(110 days)
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.
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.
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 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.
§ 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).