamol (Albuterol) | 100,000 | ND | Neg | Pos |
| Sertraline | 100,000 | ND | Neg | Pos |
| THC-COOH<br>(11-Nor-Delta-9-THC-<br>9-carboxylic acid) | 100,000 | ND | Neg | Pos |
| L-Thyroxine | 100,000 | ND | Neg | Pos |
| Tramadol | 100,000 | ND | Neg | Pos |
| Zolpidem | 100,000 | ND | Neg | Pos |

{11}------------------------------------------------

Structurally Unrelated Pharmacological Compounds, continued:

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

Structurally Unrelated Pharmacological Compounds, continued:

### Endogenous Compound Interference:

Endogenous compounds were spiked into pooled negative human urine to desired concentrations. These solutions were then split into three portions; one without methadone, and the remaining two that were further spiked with methadone standards to a final methadone concentration of 225 ng/mL or 375 ng/mL (as negative or positive controls,

±25% cutoff concentration, respectively). Samples were then evaluated against the assay's calibration curve in both qualitative and semi-quantitative modes. All samples were tested in replicates.

| Endogenous Substance | Concentration<br>Tested (ng/mL) | Spiked Methadone Concentration | | |
|-----------------------------|---------------------------------|--------------------------------|----------------------|----------------------|
| | | 0 ng/mL | 225 ng/mL<br>Control | 375 ng/mL<br>Control |
| Acetone | 1000 | Neg | Neg | Pos |
| Ascorbic Acid | 1500 | Neg | Neg | Pos |
| Bilirubin | 2 | Neg | Neg | Pos |
| Boric Acid* | 1000 | Neg | Neg | Neg |
| Calcium Chloride<br>(CaCl2) | 300 | Neg | Neg | Pos |
| Citric Acid (pH 3) | 800 | Neg | Neg | Pos |
| Creatinine | 500 | Neg | Neg | Pos |
| Ethanol | 1000 | Neg | Neg | Pos |
| Galactose | 10 | Neg | Neg | Pos |
| y-Globulin | 500 | Neg | Neg | Pos |
| Glucose | 3000 | Neg | Neg | Pos |
| Hemoglobin | 300 | Neg | Neg | Pos |
| β-hydroxybutyric Acid | 100 | Neg | Neg | Pos |
| HSA | 500 | Neg | Neg | Pos |

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

| Endogenous Substance | Concentration<br>Tested (ng/mL) | Spiked Methadone Concentration | | |
|----------------------|---------------------------------|--------------------------------|----------------------|----------------------|
| | | 0 ng/mL | 225 ng/mL<br>Control | 375 ng/mL<br>Control |
| Oxalic Acid | 100 | Neg | Neg | Pos |
| Potassium Chloride* | 6000 | Neg | Neg | Neg |
| Riboflavin | 0.3 | Neg | Neg | Pos |
| Urea | 6000 | Neg | Neg | Pos |
| Uric Acid | 10 | Neg | Neg | Pos |
| Sodium Azide | 1000 | Neg | Neg | Pos |
| Sodium Chloride | 1000 | Neg | Neg | Pos |
| Sodium Fluoride | 1000 | Neg | Neg | Pos |
| Sodium Phosphate | 300 | Neg | Neg | Pos |

### Endogenous Compound Interference, continued:

The following endogenous compounds which showed interference at ±25 % of cutoff concentrations were then spiked into negative urine and at ±50 % of cutoff concentrations (150 ng/mL and 450 ng/mL) for the assay.

Interference was observed with Boric Acid at 1 % w/v. No other significant undesired crossreactants or endogenous substance interference was observed.

| Endogenous Substance | Concentration<br>Tested (ng/mL) | Spiked Methadone Concentration | | |
|----------------------|---------------------------------|--------------------------------|----------------------|----------------------|
| | | 0 ng/mL | 150 ng/mL<br>Control | 450 ng/mL<br>Control |
| Boric Acid | 1000 | Neg | Neg | Neg |
| Potassium Chloride | 6000 | Neg | Neg | Pos |

# Specific Gravity:

Samples ranging in specific gravity from 1.003 to 1.028 were split into three portions each and either left un-spiked or further spiked to a final methadone concentration of either 225 ng/mL or 375 ng/mL (the negative and positive control concentrations, respectively). These samples were then evaluated in qualitative mode. No interference was observed.

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

### pH Interference Study:

Negative urine and urine spiked with methadone to the final methadone concentration of either 225 ng/mL or 375 ng/mL (the negative and positive control concentrations, respectively) were adjusted to the following pH levels and tested by the assay. The pH adjusted solutions were evaluated against the cutoff calibrator.

| pH | Spiked Methadone Concentration | | |
|-------|--------------------------------|-------------------|-------------------|
| | 0 ng/mL | 225 ng/mL Control | 375 ng/mL Control |
| pH 3 | Neg | Neg | Pos |
| pH 4 | Neg | Neg | Pos |
| pH 5 | Neg | Neg | Pos |
| pH 6 | Neg | Neg | Pos |
| pH 7 | Neg | Neg | Pos |
| pH 8 | Neg | Neg | Pos |
| pH 9 | Neg | Neg | Pos |
| pH 10 | Neg | Neg | Pos |
| pH 11 | Neg | Neg | Pos |

No major interference between pH 3 to pH 11. Results are summarized in the following table:

# Summary:

The information provided in this pre-market notification demonstrates that the LZI Methadone II Enzyme Immunoassay is substantially equivalent to the legally marketed predicate device for its general intended use. Substantial equivalence was demonstrated through comparison of intended use and physical properties to the commercially available predicate device as confirmed by chromatography/mass spectrometry (LC/MS), an independent analytical chemistry method. The information supplied in this pre-market notification provides reasonable assurance that the LZI Methadone II Enzyme Immunoassay is safe and effective for its stated intended use.