(166 days)
The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay intended for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers. The measurements obtained are used in monitoring levels of methotrexate to help ensure appropriate therapy. Specimens from patients who have received glucarpidase (carboxypeptidase G2) as a high dose methotrexate rescue therapy should not be tested with the ARK Methotrexate II Assay.
The ARK Methotrexate II Assay is a homogeneous immunoassay based on competition between drug in the specimen and methotrexate labeled with the recombinant enzyme glucose-6phosphate dehydrogenase (rG6PDH) for binding to the antibody reagent. 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 the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH that is measured spectrophotometrically as a rate of change in absorbance. Endogenous serum G6PDH does not interfere with the results because the coenyzme NAD functions only with the bacterial enzyme (rG6PDH) used in the assay. The ARK Methotrexate II Assay consists of reagents R1 anti-methotrexate monoclonal antibody with substrate and R2 methotrexate labeled with recombinant G6PDH enzyme. The test system includes the ARK Methotrexate II Calibrator, ARK Methotrexate II Control, and ARK Methotrexate II Dilution Buffer.
The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay for the quantitative determination of methotrexate in human serum or plasma.
Here's an analysis of the acceptance criteria and the study proving the device meets them:
1. Table of Acceptance Criteria and Reported Device Performance:
| Acceptance Criteria | Device Performance (Reported) |
|---|---|
| Limit of Quantitation (LoQ) | Established as 0.030 umol/L. Acceptable inter-assay precision (<0.010 SD) and recovery (±0.010 umol/L) observed at 0.030 umol/L. |
| Measurement Range | 0.030 - 1.300 umol/L. |
| Recovery | All concentrations tested showed ±10% recovery of the expected sample concentration. |
| Linearity | A linear relationship was demonstrated between 0.030 and 1.300 umol/L with a maximum deviation of 8.73% from linearity. |
| Clinical Accuracy (vs. LC-MS/MS) | Slope of 1.03, intercept of 0.00, and Pearson correlation (r²) of 0.98. Mean bias of 0.01 with 95% Limits of Agreement from -0.11 to 0.14. |
| Method Comparison (vs. Predicate Device) | Slope = 0.98, y-intercept = -0.02, and correlation coefficient (r²) = 0.97. |
| Precision (Total CV) | ≤10% for all tested levels of controls and human serum samples. |
| Interference by Endogenous Substances | Elevated concentrations of common endogenous substances (albumin, bilirubin, cholesterol, etc.) did not interfere significantly (<±7.48% interference). |
| Analytical Specificity (7-OH-MTX) | Not substantially affected by 7-Hydroxymethotrexate (8.72% interference at 0.050 µmol/L MTX, 0.58% at 0.500 µmol/L MTX). |
| Analytical Specificity (DAMPA) | Significant interference with DAMPA (up to 57.50% cross-reactivity at 0.040 µmol/L DAMPA). Device should not be used during glucarpidase rescue therapy. |
| Analytical Specificity (Other Compounds) | Methotrexate-selective antibody did not cross-react with various potentially co-administered drugs and folate derivatives (all within ±10% interference). |
| Sample Stability | Stable for at least 14 days refrigerated (2-8 °C), 14 days at room temperature (25 °C), 15 months frozen (-20 °C), and after 3 freeze/thaw cycles. |
| Product Stability | Shelf-life stability claim of up to 18 months when stored unopened at 2-8°C. |
| On-Board Stability | Reagents stable up to 100 days on-board the instrument. |
| Calibration Curve Stability | Effective up to at least 100 days. |
2. Sample Size Used for the Test Set and Data Provenance:
- Clinical Accuracy (vs. LC-MS/MS): 90 patient samples.
- Method Comparison (vs. Predicate Device): 123 patient samples.
- LoQ, Recovery, Linearity, Precision, Interference, Analytical Specificity: These studies involved spiked human serum samples, pooled human serum, and controls, rather than a specific number of unique patient samples for the test set. For LoQ, 40 replicates were tested. For Recovery, 6 replicates per sample were tested. For Linearity, 6 replicates per sample were tested. For Precision, 160 replicates were tested for each of the 6 control levels and 6 human serum levels. For Interference and Analytical Specificity, 6 replicates per sample were tested.
- Data Provenance: "Leftover specimens were obtained from persons receiving high-dose methotrexate therapy" for the method comparison studies. The document does not specify the country of origin of the data or whether the studies were retrospective or prospective, though "leftover specimens" suggests a retrospective collection.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:
- The ground truth for the clinical accuracy study was established by an LC-MS/MS reference method. This is an analytical laboratory technique, not based on expert consensus. Therefore, no "experts" in the traditional sense (e.g., radiologists) were used for ground truth establishment. The performance of LC-MS/MS as a reference method typically implies its results are considered the gold standard.
4. Adjudication Method for the Test Set:
- Not applicable. The ground truth for the clinical accuracy and method comparison studies was established using objective analytical methods (LC-MS/MS and the predicate device), not through expert review requiring an adjudication method.
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:
- Not applicable. This device is an in vitro diagnostic (IVD) assay for quantitative determination of a substance in human samples. It does not involve human readers interpreting medical images or data.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- Yes, the performance data presented (Limit of Quantitation, Measurement Range, Recovery, Linearity, Clinical Accuracy, Method Comparison, Precision, Interference, Analytical Specificity) reflects the standalone performance of the ARK Methotrexate II Assay (an automated immunoassay) without human intervention in the result generation. The device itself is designed to provide quantitative measurements.
7. The Type of Ground Truth Used:
- Clinical Accuracy: Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) was used as the reference method (gold standard) for ground truth.
- Method Comparison: The legally marketed predicate device, ARK™ Methotrexate Assay (K111904), was used to compare results.
- Other performance metrics (LoQ, Recovery, Linearity, Precision, Interference, Analytical Specificity): Ground truth was established by preparing samples with known concentrations of methotrexate or interfering substances, typically by spiking human serum with certified reference materials.
8. The Sample Size for the Training Set:
- Not applicable. This device is an immunoassay (laboratory test kit), not a machine learning or AI algorithm that requires a "training set" in the conventional sense of computational models. The development and optimization of the assay would involve various experiments and reagent formulations, but not a distinct "training set" like for AI models.
9. How the Ground Truth for the Training Set Was Established:
- Not applicable, as there is no "training set" in the context of an AI/ML algorithm for this type of medical device. The "ground truth" for the development of the assay components would involve standard analytical chemistry practices, such as using reference materials and established analytical methods to verify the concentration and purity of substances used in the reagents.
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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
ARK Diagnostics, Inc. Thomas Houts, Ph.D. Sr. Director, Quality, Regulatory and Planning 48089 Fremont Boulevard Fremont, California 94538
Re: K232017
Trade/Device Name: ARK Methotrexate II Assay Regulatory Class: Unclassified Product Code: LAO Dated: November 21, 2023 Received: November 22, 2023
Dear Dr. Houts:
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 (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 available at https://www.accessdata.fda.gov/scripts/cdrb/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.
Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).
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Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).
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 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 Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-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 Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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 mediation-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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Joseph A. Digitally signed by Kotarek -S Date: 2023.12.20
Joseph Kotarek Branch Chief Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics 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) K232017
Device Name ARK Methotrexate II Assay
Indications for Use (Describe)
The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay intended for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers. The measurements obtained are used in monitoring levels of methotrexate to help ensure appropriate therapy. Specimens from patients who have received glucarpidase (carboxypeptidase G2) as a high dose methotrexate rescue therapy should not be tested with the ARK Methotrexate II Assay.
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) |
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Section 5: 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 K232017.
| 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: | Thomas Houts, Ph.D.Director, Quality, Regulatory and PlanningEmail: tom@ark-tdm.comDirect phone: 510-270-6296 |
Date Prepared: June 28, 2023
807.92 (a)(2): Device name - trade name and common name, and classification
Trade Name: ARK Methotrexate II Assay
Common Name: Homogeneous Enzyme Immunoassay
Classification:
| Product Code | Classification | Regulation Section | Panel |
|---|---|---|---|
| LAO - Methotrexate enzymeimmunoassay | II | Unclassified | Toxicology(91) |
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807.92 (a)(3): Identification of the legally marketed predicate device
Predicate Device Name: ARKTM Methotrexate Assay Predicate 510(k) Number: K11904
807.92 (a)(4): Device Description
The ARK Methotrexate II Assay is a homogeneous immunoassay based on competition between drug in the specimen and methotrexate labeled with the recombinant enzyme glucose-6phosphate dehydrogenase (rG6PDH) for binding to the antibody reagent. 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 the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH that is measured spectrophotometrically as a rate of change in absorbance. Endogenous serum G6PDH does not interfere with the results because the coenyzme NAD functions only with the bacterial enzyme (rG6PDH) used in the assay.
The ARK Methotrexate II Assay consists of reagents R1 anti-methotrexate monoclonal antibody with substrate and R2 methotrexate labeled with recombinant G6PDH enzyme.
The test system includes the ARK Methotrexate II Calibrator, ARK Methotrexate II Control, and ARK Methotrexate II Dilution Buffer.
Summary and Explanation of Test
Methotrexate [N-[4][(2.4-diamino-6-pteridiny]) methylamino]benzov]]-L-glutamic acid] is an antimetabolite used in the treatment of certain neoplastic diseases, severe psoriasis, and adult rheumatoid arthritis.
807.92 (a)(5): Intended Use / Indications for Use
ARK Methotrexate II Assay
The ARK Methotrexate II Assay is a homogeneous enzyme immunoassay intended for the quantitative determination of methotrexate in human serum or automated clinical chemistry analyzers. The measurements obtained are used in monitoring levels of methotrexate to help ensure appropriate therapy. Specimens from patients who have received glucarpidase (carboxypeptidase G2) as a high dose methotrexate rescue therapy should not be tested with the ARK Methotrexate II Assay.
807.92 (a)(6): Technological Similarities and Differences to the Predicate
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SUBSTANTIAL EQUIVALENCE COMPARATIVE TABLES
| Comparison between ARK™ Methotrexate Assay and ARK Methotrexate II Assay | ||
|---|---|---|
| Characteristic | Predicate Device | Candidate Device |
| ARK™ Methotrexate Assay (K111904) | ARK™ Methotrexate II Assay | |
| Intended Use | The ARK™ Methotrexate Assay isintended for the quantitative determinationof methotrexate in human serum or plasmaon automated clinical chemistry analyzers. | Same |
| Indications forUse | The results obtained are used in monitoringlevels of methotrexate to help ensureappropriate therapy. | Same |
| Sample Matrix | Human serum or plasma | Same |
| Two (2) reagent system: | Two (2) reagent system: | |
| ReagentComponents | Anti-methotrexate Antibody/SubstrateReagent (R1) containing rabbit polyclonalantibodies to methotrexate, glucose-6-phosphate, nicotinamide adeninedinucleotide, bovine serum albumin,preservatives, and stabilizersEnzyme Reagent (R2) containingmethotrexate labeled with bacterialG6PDH, buffer, bovine serum albumin,preservatives, and stabilizers | Anti-methotrexate Antibody/SubstrateReagent (R1) containing rabbit monoclonalantibody to methotrexate, glucose-6-phosphate, nicotinamide adeninedinucleotide, bovine serum albumin,sodium azide, and stabilizersEnzyme Reagent (R2) containingmethotrexate labeled with recombinantG6PDH, buffer, bovine serum albumin,sodium azide, and stabilizers |
| Methodology | Homogeneous Enzyme Immunoassay(EIA) | Same |
| Platform Required | Automated Clinical Chemistry Analyzer | Same |
| User Environment | Professional Clinical Laboratory:Prescription Use Only | Same |
| Reagents Form | Liquid - Ready to use | Same |
| Storage | 2-8° C until expiration date | Same |
| Analyte | Methotrexate | Same |
Comparison between ARK™ Methotrexate Assay and ARK Methotrexate II Assay
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.
Limit of Quantitation (LoQ)
ARK Diagnostics, Inc. – 510(k) SummaryPage 5-3 of 5-11 ARK Methotrexate II Assay
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The LoQ of the ARK Methotrexate II Assay was established as 0.030 umol/L and may depend on analyzer specific performance. The LoQ was determined according to CLSI EP17-A2 and is defined as the lowest concentration for which acceptable inter-assay precision (<0.010 SD) and recovery (±0.010 umol/L) is observed. Pooled human serum was supplemented with methotrexate to give concentrations of 0.030, 0.040, and 0.050 umol/L. Eight (8) replicates of each sample were tested in each of five (5) runs to give a minimum of 40 replicates of each LoQ sample tested.
| NominalConcentration(umol/L) | Grand Mean(umol/L) | SD | CV (%) |
|---|---|---|---|
| 0.030 | 0.034 | 0.002 | 4.87 |
| 0.040 | 0.043 | 0.002 | 4.01 |
| 0.050 | 0.052 | 0.003 | 4.00 |
Measurement Range
The measurement range of the ARK Methotrexate II Assay is 0.030 - 1.300 umoVL. Specimens containing methotrexate in higher concentrations (>1.300 umol/L) may be assayed by dilution of the specimen into the measurement range for a quantitative result or otherwise reported as detected above the measurement range. Multiply the assay result by the dilution factor to obtain the concentration of methotrexate in the undiluted specimen.
Recovery
Analytical recovery throughout the measurement range was performed by adding concentrated methotrexate drug into human serum negative for methotrexate. A certified stock concentrate of highly pure methotrexate was added volumetrically to human serum negative for methotrexate, representing drug concentrations across the assay range. Two analytical runs of three replicates of each sample were assayed on an automated clinical chemistry analyzer. The results of the six replicates of each sample were averaged and compared to the target concentration and percent recovery calculated. Recovery at all concentrations tested was ±10% of the expected sample concentration.
| Theoretical ConcentrationTested (umol/L) | Mean (umol/L) | %Recovery |
|---|---|---|
| 0.060 | 0.063 | 104.4 |
| 0.100 | 0.105 | 105.2 |
| 0.300 | 0.322 | 107.2 |
| 0.600 | 0.628 | 104.7 |
| 1.000 | 1.079 | 107.9 |
| 1.200 | 1.293 | 107.8 |
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Linearity
Linearity studies were performed as suggested in CLSI Protocol EP06-Ed2. A methotrexate serum sample was prepared to contain 1.600 umol/L, and dilutions were made proportionally with human serum negative for methotrexate. Methotrexate concentrations ranged from 0.030 to 1.300 umol/L. Two analytical runs of three replicates of each sample were assayed on an automated clinical chemistry analyzer. The results of the six replicates of each sample were averaged. A weighted linear regression analysis (intercept set to zero) was performed in which the varying observed standard deviations were weighed into the calculation. A fitted slope was generated (1.087) and used to calculate the predicated results of which the observed results were compared to. A ±10% deviation from linearity was allowable. A linear relationship was demonstrated between 0.030 and 1.300 umol/L. Results are shown below.
| NominalConcentration(µmol/L) | ObservedResults(µmol/L) | PredictedResults(µmol/L) | % Deviation |
|---|---|---|---|
| 0.000 | 0.000 | NA | NA |
| 0.030 | 0.035 | 0.033 | 5.78 |
| 0.060 | 0.062 | 0.065 | -4.96 |
| 0.130 | 0.129 | 0.141 | -8.73 |
| 0.260 | 0.296 | 0.283 | 4.66 |
| 0.390 | 0.399 | 0.424 | -5.98 |
| 0.520 | 0.549 | 0.565 | -2.89 |
| 0.650 | 0.721 | 0.707 | 2.07 |
| 0.780 | 0.877 | 0.848 | 3.36 |
| 0.910 | 1.012 | 0.989 | 2.32 |
| 1.040 | 1.157 | 1.131 | 2.34 |
| 1.170 | 1.261 | 1.272 | -0.87 |
| 1.300 | 1.380 | 1.413 | -2.40 |
Method Comparison
Measurement procedure comparison studies were performed using CLSI Protocol EP09-A3. Results from the ARK Methotrexate II Assay on the Beckman AU680 were compared with (1) methotrexate determinations by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and (2) results from the predicate ARK Methotrexate Assay on the Beckman AU680. Leftover specimens were obtained from persons receiving high-dose methotrexate therapy.
ARK Methotrexate II Assay vs Reference Method LC-MS/MS
ARK Diagnostics, Inc. - 510(k) SummaryPage 5-5 of 5-11 ARK Methotrexate II Assay
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Clinical accuracy of the ARK Methotrexate II Assay was demonstrated versus the LC-MS/MS reference method. Ninety (90) patient samples were tested. Methotrexate levels ranged from 0.026 to 1.280 umol/L by LC-MS/MS and methotrexate levels ranged from 0.033 to 1.294 umol/L by the ARK Methotrexate II Assay. Comparative analysis gave a slope of 1.03, intercept of 0.00, and a correlation of 0.98 calculated using Pearson's Correlation (r²). The Passing-Bablok regression plot is shown Figure 1 and the Bland Altman analysis is shown in Figure 2.
| Sample range: 0.026 to 1.280 µmol/L | |||
|---|---|---|---|
| Method | Description | 95 % Confidence Interval | |
| Passing Bablok | Number of samples | 90 | |
| Sample range (µmol/L) | 0.026 to 1.280 | ||
| Constant Bias | 0.00 | -0.01 to 0.01 | |
| Proportional Bias | 1.03 | 1.00 to 1.06 | |
| Pearson Correlation | Correlation Coefficient (r²) | 0.98 | 0.96 to 0.98 |
| Bland Altman | Mean Bias | 0.01 | 0.00 to 0.03 |
| SE | 0.007 | ||
| SD of difference | 0.06 | ||
| 95 % Limits of Agreement | |||
| Lower | -0.11 | -0.14 to -0.09 | |
| Upper | 0.14 | 0.11 to 0.16 |
| ARK Methotrexate II Assay vs LC-MS/MS | ||
|---|---|---|
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Image /page/9/Figure/0 description: The image is a scatter plot titled "ARK Methotrexate II Assay vs. LC-MS/MS Scatter Plot with Passing & Bablok Fit". The x-axis represents LC-MS/MS (µmol/L) ranging from 0.000 to 1.400, and the y-axis represents ARK MTX II Assay (µmol/L) ranging from 0.000 to 1.400. The plot includes data points, a gray line labeled "Identity", and a blue line labeled "Passing & Bablok (I) fit (0.00 + 1.03x)".
Figure 1. Method Comparison -ARK Methotrexate II Assay versus LC-MS/MS
Image /page/9/Figure/2 description: The image is a difference plot titled "LC-MS/MS & ARK Methotrexate II Assay Difference Plot". The y-axis is labeled "Difference (ARK MTX II Assay (µmol/L) - LC-MS/MS (µmol/L))" and ranges from -0.400 to 0.400. The x-axis is labeled "Mean of All (µmol/L)" and ranges from 0.000 to 1.400. The plot shows scattered data points, a blue line indicating a bias of 0.0, and two dashed lines indicating 95% limits of agreement from -0.1 to 0.1.
Figure 2. Bland-Altman Difference Plot
ARK Diagnostics, Inc. - 510(k) SummaryPage 5-7 of 5-11 ARK Methotrexate II Assay
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Method comparison was also performed against the original ARK Methotrexate Assay for 123 patient samples with methotrexate values ranging from 0.054 to 1.168. Statistics with confidence intervals from the Passing-Bablok comparison are slope = 0.98 (0.95 to 1.01); y-intercept = - 0.02 (-0.03 to -0.01); and correlation coefficient (r2) = 0.97 (0.96 to 0.98).
Precision
Precision was assessed as described in CLSI Protocol EP05-A3. Six-level controls and six samples of methotrexate in pooled human serum were used in the study. Data were collected on a single analyzer over twenty (20) non-consecutive days. One (1) calibration was performed according to requirements for quality control. Each level was assayed in quadruplicate twice a day for 20 days. Each of the runs per day was separated by at least two hours. The within run, between day, total SD, and percent CVs were calculated. Acceptance criteria: ≤10% total CV.
| Within Run | Between Day | Total | ||||||
|---|---|---|---|---|---|---|---|---|
| Sample | N | Mean(µmol/L) | SD | CV(%) | SD | CV(%) | SD | CV(%) |
| ARK Methotrexate II Control | ||||||||
| LOW | 160 | 0.069 | 0.002 | 2.84 | 0.001 | 1.23 | 0.002 | 3.00 |
| MID | 160 | 0.411 | 0.006 | 1.40 | 0.002 | 0.43 | 0.006 | 1.40 |
| HIGH | 160 | 0.811 | 0.014 | 1.79 | 0.008 | 0.97 | 0.017 | 2.05 |
| 5 | 160 | 4.868 | 0.070 | 1.44 | 0.036 | 0.74 | 0.077 | 1.58 |
| 50 | 160 | 49.660 | 1.108 | 2.23 | 0.397 | 0.80 | 1.141 | 2.30 |
| 500 | 160 | 493.769 | 8.012 | 1.62 | 2.483 | 0.50 | 8.012 | 1.62 |
| Human Serum | ||||||||
| LOW | 160 | 0.070 | 0.002 | 2.50 | 0.001 | 1.49 | 0.002 | 2.88 |
| MID | 160 | 0.404 | 0.008 | 1.86 | 0.003 | 0.65 | 0.008 | 1.92 |
| HIGH | 160 | 0.846 | 0.016 | 1.93 | 0.008 | 0.95 | 0.017 | 2.06 |
| 5 | 160 | 5.247 | 0.076 | 1.45 | 0.028 | 0.54 | 0.078 | 1.49 |
| 50 | 160 | 51.614 | 0.723 | 1.40 | 0.285 | 0.55 | 0.777 | 1.51 |
| 500 | 160 | 507.988 | 7.632 | 1.50 | 4.240 | 0.83 | 8.538 | 1.68 |
Interference by Endogenous Substances
Interference studies were conducted using CLSI EP07-A3 as a guideline. Clinically high concentrations of the following potentially interfering substances in serum with known levels of methotrexate (approximately 0.050 and 0.500 umol/L) were evaluated. Two analytical runs of three replicates of each sample (6 replicates total) were assayed using the ARK Methotrexate II Assay, along with a serum control of methotrexate. The mean results of methotrexate were calculated and the percentage recovery relative to the serum control mean result was determined. Elevated concentrations of endogenous substances did not interfere with the measurement of methotrexate at the concentrations tested.
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| EndogenousSubstance | SuggestedConcentrationto Test | ConcentrationTested | ± umol/L fromControl(0.050 umol/LMethotrexate) | % Interference(0.500 µmol/LMethotrexate) |
|---|---|---|---|---|
| Human Albumin | 12 g/dL | 12 g/dL | 0.002 | -1.04 |
| Conj. - Bilirubin | 70 mg/dL | 72 mg/dL | 0.001 | 1.96 |
| Unconj. - Bilirubin | 70 mg/dL | 72 mg/dL | 0.003 | 0.23 |
| Cholesterol | 500 mg/dL | 500 mg/dL | 0.005 | 3.49 |
| Human IgG | 12 g/dL | 12 g/dL | 0.003 | 2.42 |
| Hemoglobin | 1000 mg/dL | 1000 mg/dL | -0.006 | -2.72 |
| Rheumatoid Factor | 1000 IU/mL | 1080 IU/mL | 0.001 | 3.52 |
| Triglycerides | 1000 mg/dL | 1000 mg/dL | -0.007 | 7.48 |
| Uric Acid | 30 mg/dL | 30 mg/dL | 0.000 | 1.60 |
Analytical Specificity
Methotrexate's metabolites, structurally similar compounds, folate derivatives, and potentially coadministered medications were tested to determine whether these compounds affect the quantitative measurement of methotrexate using the ARK™ Methotrexate II Assay.
7-Hydroxymethotrexate (7-OH-MTX) is the main metabolite in serum following high-dose methotrexate (HDMTX) treatment. The concentration of 7-OH-MTX may exceed that of the parent compound by up to 100-fold in plasma shortly after MTX infusion. Methotrexate is also metabolized by intestinal bacteria to the minor, inactive metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA).
Pooled human serum was supplemented with methotrexate prior to addition of potentially crossreacting metabolites (7-OH-MTX and DAMPA) or other compounds with structural similarity. Preparation of serum pools with 0.050 and 0.500 umol/L methotrexate were identical to the interference study above. Then the potentially cross-reactive compounds at their respectively solvated concentrations were added to serum containing methotrexate. Appropriate Serum/Solvent Controls containing methotrexate for each potentially cross-reacting interferent sample were also prepared.
Metabolites
7-Hydroxymethotrexate (7-OH-MTX)
Measurement of methotrexate by the ARK Methotrexate II Assay was not substantially affected by the presence of its major metabolite, 7-Hydroxymethotrexate (7-OH-MTX), when tested at 50 umol/L.
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| Metabolite | 7-OH-MTX(µmol/L) | (Percent Interference) | |
|---|---|---|---|
| Methotrexate0.050 µmol/L | Methotrexate0.500 µmol/L | ||
| 7-OH-MTX:7-Hydroxymethotrexate | 50 | 8.72% | 0.58% |
2, 4-Diamino-N(10)-methylpteroic acid (DAMPA)
Minor, inactive metabolite DAMPA is not expected to circulate at concentrations that would interfere in measurements of methotrexate. However, for patients at risk of renal toxicity, glucarpidase rescue therapy may be administered to rapidly convert extracellular methotrexate to DAMPA. This can cause the serum concentration of DAMPA to be significant and interfere with the ARK Methotrexate II Assay substantially. The assay should not be used during glucarpidase (carboxypeptidase G2) rescue therapy.
| Metabolite | DAMPA(µmol/L) | (Percent Cross-reactivity)Methotrexate0.000 µmol/L |
|---|---|---|
| DAMPA: | 0.040 | 57.50% |
| 0.100 | 51.50% | |
| 2, 4-Diamino-N(10)-methylpteroic acid | 0.500 | 42.93% |
| 0.800 | 23.42% | |
| 1.000 | 18.80% |
Other Compounds
Methotrexate-selective antibody did not cross-react with potentially co-administered drugs, folate derivatives, and other compounds of similar structure. The interference by trimethoprim and triamterene seen in the predicate assay was avoided by selection of an improved antibody. A high concentration of each compound was spiked into normal human serum with known levels of methotrexate (approximately 0.050 and 0.500 umol/L) and assayed along with a serum control of methotrexate. All compounds tested were within ±10% interference.
| Compound | Conc.Tested(µmol/L) | % Interference(0.050 µmol/LMTX) | % Interference(0.500 µmol/LMTX) |
|---|---|---|---|
| Adriamycin | 1000 | -3.92 | -0.03 |
| Cyclophosphamide | 2200 | 0.00 | -1.24 |
| Cytosine | 1000 | -0.66 | -0.78 |
| Dihydrofolic Acid | 1000 | 8.60 | 1.15 |
| Tetrahydrofolic Acid | 1000 | 6.79 | -0.64 |
| DL-6-Methyl-5,6,7,8-Tetrahydropterine | 1000 | -0.71 | -1.03 |
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| Compound | Conc.Tested(umol/L) | % Interference(0.050 µmol/LMTX) | % Interference(0.500 µmol/LMTX) |
|---|---|---|---|
| Folic Acid | 1000 | 2.50 | 5.05 |
| Folinic Acid | 1000 | -0.65 | 0.15 |
| 5-Fluorouracil | 3000 | -0.33 | 0.90 |
| 6-Mercaptopurine | 1000 | 2.31 | -1.99 |
| 5-Methyltetrahydrofolic Acid | 1000 | 0.00 | -0.25 |
| Prednisolone | 1000 | -2.50 | 1.35 |
| Pyrimethamine | 1000 | -2.14 | -3.02 |
| Sulfamethoxazole | 1600 | 0.36 | -0.29 |
| Vinblastine | 1000 | -3.57 | -0.45 |
| Vincristine | 1000 | -0.42 | -0.24 |
| Trimethoprim | 150 | 0.97 | -0.95 |
| Triamterene | 25 | -0.65 | 0.79 |
Sample Stability
Serum specimens were shown to be stable for at least fourteen (14) days when refrigerated (2-8 °C), fourteen (14) days at room temperature (25 °C), frozen (-20 °C) for at least 15 months (K111904), and after three (3) successive freeze/thaw cycles based on supporting data.
Product Stability
Accelerated stability studies and real time stability studies support a shelf-life stability claim of up to 18 months for the ARK Methotrexate II Reagents when stored unopened at 2-8°C.
On-Board Stability
Reagents were stable up to 100 days when stored on-board the instrument based on supporting data.
Calibration Curve Stability
A stored calibration curve was effective up to at least 100 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 Methotrexate II Assay is substantially equivalent to the legally marketed predicate device K111904. Reasonable assurance of safety and effectiveness for its intended use was shown for the ARK Methotrexate II Assay based on performance studies.
N/A