The ARK Tramadol Assay is an immunoassay intended for the qualitative and/or semiquantitative determination of tramadol in human urine at a cutoff concentration of 100 ng/mL. The assay is intended for use in laboratories with automated clinical chemistry analyzers. This in vitro diagnostic device is for prescription use only.

The semiquantitative mode is for the purpose of (1) 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 Liquid Chromatography/tandem Mass Spectrometry (LC-MS/MS), or (2) permitting laboratories to establish quality control procedures.

The ARK Tramadol Assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used in order to obtain a confirmed positive analytical result. Gas Chromatography/Mass Spectrometry (GC/MS) or Liquid Chromatography/tandem Mass Spectrometry (LC-MS/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be exercised with any drug test result, particularly when the preliminary test result is positive.

Device Description

The ARK Tramadol Assay is a homogeneous enzyme immunoassay technique used for the analysis of tramadol in human urine. The assay is based on competition between drug in the specimen and drug labeled with recombinant glucose-6-phosphate dehydrogenase (rG6PDH) for antibody binding sites. As the latter binds antibody, enzyme activity decreases. In the presence of drug from the specimen, enzyme activity increases and is directly related to the drug concentration. Active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate (G6P), resulting in an absorbance change that is measured spectrophotometrically. Endogenous G6PDH does not interfere because the coenzyme NAD functions only with the bacterial enzyme used in the assay.

The ARK Tramadol Assay consists of reagents R1 anti-tramadol rabbit polyclonal antibody with substrate and R2 tramadol derivative labeled with bacterial recombinant G6PDH enzyme.

AI/ML Overview

The provided text describes the performance characteristics of the ARK Tramadol Assay, an immunoassay for detecting tramadol in human urine. Here's a breakdown of the acceptance criteria and study details:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a bulleted or numbered list with corresponding "reported performance." However, the performance parameters evaluated and the results presented implicitly define the acceptance criteria for the device to be considered substantially equivalent to the predicate. Based on the provided data, the implicit acceptance criteria and the device's performance are:

Acceptance Criteria (Implicit)	Reported Device Performance
Precision (Qualitative): Reliable classification of samples as negative below -25% cutoff, positive above +25% cutoff, and expected indeterminate results around the cutoff.	At 0.0, 25.0, 50.0, and 75.0 ng/mL (below -25% cutoff), all 160 determinations were Negative. At 125.0, 150.0, 175.0, and 200.0 ng/mL (above +25% cutoff), all 160 determinations were Positive. At the 100 ng/mL Cutoff, results were 83 Negative/77 Positive (demonstrating expected variability around the cutoff).
Precision (Semiquantitative): Accurate mean concentration values for different spiked levels and reliable classification (negative below -25% cutoff, positive above +25% cutoff).	Mean concentrations closely matched nominal values (e.g., 29.2 ng/mL for 25.0 ng/mL samples, 189.0 ng/mL for 200.0 ng/mL samples). At 0.0, 25.0, 50.0, and 75.0 ng/mL, all 160 determinations were Negative. At 125.0, 150.0, 175.0, and 200.0 ng/mL, all 160 determinations were Positive. At the 100 ng/mL Cutoff, results were 97 Negative/63 Positive (demonstrating expected variability around the cutoff).
Analytical Recovery: Percentage recovery for various tramadol concentrations should be within an acceptable range (e.g., likely 80-120% or 90-110%, though not explicitly stated).	Recovery percentages ranged from 90.4% to 109.1% across concentrations from 50.0 to 1000.0 ng/mL, indicating good analytical recovery.
Analytical Specificity (Tramadol Metabolites): Known metabolites should exhibit a specific level of cross-reactivity, allowing detection without excessive interference from non-target compounds at the cutoff.	O-Desmethyltramadol: 16.67% cross-reactivity (positive at 600 ng/mL for 100 ng/mL cutoff). N-Desmethyltramadol: 66.67% cross-reactivity (positive at 150 ng/mL for 100 ng/mL cutoff). This demonstrates the assay's ability to react with key metabolites.
Analytical Specificity (Structurally-Related Compounds): Structurally related compounds (not metabolites) should not cause false positives at relevant concentrations, demonstrating assay specificity.	All listed structurally related compounds (e.g., 6-Acetyl Morphine, Amphetamine, Morphine, Fentanyl, etc.) were negative at the high concentrations tested (e.g., 10-500 µg/mL), confirming minimal to no cross-reactivity and high specificity for tramadol.
Interference (Structurally Unrelated Compounds): High concentrations of common medications/substances should not cause false positive or false negative results with tramadol present at ±25% of the cutoff.	Over 100 structurally unrelated compounds (e.g., Acetaminophen, Alprazolam, Caffeine, Cocaine, Ibuprofen, etc.) at very high concentrations (up to 500,000 ng/mL) did not yield false results (i.e., remained Negative at 75 ng/mL tramadol and Positive at 125 ng/mL tramadol). This indicates robust resistance to interference.
Interference (Endogenous Substances): High concentrations of common endogenous urine components should not cause false positive or false negative results.	All listed endogenous substances (e.g., Acetone, Bilirubin, Creatinine, Glucose, Hemoglobin, Urea, etc.) at physiological or high concentrations did not cause interference, maintaining expected negative or positive results at ±25% of the cutoff.
Interference (Specific Gravity and pH): Assay performance should be stable across a wide range of specific gravity and pH values expected in human urine.	No interference was observed across specific gravity values from 1.000 to 1.030 and pH values from 3.0 to 11.0, demonstrating robustness to variations in urine characteristics.
Interference (Boric Acid): The assay should either not be interfered with by common preservatives, or any interference should be clearly identified and communicated.	1% w/v Boric Acid did interfere by causing a negative result at 125 ng/mL in the qualitative mode (expected positive). This is explicitly noted, and a warning is provided: "Boric acid interferes with results from this device. Do not test samples that have boric acid as a preservative." This demonstrates that the interference was identified and addressed with a clear instruction.
Method Comparison with Confirmatory Method (LC-MS/MS): A high degree of concordance with the gold standard confirmatory method (LC-MS/MS) for samples far from the cutoff, and clear explanation of discordant results near the cutoff, demonstrating clinical utility.	Concordant Results: - 50 samples with LC-MS/MS < 50 ng/mL were Negative. - 56 samples with LC-MS/MS > 150 ng/mL were Positive. - 4 samples with LC-MS/MS 100-150 ng/mL were Positive. Discordant Results: - 5 samples with LC-MS/MS 50-99 ng/mL were shown as Positive by the ARK Immunoassay but were Negative by LC-MS/MS according to the cutoff. The cause of discordance for these 5 samples was identified as the presence of O-desmethyltramadol, which cross-reacts with the assay. This provides a clear explanation for expected discrepancies.
Calibration Curve Stability: Calibration should remain stable for a reasonable period.	A stored calibration curve was effective for at least 30 days.

2. Sample Size Used for the Test Set and the Data Provenance

Precision Studies: 160 determinations for each of 9 concentration levels (0.0 to 200.0 ng/mL) for both qualitative and semiquantitative modes. This totals 160 * 9 = 1440 individual measurements across 20 days. The samples were
- Data Provenance: Drug-free, negative human urine that was spiked with tramadol to create the test concentrations. The geographic origin of the human urine or whether it was retrospective/prospective is not specified beyond "human urine."
Analytical Recovery: Not explicitly stated, but "N=6" is mentioned for the mean concentration calculation at each of the 11 tested levels. So, 6 samples for each of 11 concentrations (50.0 to 1000.0 ng/mL) were used. These were also dilutions from drug-free, negative human urine spiked with tramadol.
Analytical Specificity (Metabolites and Structurally Related Compounds): Not explicitly stated, but compounds were "spiked into drug-free, negative human urine." The number of samples per compound is not specified, but each compound was tested.
Interference (Structurally Unrelated Compounds & Endogenous Substances): Not explicitly stated for each concentration or substance, but involved "High concentrations of the following structurally unrelated compounds/endogenous substances were added into urine spiked with tramadol (± 25% of the cutoff concentration) and tested."
Interference (Specific Gravity, pH, Boric Acid): Samples were spiked urine (± 25% of cutoff) manipulated for specific gravity/pH or with boric acid added. The number of samples is not explicitly stated per condition.
Method Comparison: A total of 115 unaltered clinical human urine specimens.
- Data Provenance: "clinical human urine specimens that are not individually identifiable." No country of origin is specified, but "unaltered clinical human urine specimens" suggests real-world samples. Whether retrospective or prospective is not explicitly stated.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts

This is an in vitro diagnostic (IVD) device for chemical analysis, not an imaging or clinical interpretation device. Therefore, the concept of "experts" establishing ground truth in the way a radiologist would for an image is not applicable.
For the method comparison study, the ground truth was established by a "licensed reference laboratory" using Gas Chromatography/Mass Spectrometry (GC/MS) or Liquid Chromatography/tandem Mass Spectrometry (LC-MS/MS), which are considered the gold standard confirmatory methods for drug testing. The qualifications of the personnel performing these confirmatory tests are inherent to the "licensed reference laboratory" performing a gold-standard chemical analysis, rather than subjective expert opinion.

4. Adjudication Method for the Test Set

Not applicable as this is a chemical assay with objective measurements (spectrophotometric absorbance changes) and comparison to a definitive analytical method (LC-MS/MS) for ground truth. Discrepancies in the method comparison were analytically explained (e.g., cross-reactivity with O-desmethyltramadol) rather than requiring human adjudication of subjective interpretations.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was Done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This type of study design is typically used for medical imaging devices or other diagnostic tools where human interpretation plays a significant role. The ARK Tramadol Assay is an automated immunoassay for chemical analysis, not a device requiring human "readers" in the context of an MRMC study.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, this entire study represents a standalone performance evaluation. The ARK Tramadol Assay is an automated immunoassay performed on "automated clinical chemistry analyzers." Its performance is directly compared to an established analytical method (LC-MS/MS), without "human-in-the-loop" interpretation as a primary output of the device itself. The results are quantitative (in semiquantitative mode) or qualitative (positive/negative), derived directly from the instrument's measurements.

7. The Type of Ground Truth Used

The ground truth used was confirmatory analytical methods, specifically Gas Chromatography/Mass Spectrometry (GC/MS) or Liquid Chromatography/tandem Mass Spectrometry (LC-MS/MS), which are considered the gold standard for drug detection and quantification in biological samples.

8. The Sample Size for the Training Set

The document does not provide information about a "training set" in the context of machine learning or AI. This device is an immunoassay, whose performance is based on established biochemical principles and reagents, not a machine learning algorithm that is "trained" on data. The studies described are method development and validation studies to establish the assay's analytical performance.

9. How the Ground Truth for the Training Set Was Established

As noted in point 8, there is no "training set" in the context of this immunoassay. The concept of "ground truth for a training set" is therefore not applicable. The assay's design and reagents are based on antibody-antigen binding principles rather than data-driven machine learning.

Summary

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: on the left, there is a symbol representing the Department of Health & Human Services - USA, and on the right, there is the text "FDA U.S. FOOD & DRUG ADMINISTRATION" in blue. The FDA logo is a recognizable symbol associated with the agency responsible for regulating and supervising the safety of food, drugs, and other products in the United States.

December 10, 2018

ARK Diagnostics, Inc. Cherry Mun Manager, Quality and Regulatory Affairs 48089 Fremont Boulevard Fremont, CA 94538

Re: K182280

Trade/Device Name: ARK Tramadol Assay Regulation Number: 21 CFR 862.3650 Regulation Name: Opiate test system Regulatory Class: Class II Product Code: DJG Dated: November 12, 2018 Received: November 13, 2018

Dear Cherry Mun:

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 statutes and regulations administered by other Federal agencies. You must comply with all the Act's

{1}------------------------------------------------

Page 2

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/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.htm); good manufacturing practice requirements as set forth in the quality systems (OS) 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 http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

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/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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

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

{2}------------------------------------------------

Indications for Use

510(k) Number (if known)

Form Approved: OMB No. 0910-0120 Expiration Date: 06/30/2020 See PRA Statement below.

Device Name ARK Tramadol Assay

Indications for Use (Describe)

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

Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

510(k) SUMMARY

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

The assigned 510(k) number is K182280.

807.92 (a)(1): Name:	ARK Diagnostics, Inc.
Address:	48089 Fremont BlvdFremont, CA 94538 USA
Owner Operator Number:	10027663
Establishment Registration:	3005755244
Phone:	(510) 270-6270
FAX:	(510) 270-6298
Contact:	Cherry Mun – (510) 270-6288Manager of Quality and Regulatory Affairs

Date Prepared: November 27th, 2018

807.92 (a)(2): Device Name – Trade Name, Common Name, and Classification

Trade Name:	ARKTM Tramadol Assay
Common Name:	Homogeneous Enzyme Immunoassay, Opiate Test System

Classification:

Product Code	Classification	Regulation Section	Panel
DJG	Class II	21 CFR 862.3650Opiate Test System	Toxicology(91)

807.92 (a)(3): Identification of the Legally Marketed Predicate Device

Immunalysis Tramadol Urine Enzyme Immunoassay K141803

{4}------------------------------------------------

807.92 (a)(4): Device Description

The ARK Tramadol Assay consists of reagents R1 anti-tramadol rabbit polyclonal antibody with substrate and R2 tramadol derivative labeled with bacterial recombinant G6PDH enzyme.

807.92 (a)(5): Intended Use / Indications for Use

ARK Tramadol Assay

{5}------------------------------------------------

807.92 (a)(6): Technological Similarities and Differences to the Predicate

SUBSTANTIAL EQUIVALENCE COMPARATIVE TABLE

Comparison between the Immunalysis Tramadol Urine Enzyme Immunoassay and the ARK™ Tramadol Assay

Characteristic	Predicate DeviceImmunalysis Tramadol Urine EnzymeImmunoassay (K141803)	Candidate DeviceARK™ Tramadol Assay
Similarities
Test System	Homogenous enzyme immunoassay(EIA)	Same
Intended Use	For the qualitative and semiquantitativedetermination of tramadol in humanurine; For in vitro diagnostic use	Same
Sample Matrix	Human urine	Same
User Environment	Clinical laboratories; Prescription useonly	Same
Mass SpectrometryConfirmation	Required to confirm preliminary positiveanalytical results	Same
Platform Required	Automated clinical chemistry analyzer	Same
Reagents Form	Liquid - Ready to use	Same
Reagent Materials	Two (2) reagent system:Antibody/substrate reagent and enzymelabeled conjugateSodium azide preservative	Same
Storage	2-8°C until expiration date	Same
Measured Analyte	Tramadol	Same
Antibody	Polyclonal antibodies to tramadol	Same
Detection	Absorbance change measuredspectrophotometrically at 340 nm	Same
Characteristic	Predicate DeviceImmunalysis Tramadol Urine EnzymeImmunoassay (K141803)	Candidate DeviceARK™ Tramadol Assay
Differences

200 ng/mL

Cutoff Level

100 ng/mL

{6}------------------------------------------------

807.92 (b)(1) and 807.92 (b)(2): Brief Description of Nonclinical and Clinical Data

The following performance characteristics were obtained on the Beckman Coulter AU680® automated clinical chemistry analyzer.

Precision

Precision studies were performed using CLSI EP05-A3 as a guideline. Drug-free, negative human urine was supplemented with tramadol (0.0 to 200.0 ng/mL). Each level was assayed in quadruplicate twice a day for 20 days (N=160) in both qualitative and semiquantitative modes. Results are summarized in the tables below.

Qualitative Precision

Human Urine(ng/mL)	% Cutoff	# ofDeterminations	QualitativePrecision Results
0.0	-100	160	160 Negative
25.0	-75	160	160 Negative
50.0	-50	160	160 Negative
75.0	-25	160	160 Negative
100.0	Cutoff	160	83 Negative/77 Positive
125.0	+25	160	160 Positive
150.0	+50	160	160 Positive
175.0	+75	160	160 Positive
200.0	+100	160	160 Positive

Semiquantitative Precision

HumanUrine(ng/mL)	Relative %Cutoff	# of Results	Mean(ng/mL)	SemiquantitativePrecision Results
0.0	-100	160	1.7	160 Negative
25.0	-75	160	29.2	160 Negative
50.0	-50	160	53.7	160 Negative
75.0	-25	160	76.7	160 Negative
100.0	Cutoff	160	98.5	97 Negative/63 Positive
125.0	+25	160	120.5	160 Positive
150.0	+50	160	142.6	160 Positive
175.0	+75	160	165.3	160 Positive
200.0	+100	160	189.0	160 Positive

{7}------------------------------------------------

Analytical Recovery

Recovery across the assay range was assessed using the semiquantitative mode. Drug-free, negative human urine was supplemented with tramadol (1100.0 ng/mL) and dilutions were made proportionally with drug-free human urine. Tramadol concentrations ranged from 50.0 to 1000.0 ng/mL. At each level, percentage recovery was calculated based on the mean concentration (N=6) compared to the expected concentration. Results are summarized in the table below.

TheoreticalConcentration(ng/mL)	MeanConcentration(ng/mL)	Recovery(%)
50.0	52.8	105.6
100.0	107.3	107.3
200.0	191.2	95.6
300.0	277.1	92.4
400.0	361.5	90.4
500.0	490.7	98.1
600.0	654.8	109.1
700.0	724.4	103.5
800.0	872.5	109.1
900.0	917.9	102.0
1000.0	984.1	98.4

Analytical Specificity

All compounds tested were added to drug-free, negative human urine and tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes.

Tramadol Metabolites

The cross-reactivity of the following metabolites of tramadol was evaluated by spiking these compounds into drug-free, negative human urine to determine the minimum concentration that would give a positive result approximately equivalent to the 100 ng/mL tramadol cutoff. These concentrations were used to determine the percent cross-reactivity according to the formula:

% Cross-reactivity = (Cutoff concentration / Lowest concentration of cross-reactant causing a positive result) X 100

Compound	Lowest ConcentrationTested That Produceda ResponseApproximatelyEquivalent to theCutoff(ng/mL)	Percent Cross-reactivity (%)
O-Desmethyltramadol	600	16.67
N-Desmethyltramadol	150	66.67

{8}------------------------------------------------

Structurally Related Compounds

The following structurally related compounds were negative at the concentrations tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes.

Compound	Concentration Tested (µg/mL)
6-Acetyl Morphine	10
Amitriptyline	100
Amphetamine	100
Chlorpromazine	50
Clomipramine	50
Cyclobenzaprine	10
Desipramine	50
Dextromethorphan	100
Diphenhydramine	500
Doxepin	50
EDDP	100
EMDP	50
Fentanyl	100
Fluoxetine	50
Imipramine	30
Ketamine	100
MDEA	75
Meperidine	100
Methadone	100
Methapyrilene	10
Methylphenidate	100
Methylphenidate Metabolite	100
Morphine	50
Morphine-3-glucuronide	50
N-Desmethyltapentadol	100
Norcodeine	100
NorFentanyl	100
Norketamine	100
Normeperidine	50
Normorphine	50
Noroxycodone	25
Nortriptyline	10
Pentazocine (Talwin)	100
PCP	100
Propranolol	15
Quinine	450
Risperidone	50
Tapentadol	100
Thioridazine	100
Trazodone	10
Venlafaxine	100

{9}------------------------------------------------

Interference – Structurally Unrelated Compounds

High concentrations of the following structurally unrelated compounds were added into urine spiked with tramadol (± 25% of the cutoff concentration) and tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes. The substances listed at the concentrations below did not yield a false result relative to the 100 ng/mL cutoff.

Compound	Concentration(ng/mL)	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
6-Acetylcodone	100,000	Negative	Positive
6-Acetylmorphine	100,000	Negative	Positive
7-Aminoclonazepam	100,000	Negative	Positive
7-Aminoflunitrazepam	100,000	Negative	Positive
7-Aminonitrazepam	100,000	Negative	Positive
Albuterol	100,000	Negative	Positive
Acetaminophen	500,000	Negative	Positive
Acetylsalicylic Acid	500,000	Negative	Positive
Alprazolam	50,000	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
Bromazepam	100,000	Negative	Positive
4-Bromo-2,5,Dimethoxyphenethylamine	100,000	Negative	Positive
Buprenorphine	100,000	Negative	Positive
Buprenorphine Glucuronide	50,000	Negative	Positive
Bupropion	25,000	Negative	Positive
Butabarbital	100,000	Negative	Positive
Caffeine	500,000	Negative	Positive
Cannabidiol	100,000	Negative	Positive
Cannabinol	100,000	Negative	Positive
Carbamazepine	100,000	Negative	Positive
Carisoprodol	100,000	Negative	Positive
Chlordiazepoxide	100,000	Negative	Positive
Chlorpromazine	100,000	Negative	Positive
Clobazam	100,000	Negative	Positive
Clomipramine	100,000	Negative	Positive
Clonazepam	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
Demoxepam	100,000	Negative	Positive
Desakylflurazepam	100,000	Negative	Positive
Desipramine	100,000	Negative	Positive
Dextromethorphan	100,000	Negative	Positive
Diazepam	50,000	Negative	Positive
Compound	Concentration(ng/mL)	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
Dihydrocodeine	100,000	Negative	Positive
Diphenhydramine	100,000	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
EtG	100,000	Negative	Positive
Ethylmorphine	100,000	Negative	Positive
R-Fenfluramine	100,000	Negative	Positive
S-Fenfluramine	100,000	Negative	Positive
Fentanyl	100,000	Negative	Positive
Flunitrazepam	100,000	Negative	Positive
Fluoxetine	50,000	Negative	Positive
Fluphenazine	100,000	Negative	Positive
Flurazepam	100,000	Negative	Positive
Heroin	100,000	Negative	Positive
Hexobarital	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	75,000	Negative	Positive
Lidocaine	100,000	Negative	Positive
Lorazepam	100,000	Negative	Positive
Lorazepam Glucuronide	50,000	Negative	Positive
Lormetazepam	100,000	Negative	Positive
LSD	100,000	Negative	Positive
Maprotiline	100,000	Negative	Positive
MDA	100,000	Negative	Positive
MDEA	10,000	Negative	Positive
MDMA	50,000	Negative	Positive
Meperidine	100,000	Negative	Positive
Meprobamate	100,000	Negative	Positive
Meprotiline	50,000	Negative	Positive
Methadone	500,000	Negative	Positive
S(+)-methamphetamine	500,000	Negative	Positive
Methaqualone	100,000	Negative	Positive
Methylphenidate	25,000	Negative	Positive
Metronidazole	300,000	Negative	Positive
Midazolam	100,000	Negative	Positive
Morphine	100,000	Negative	Positive
Morphine-3-beta-glucuronide	100,000	Negative	Positive
Compound	Concentration(ng/mL)	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
Morphine-6-beta-glucuronide	100,000	Negative	Positive
Nalorphine	100,000	Negative	Positive
Naloxone	100,000	Negative	Positive
Naltrexone	100,000	Negative	Positive
Naproxen	100,000	Negative	Positive
N-desmethyltapentadol	25,000	Negative	Positive
Nicotine	10,000	Negative	Positive
Nitrazepam	100,000	Negative	Positive
Norbuprenorphine	100,000	Negative	Positive
Norcodeine	100,000	Negative	Positive
Nordiazepam	100,000	Negative	Positive
Normorphine	100,000	Negative	Positive
Norproproxyphene	100,000	Negative	Positive
Norpseudoephedrine	100,000	Negative	Positive
Nortriptyline	100,000	Negative	Positive
Oxazepam	100,000	Negative	Positive
Oxazepam Glucuronide	10,000	Negative	Positive
Oxycodone	100,000	Negative	Positive
Oxymorphone	100,000	Negative	Positive
PCP	10,000	Negative	Positive
Pentazocine	50,000	Negative	Positive
Pentobarbital	100,000	Negative	Positive
Phenobarbital	100,000	Negative	Positive
Phentermine	100,000	Negative	Positive
Phenylephrine	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	2,000	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	50,000	Negative	Positive
Sufentanil Citrate	10,000	Negative	Positive
Tapentadol	25,000	Negative	Positive
Temazepam	100,000	Negative	Positive
11-nor-9-carboxy THC	100,000	Negative	Positive
Theophylline	100,000	Negative	Positive
Thioridazine	25,000	Negative	Positive
Tilidine	50,000	Negative	Positive
Trazodone	100,000	Negative	Positive
Triazolam	100,000	Negative	Positive
Compound	Concentration(ng/mL)	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
Trifluoromethylphenylpiperazine	100,000	Negative	Positive
Trimipramine	100,000	Negative	Positive
Valproic Acid	250,000	Negative	Positive
Zolpidem Tartrate	100,000	Negative	Positive

ARK Diagnostics, Inc. - 510(k) Summary ARK Tramadol Assay

Page 5-7 of 5-12

{10}------------------------------------------------

ARK Diagnostics, Inc. – 510(k) Summary ARK Tramadol Assay

Page 5-8 of 5-12

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

ARK Diagnostics, Inc. – 510(k) Summary ARK Tramadol Assay

Page 5-9 of 5-12

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

Interference – Endogenous Substances

Interference studies were performed using CLSI EP07-A2 as a guideline. High concentrations of the following endogenous substances were added into urine spiked with tramadol (± 25% of the cutoff concentration). No interference was observed when tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes.

Compound	ConcentrationTested	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
Acetone	1000 mg/dL	Negative	Positive
Ascorbic Acid	1500 mg/dL	Negative	Positive
Bilirubin	2 mg/dL	Negative	Positive
Creatinine	500 mg/dL	Negative	Positive
Ethanol	1000 mg/dL	Negative	Positive
Galactose	10 mg/dL	Negative	Positive
Gamma Globulin	500 mg/dL	Negative	Positive
Glucose	3000 mg/dL	Negative	Positive
Hemoglobin	300 mg/dL	Negative	Positive
Human Albumin	500 mg/dL	Negative	Positive
Oxalic Acid	100 mg/dL	Negative	Positive
Riboflavin	7.5 mg/dL	Negative	Positive
Sodium Azide	1% w/v	Negative	Positive
Sodium Chloride	6000 mg/dL	Negative	Positive
Sodium Fluoride	1% w/v	Negative	Positive
Urea	6000 mg/dL	Negative	Positive

Interference – Specific Gravity and pH

Urine samples with specific gravity values from 1.000 to 1.030 and pH values ranging from 3.0 to 11.0 were tested in the presence of the two levels of tramadol at ± 25% of the cutoff concentration. No interference was observed when tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes.

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

Interference – Boric Acid

One percent (1%) w/v of boric acid was added into urine spiked with tramadol (± 25% of the cutoff concentration) and tested with the ARK Tramadol Assay in both qualitative and semiquantitative modes. Results are provided in the table below.

		Semiquantitative Mode		Qualitative Mode
Compound	ConcentrationTested	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)	75 ng/mL(-25% Cutoff)	125 ng/mL(+25% Cutoff)
Boric Acid	1% w/v	Negative	Positive	Negative	Negative

Boric acid interferes with results from this device. Do not test samples that have boric acid as a preservative.

Method Comparison

A total of one hundred fifteen (115) unaltered clinical human urine specimens that are not individually identifiable were analyzed for tramadol with the ARK Tramadol Assay in both qualitative and semiquantitative modes and the results were compared to LC-MS/MS. The LC-MS/MS confirmatory method was performed by a licensed reference laboratory. Results are summarized in the tables below.

ARKImmunoassayResult	Low NegativeLess than50% belowthe Cutoff(< 50 ng/mLby LC-MS/MS)	Near CutoffNegativeBetween 50%below theCutoff and theCutoff(50 – 99ng/mL by LC-MS/MS)	Near CutoffPositiveBetween theCutoff and50% abovethe Cutoff(100 - 150ng/mL by LC-MS/MS)	High PositiveGreater than50% abovethe Cutoff(> 150 ng/mLby LC-MS/MS)
Negative	50	0	0	0
Positive	0	5*	4	56

*Discordant Results

Sample ID Number	ARK ImmunoassayResult	Tramadol(ng/mL by LC-MS/MS)
01	Positive	74.0
05	Positive	98.7
06	Positive	98.9
51	Positive	75.0
52	Positive	79.0

O-desmethyltramadol was detected in these samples and contributed to the positive result obtained with the ARK Tramadol Assay.

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

Traceability and Value Assignment

ARK Tramadol Calibrators and Controls are prepared by volumetric dilution of high purity tramadol (certified solution traceable to HPLC) into non-sterile, processed human urine free of tramadol. Testing is performed with the ARK Tramadol Assay on the Beckman Coulter AU680 automated clinical chemistry analyzer, calibrated with the ARK Tramadol Calibrator.

Calibration Curve Stability

A stored calibration curve was effective up to at least 30 days based on supporting data. Calibration curve stability may depend on individual laboratory performance.

807.92 (b)(3): Conclusions from Nonclinical Testing

As summarized above, the ARK Tramadol Assay is substantially equivalent to the legally marketed predicate device, Immunalysis Tramadol Urine Enzyme Immunoassay (K141803).

Regulation Number and Section

§ 862.3650 Opiate test system.

(a)
Identification. An opiate test system is a device intended to measure any of the addictive narcotic pain-relieving opiate drugs in blood, serum, urine, gastric contents, and saliva. An opiate is any natural or synthetic drug that has morphine-like pharmocological actions. The opiates include drugs such as morphine, morphine glucoronide, heroin, codeine, nalorphine, and meperedine. Measurements obtained by this device are used in the diagnosis and treatment of opiate use or overdose and in monitoring the levels of opiate administration to ensure appropriate therapy.(b)
Classification. Class II (special controls). An opiate 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).