LZI Cotinine II Enzyme Immunoassay

K192299 · Lin-Zhi International, Inc. · MKU · Nov 21, 2019 · Clinical Toxicology

Device Facts

• Record ID: K192299
• Device Name: LZI Cotinine II Enzyme Immunoassay
• Applicant: Lin-Zhi International, Inc.
• Product Code: MKU · Clinical Toxicology
• Decision Date: Nov 21, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3220
• Device Class: Class 1

Indications for Use

The LZI Cotinine II Enzyme Immunoassay is intended for the qualitative and semi-quantitative determination of cotinine in human urine at the cutoff value of 200 ng/mL when calibrated against cotinine. The assay is intended as an aid in the detection of cotinine after use or exposure to tobacco products. The assay is designed for prescription use with a number of 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 chemical method (e.g., gas or liquid chromatography and mass spectrometry) must be used in order to obtain a confirmed analytical result. 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 Cotinine II Enzyme Immunoassay is a homogeneous enzyme immunoassay for urine cotinine detection. Input: human urine sample. Principle: competitive binding between sample cotinine and cotinine-labeled G6PDH enzyme for fixed antibody; drug presence inhibits antibody binding, allowing G6PDH to remain active. Active enzyme converts NAD to NADH, measured spectrophotometrically at 340 nm. Output: qualitative or semi-quantitative cotinine concentration. Used in clinical laboratories; operated by trained personnel (technicians/medical technologists). Results are preliminary; require confirmation by GC/MS or LC/MS. Clinical decision-making relies on professional judgment; aids in identifying tobacco use/exposure.

Clinical Evidence

Bench testing only. Precision/reproducibility evaluated over 22 days (N=88) on AU480 analyzer. Linearity confirmed across 0-1000 ng/mL range. Method comparison performed on 104 clinical samples against LC/MS reference method; demonstrated 96.4% positive agreement and 98.7% negative agreement. Interference studies evaluated endogenous compounds, pH (3-11), and specific gravity (1.005-1.028). Cross-reactivity tested against various drugs and metabolites.

Technological Characteristics

Homogeneous enzyme immunoassay; liquid reagents (R1: mouse monoclonal anti-cotinine antibody, G6P, NAD; R2: G6PDH-labeled cotinine). Preservative: 0.09% sodium azide. Sensing: spectrophotometric absorbance at 340 nm. Connectivity: compatible with automated clinical chemistry analyzers. Traceable to Cerilliant Cotinine standards.

Indications for Use

Indicated for the qualitative and semi-quantitative detection of cotinine in human urine at a 200 ng/mL cutoff to aid in identifying tobacco use or exposure. For prescription use in clinical laboratories using automated chemistry analyzers. Requires confirmation by GC/MS or LC/MS.

Regulatory Classification

Identification

A carbon monoxide test system is a device intended to measure carbon monoxide or carboxyhemoglobin (carbon monoxide bound to the hemoglobin in the blood) in blood. Measurements obtained by this device are used in the diagnosis and treatment of or confirmation of carbon monoxide poisoning.

Predicate Devices

• Niccheck I (k963733)

Submission Summary (Full Text)

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Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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

## I Background Information:

A 510(k) Number
k192299

B Applicant
Lin-Zhi International, Inc.

C Proprietary and Established Names
LZI Cotinine II Enzyme Immunoassay

D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| MKU | Class I | 21 CFR 862.3220 - Carbon Monoxide Test System | TX - Clinical Toxicology |

## II Submission/Device Overview:

A Purpose for Submission:
New Device

B Measurand:
Cotinine

C Type of Test:
Immunoassay

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III Intended Use/Indications for Use:

A Intended Use(s):
See Indications for Use below

B Indication(s) for Use:
The LZI Cotinine II Enzyme Immunoassay is intended for the qualitative and semi-quantitative determination of cotinine in human urine at the cutoff value of 200 ng/mL when calibrated against cotinine. The assay is intended as an aid in the detection of cotinine after use or exposure to tobacco products. The assay is designed for prescription use with a number of 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 chemical method (e.g., gas or liquid chromatography and mass spectrometry) must be used in order to obtain a confirmed analytical result. Clinical consideration and professional judgment should be exercised with any drug of abuse test result, particularly when the preliminary test result is positive.

C Special Conditions for Use Statement(s):
Prescription use only

D Special Instrument Requirements:
Validation studies were conducted using the AU480 Chemistry Analyzer.

IV Device/System Characteristics:

A Device Description:
The LZI Cotinine Enzyme Immunoassay is a homogeneous enzyme immunoassay with ready-to use liquid reagents. The LZI Cotinine Enzyme Immunoassay is a kit comprised of two reagents, an R1 and R2, which are bottled separately but sold together within the kit. The R1 solution contains mouse monoclonal anti-cotinine 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 cotinine in buffer with sodium azide (0.09 %) as a preservative.

B Principle of Operation:
The assay is based on competition between drug in the sample and drug 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 drug concentration in the sample is measured in terms of enzyme activity. In the absence of drug in the sample, cotinine-

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labeled G6PDH conjugate is bound to antibody, and the enzyme activity is inhibited. On the other hand, when free drug is present in the sample, antibody would bind to free drug; the unbound cotinine-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.

V Substantial Equivalence Information:

A Predicate Device Name(s):

Niccheck I

B Predicate 510(k) Number(s):

k963733

C Comparison with Predicate(s):

| Device & Predicate Device(s): | k192299 | k963733 |
| --- | --- | --- |
| Device Trade Name | LZI Cotinine II Enzyme Immunoassay | NicCheckI |
| General Device Characteristic Similarities | | |
| Analyte | Cotinine | same |
| Matrix | Urine | same |
| Cutoff | 200 ng/mL | same |
| Intended Use/Indications For Use | For the qualitative and semi-quantitative determination of cotinine in human urine at the cutoff value of 200 ng/mL when calibrated against cotinine. The assay is intended as an aid in the detection of cotinine after use or exposure to tobacco products. | same |
| General Device Characteristic Differences | | |
| Storage | 2-8°C until expiration date | 2-8°C until 2 years from date of manufacture. Test strips are susceptible to conditions of high humidity, the canister must be kept tightly closed after removal of the required number of strips. |

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VI Standards/Guidance Documents Referenced:

CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:
Cotinine samples (cotinine spiked negative urine pools) were run in replicates of 2, two runs per day (one in the morning and one in the afternoon) for 22 days on one AU480 automatic clinical analyzer for a total of 88 runs, in both qualitative and semi-quantitative modes. One single lot of reagents and calibrators and controls were used. Cotinine was spiked into samples at the concentrations shown in the tables below, and all sample cotinine concentrations were confirmed by LC/MS.

Semi-Quantitative Positive/Negative Results:

| 200 ng/mL Cutoff Result: | | Within Run (N=22) | | Total Precision (N=88) | |
| --- | --- | --- | --- | --- | --- |
| Cotinine Concentration | % of Cutoff | # of Determinatio n | EIA Result | # of Determinatio n | EIA Result |
| 0 ng/mL | 0.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 50 ng/mL | 25.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 100 ng/mL | 50.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 150 ng/mL | 75.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 200 ng/mL | 100.0 % | 22 | 14 Neg/8 Pos | 88 | 55 Neg/33 Pos |
| 250 ng/mL | 125.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 300 ng/mL | 150.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 350 ng/mL | 175.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 400 ng/mL | 200.0 % | 22 | 22 Positive | 88 | 88 Positive |

Qualitative Positive/Negative Results:

| 200 ng/mL Cutoff Result: | | Within Run (N=22) | | Total Precision (N=88) | |
| --- | --- | --- | --- | --- | --- |
| Cotinine Concentration | % of Cutoff | # of Determinatio n | EIA Result | # of Determinatio n | EIA Result |
| 0 ng/mL | 0.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 50 ng/mL | 25.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 100 ng/mL | 50.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 150 ng/mL | 75.0 % | 22 | 22 Negative | 88 | 88 Negative |
| 200 ng/mL | 100.0 % | 22 | 10 Neg/12 Pos | 88 | 51 Neg/37 Pos |
| 250 ng/mL | 125.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 300 ng/mL | 150.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 350 ng/mL | 175.0 % | 22 | 22 Positive | 88 | 88 Positive |
| 400 ng/mL | 200.0 % | 22 | 22 Positive | 88 | 88 Positive |

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2. Linearity:
A recovery/linearity study was performed by spiking cotinine into a pool of negative urine to create a 1000 ng/mL high concentration. The high concentration was then diluted to reach the final concentrations listed in the table below. Each sample was run in 10 replicates on the AU480 automated clinical analyzer in semi-quantitative mode with a calibration curve established with the five Cotinine calibrators (0, 100, 200, 400, and 1000 ng/mL). Recovery values (expected value divided by average observed value) were calculated and are presented in the table below.

| Target Concentration (ng/mL) | Determined (ng/mL) | % Recovery |
| --- | --- | --- |
| 1000 | 978.9 | 97.9% |
| 900 | 919.6 | 102.2% |
| 800 | 851.7 | 106.5% |
| 700 | 766.0 | 109.4% |
| 600 | 664.8 | 110.8% |
| 500 | 539.1 | 107.8% |
| 400 | 395.0 | 98.8% |
| 300 | 302.4 | 100.8% |
| 200 | 199.9 | 100.0% |
| 100 | 109.0 | 109.0% |
| 20 | 33.5 | 167.5% |
| 0 | 19.3 | N/A |

3. Analytical Specificity/Interference:
The compounds listed below were spiked into urine to the desired concentrations. These solutions were then split into three portions; one without cotinine, and the remaining two that were further spiked with cotinine standards obtained from Cerilliant to a final cotinine concentration of 150 ng/mL or 250 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, and the results are as shown below (results were identical for both qualitative and semi-quantitative modes). Results indicated that there is interference from boric acid at 1 % w/v and citric acid. No other compounds showed interference concentrations at the concentrations tested. Data collected on the AU480 automated clinical analyzer is summarized in the following table:

Endogenous Compounds Interference

| Endogenous Substance | Concentration Tested (ng/mL) | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- | --- |
| | | 0 ng/mL | 150 ng/mL Control | 250 ng/mL 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 (CaCl2) | 300 | Neg | Neg | Pos |
| Citric Acid (pH 3) | 800 | Neg | Neg | Neg |
| Creatinine | 500 | Neg | Neg | Pos |
| Ethanol | 1000 | Neg | Neg | Pos |
| Galactose | 10 | Neg | Neg | Pos |
| γ-Globulin | 500 | Neg | Neg | Pos |

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| Glucose | 3000 | Neg | Neg | Pos |
| --- | --- | --- | --- | --- |
| Hemoglobin | 300 | Neg | Neg | Pos |
| β-hydroxybutyric Acid | 100 | Neg | Neg | Pos |
| HAS | 500 | Neg | Neg | Pos |
| Oxalic Acid | 100 | Neg | Neg | Pos |
| Potassium Chloride | 6000 | Neg | Neg | Pos |
| 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 | 6000 | Neg | Neg | Pos |
| Sodium Fluoride | 1000 | Neg | Neg | Pos |
| Sodium Phosphate | 300 | Neg | Neg | Pos |
| Endogenous Substance | Concentration Tested (ng/mL) | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- | --- |
| | | 0 ng/mL | 100 ng/mL Control | 300 ng/mL Control |
| Boric Acid | 1000 | Neg | Neg | Neg |
| Citric Acid (pH 3) | 800 | Neg | Neg | Neg |

## pH Interference Study

Drug free urine and urine spiked with cotinine to the final cotinine concentration of either 150 ng/mL or 250 ng/mL (as negative or positive controls, ±25% cutoff concentration, respectively) were adjusted to the pH levels reported in the table below and tested by the assay. The pH adjusted solutions were evaluated against the cutoff calibrator. No major interference between pH 3 to pH 11. Results are summarized in the table below.

| pH | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- |
| | 0 ng/mL | 150 ng/mL Control | 250 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 |

## Specific Gravity Interference Study

Samples ranging in specific gravity from 1.005 to 1.028 were split into three portions each and either left un-spiked or further spiked to a final cotinine concentration of either 150 ng/mL or 250 ng/mL (as negative or positive controls, ±25% cutoff concentration, respectively). These samples were then evaluated in qualitative mode. No interference was observed at any specific gravity.

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# Cross-Reactivity Study

The cross-reactivity of various potentially interfering drugs were tested by spiking various concentrations of each substance listed in the table below into drug free urine and then evaluating the sample against the assay. The results are summarized in the tables below.

Cotinine

| Compound | Target Concentration (ng/mL) | % Cross-reactivity |
| --- | --- | --- |
| (-)-Cotinine | 200 | 100.00% |

Cotinine Metabolites

| Compound | Target Concentration (ng/mL) | % Cross-reactivity |
| --- | --- | --- |
| (+)-Anabasine | 250,000 | 0.08% |
| S(-)-Nicotine | 25,000 | 0.80% |
| Nicotinic Acid (Niacin) | 100,000 | 0.20% |
| (-) Norcotinine | 1000 | 20.00% |
| (+) Norcotinine | 100,000 | 0.20% |
| (R, S)-Norcotinine | 850 | 23.53% |
| (±)-Nornicotine | 250,000 | 0.08% |
| trans-3'-hydroxycotinine | 10,000 | 2.00% |

Cross-reactivity of various potentially interfering drugs were tested by spiking 100,000 ng/mL (or as listed in the table below) of each substance into drug free urine. These solutions were then split into three portions; one without Cotinine, and two that were further spiked with Cotinine to a final Cotinine concentration of 150 ng/mL or 250 ng/mL (as negative or positive controls, ±25 % Cutoff Concentration, respectively). Samples were then evaluated with the assay. The results are as shown below.

Structurally Unrelated Pharmacological Compounds

| Cross-reactant | Spiked [ ] (ng/mL) | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- | --- |
| | | 0 ng/mL | 150 ng/mL Control | 250 ng/mL Control |
| Acetaminophen | 100,000 | ND | Neg | Pos |
| 6-Acetylmorphine | 100,000 | ND | Neg | Pos |
| Amitriptyline | 100,000 | ND | Neg | Pos |
| Amlodipine Besylate | 100,000 | ND | Neg | Pos |
| Amoxicillin | 100,000 | ND | Neg | Pos |
| Azelastine | 100,000 | ND | Neg | Pos |
| d-Amphetamine | 100,000 | ND | Neg | Pos |
| Atorvastatin | 20,000 | ND | Neg | Pos |
| Benzoylecgonine | 100,000 | ND | Neg | Pos |

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Structurally Unrelated Pharmacological Compounds, continued

| Cross-reactant | Spiked [ ] (ng/mL) | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- | --- |
| | | 0 ng/mL | 150 ng/mL Control | 250 ng/mL Control |
| Loratidine | 100,000 | ND | Neg | Pos |
| MDA (3,4-methylenedioxyamphetamine) | 100,000 | ND | Neg | Pos |
| MDEA | 100,000 | ND | Neg | Pos |
| MDMA (3,4-methylenedioxymethamphetamine) | 100,000 | ND | Neg | Pos |

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| Cross-reactant | Spiked [ ] (ng/mL) | Spiked Cotinine Concentration | | |
| --- | --- | --- | --- | --- |
| | | 0 ng/mL | 150 ng/mL Control | 250 ng/mL Control |
| Meperidine | 100,000 | ND | Neg | Pos |
| Metformin | 100,000 | ND | Neg | Pos |
| Methapyrilene | 100,000 | ND | Neg | Pos |
| Metoprolol | 100,000 | ND | Neg | Pos |
| Methadone | 100,000 | ND | Neg | Pos |
| d-Methamphetamine | 100,000 | ND | Neg | Pos |
| Morphine | 100,000 | ND | Neg | Pos |
| Nortriptyline | 100,000 | ND | Neg | Pos |
| Omeprazole | 100,000 | ND | Neg | Pos |
| Oxazepam | 100,000 | ND | Neg | Pos |
| Oxycodone | 100,000 | ND | Neg | Pos |
| Oxymorphone | 100,000 | ND | Neg | Pos |
| Phenobarbital | 100,000 | ND | Neg | Pos |
| Promethazine | 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 |
| Terfenadine | 100,000 | ND | Neg | Pos |
| THC-COOH (11-Nor-Delta-9-THC-9-carboxylic acid) | 1,000 | ND | Neg | Pos |
| l-Thyroxine | 10,000 | ND | Neg | Pos |
| Tramadol | 100,000 | ND | Neg | Pos |
| Triprolidine | 100,000 | ND | Neg | Pos |
| Zolpidem | 10,000 | ND | Neg | Pos |

4. Assay Reportable Range:

Not Applicable.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

The device is traceable to certified Cerilliant Cotinine, verified by HPLC and GC/MS. Purity determination and gravimetric preparation using balances calibrated with NIST traceable weights ensured the accuracy of the stock standard solution.

6. Detection Limit:

See section VII.A.1 above

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7. Assay Cut-Off:

See section VII.A.1 above

B Comparison Studies:

1. Method Comparison with Predicate Device

A total of one-hundred and four (104) unaltered clinical samples were tested with the LZI Cotinine II Enzyme Immunoassay on the AU480 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 cotinine concentrations, and the accuracy of the assay to LC/MS is provided in the table below.

Semi-Quantitative Results

| Candidate Device Results 200 ng/mL Cutoff | Negati ve | < 50 % of the cutoff concentration by GC/MS analysis | Near Cutoff Negative (Between 50 % below the cutoff and the cutoff concentration) | Near Cutoff Positive (Between the cutoff and 50 % above the cutoff concentration) | High Positive (Greater than 50 % above the cutoff concentration) | % Agreement t |
| --- | --- | --- | --- | --- | --- | --- |
| Positive | 0 | 0 | 1* | 11 | 16 | 96.4 % |
| Negati ve | 20 | 32 | 23 | 1** | 0 | 98.7 % |
| Sample # | GC/MS Cotinine (ng/mL) | Pos/ Neg Result |
| --- | --- | --- |
| 57* | 128.6 | - |
| 78** | 204.2 | + |

Qualitative Results

| Candidate Device Results 200 ng/mL Cutoff | Negati ve | < 50 % of the cutoff concentration by GC/MS analysis | Near Cutoff Negative (Between 50 % below the cutoff and the cutoff concentration) | Near Cutoff Positive (Between the cutoff and 50 % above the cutoff concentration) | High Positive (Greater than 50 % above the cutoff concentration) | % Agreement t |
| --- | --- | --- | --- | --- | --- | --- |
| Positive | 0 | 0 | 1* | 11 | 16 | 96.4 % |
| Negati ve | 20 | 32 | 23 | 1** | 0 | 98.7 % |

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| Sample # | GC/MS Cotinine (ng/mL) | Pos/ Neg Result |
| --- | --- | --- |
| 57* | 128.6 | - |
| 78** | 204.2 | + |

2. Matrix Comparison:
Not applicable.

C Clinical Studies:
Not Applicable.

D Clinical Cut-Off:
See section VII.A.1. above

E Expected Values/Reference Range:
Not Applicable.

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

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

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