(123 days)
ONLINE TDM Methotrexate is an in vitro test for the quantitative determination of methotrexate in human serum and plasma on cobas c systems. The determination of methotrexate is used for monitoring levels of methotrexate to ensure appropriate therapy.
The ONLINE TDM Methotrexate assay is an in vitro test for the quantitative determination of methotrexate in human serum and plasma on cobas c systems. The determination of methotrexate is used for monitoring levels of methotrexate to ensure appropriate therapy.
The ONLINE TDM MTX assay is a two-reagent system used for the detection of methotrexate in serum and plasma. In this technology drug hapten attached to the enzyme glucose 6 phosphate dehydrogenase (G6PDH) serves as the binding partner to anti-methotrexate antibody. A competitive reaction to a limited amount of specific anti-methotrexate antibody takes place between the enzyme bound hapten and free methotrexate in the sample. Enzyme activity is reduced with bound antibody. Only active enzymes reduce NAD+ to NADH. The rate of NADH formation during the reaction correlates to the methotrexate concentration and is measured photometrically.
The ONLINE TDM MTX assay is a homogeneous enzyme-immunoassay.
Reagents - working solutions
R1: Anti-methotrexate antibody (rabbit monoclonal), 3 µg/mL; NAD, G6P, bovine serum albumin in water, pH 6.3; preservative
R3: Methotrexate hapten conjugated to G6PDH, 0.3 µg/mL; bovine serum albumin in buffer, pH 7.8; preservative
The provided document describes the analytical performance evaluation of the "ONLINE TDM Methotrexate" assay. This is an in vitro diagnostic device used for the quantitative determination of methotrexate in human serum and plasma. The study aims to demonstrate that the device meets predefined acceptance criteria.
Here's an analysis of the acceptance criteria and the study proving the device meets them:
1. Table of Acceptance Criteria & Reported Device Performance:
The document doesn't explicitly present a single table of "acceptance criteria" alongside "reported device performance" in a comparative format for all tests. Instead, it states for each section: "All acceptance criteria were met," and then provides the results. We can infer the acceptance criteria from the context and the reported successful outcomes.
Here is a reconstructed table based on the provided text, indicating the acceptance criteria (inferred from the "All acceptance criteria were met" statements and industry standards implicitly followed) and the reported performance.
| Performance Characteristic | Acceptance Criteria (inferred/implied) | Reported Device Performance |
|---|---|---|
| Precision | CV (Coefficient of Variation) within acceptable limits for repeatability & intermediate precision (e.g., generally lower CVs for higher concentrations). | Repeatability:- Control 1 (0.0863 µmol/L): 4.4% CV- Control 2 (0.485 µmol/L): 0.9% CV- Control 3 (0.849 µmol/L): 0.7% CV- Human Serum 1 (0.0872 µmol/L): 4.0% CV- Human Serum 2 (0.526 µmol/L): 0.8% CV- Human Serum 3 (0.889 µmol/L): 0.7% CV- Human Serum 4 (4.85 µmol/L): 1.0% CV- Human Serum 5 (44.2 µmol/L): 4.0% CV- Human Serum 6 (449 µmol/L): 3.7% CV- Human Serum 7 (1334 µmol/L): 3.1% CVIntermediate Precision:- Control 1 (0.0737 µmol/L): 10.9% CV- Control 2 (0.487 µmol/L): 1.2% CV- Control 3 (0.841 µmol/L): 0.8% CV- Human Serum 1 (0.0752 µmol/L): 11.2% CV- Human Serum 2 (0.526 µmol/L): 1.3% CV- Human Serum 3 (0.889 µmol/L): 1.1% CV- Human Serum 4 (4.91 µmol/L): 2.0% CV- Human Serum 5 (44.2 µmol/L): 5.3% CV- Human Serum 6 (449 µmol/L): 6.3% CV- Human Serum 7 (1316 µmol/L): 5.1% CVAll stated to have met acceptance criteria. |
| Analytical Sensitivity | LoB, LoD, LoQ within predefined thresholds to ensure accurate measurement at low concentrations. | LoB: ≤ 0.0250 µmol/L (claim in labeling)LoD: < 0.0350 µmol/L (claim in labeling)LoQ: 0.0400 µmol/L (claim in labeling)All stated to have met acceptance criteria. |
| Linearity/Reportable Range | Assay results must be linear across the claimed measuring range. | Confirmed for the measuring range of 0.0400 – 1.20 µmol/L.All stated to have met acceptance criteria. |
| Dilution | Expected recovery of concentration from diluted samples. | Post-dilution checks performed, supporting instrument and/or manual dilution for samples above the measuring range. (Implied acceptance criteria met, as no issues reported). |
| Endogenous Interferences | No significant interference from common endogenous substances at specified concentrations. | No interference claims for: Icterus (up to I index of 60/~1026 µmol/L bilirubin), Hemolysis (up to H index of 1000/~621 µmol/L hemoglobin), Lipemia (up to L index of 1000), Albumin (60 g/L), Immunoglobulin G (60 g/L), Total protein (2-12 g/dL), Rheumatoid factors (1000 IU/mL).All predefined acceptance criteria were met. |
| Analytical Specificity/ Cross-Reactivity | Expected cross-reactivity with structurally similar compounds. | DAMPA cross-reactivity: 87.6% at 0.1 µmol/L MTX, 64.6% at 1.0 µmol/L MTX.DAMPA can cross-react between 50% and 150% (This range implies the acceptance criteria for DAMPA cross-reactivity). |
| Exogenous Interferences (Drugs) | No significant interference from commonly used pharmaceuticals. | A long list of common and special pharmaceuticals were tested with no significant interference (implicitly meaning acceptance criteria were met). |
| Sample Matrix Comparison | Acceptable correlation between different sample matrices (serum vs. various plasma types). | Correlation (Pearson r) with Serum vs.:- Li-Heparin plasma: 0.998 - K2-EDTA plasma: 0.999 - K3-EDTA plasma: 0.998 - Na-Heparin plasma: 0.998 Slopes are close to 1, intercepts close to 0.All predefined acceptance criteria were met, supporting the claims for acceptable sample types. |
| Method Comparison | Acceptable correlation/agreement with a validated comparator method (LC-MS/MS). | Deming Regression: y = 1.032x + 0.000831 µmol/LCorrelation (r): 0.997(Implied acceptance criteria met, as close agreement shown). |
| Stability | Device must maintain performance throughout its claimed shelf life and on-board stability. | Stability studies were conducted to support Roche Diagnostic's claims as reported in the package labeling. (Implied acceptance criteria met). |
2. Sample Sizes Used for the Test Set and Data Provenance:
- Precision (Repeatability & Intermediate Precision):
- Repeatability: n = 84 measurements (total across all samples/controls).
- Intermediate Precision: Not explicitly stated as a single 'n'. It involved 2 aliquots per run, 2 runs per day, over 21 days for controls and human serum samples.
- Analytical Sensitivity (LoB, LoD, LoQ):
- LoB: One analyte-free sample measured with three reagent lots, in 6 runs, each with 10-fold determination, distributed over 3 days.
- LoD: 5 serum samples measured on three lots with 2-fold determination per run, across 6 runs distributed over 3 days.
- LoQ: 5 serum samples measured with three reagent lots, across 6 runs distributed over 3 days.
- Linearity/Assay Reportable Range:
- A dilution series spanning ≥ 9 levels, assayed on one analyzer using 3 reagent lots and 4 replicates per sample.
- Endogenous Interferences: Not explicitly stated as a numerical sample size but implies a study design for each substance.
- Analytical Specificity/Cross-Reactivity (DAMPA):
- Two human serum sample pools.
- Exogenous Interferences (Drugs): Not explicitly stated as a numerical sample size but implies a study design for each drug.
- Sample Matrix Comparison:
- ≥ 50 samples compared across serum and various plasma types.
- Method Comparison:
- 105 native human plasma and serum samples.
Data Provenance: The studies were conducted on "human serum" and "human plasma" samples. No specific country of origin is mentioned for the samples. The studies are retrospective in nature, as they involve testing existing samples (or creating spiked samples from existing matrices) within a laboratory setting to characterize the device performance. This is a typical approach for IVD device validation.
3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications:
This information is not provided in the document. The type of device (in vitro diagnostic for quantitative determination of a drug) means that "ground truth" is typically established via a reference method (like LC-MS/MS), not by expert human graders.
4. Adjudication Method for the Test Set:
This information is not applicable for this type of in vitro diagnostic device study. Adjudication methods (like 2+1, 3+1) are typically used in clinical studies where human readers evaluate medical images and their interpretations need to be reconciled. For a quantitative assay like this, the 'ground truth' is established by a reference analytical method.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done and Effect Size:
No, a MRMC comparative effectiveness study was not done. This type of study is relevant for AI-powered devices that assist human interpretation (e.g., radiologists reading images). This device is a fully automated in vitro diagnostic assay, so human interpretation assistance is not part of its function.
6. If a Standalone (algorithm only without human-in-the-loop performance) was done:
Yes, this entire study describes the standalone performance of the "ONLINE TDM Methotrexate" assay. It is an automated laboratory test that measures methotrexate levels, meaning it operates "without human-in-the-loop performance" during the measurement process. The results are quantitative values generated directly by the instrument using the assay's reagents.
7. The Type of Ground Truth Used:
The primary type of "ground truth" used for performance evaluation, particularly for method comparison and potentially for linearity/dilution studies, is a validated comparator method, specifically LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry). This is a highly accurate and precise analytical technique often considered a reference method for drug quantification. The document explicitly states: "The target value was determined with the LC-MS/MS" for dilution checks and "A method comparison of the ONLINE TDM Methotrexate on the cobas c 503 analyzer versus a validated comparator method on LC-MS/MS was completed."
For other tests like precision, sensitivity, interference, matrix comparison, ground truth is established by the known concentrations of controls, spiked samples, or comparison against established acceptable ranges as per CLSI (Clinical and Laboratory Standards Institute) guidelines.
8. The Sample Size for the Training Set:
The document does not provide any details about a training set. This is expected because this device is an in vitro diagnostic reagent assay, not an AI/Machine Learning model that undergoes a "training" phase. The assay relies on established biochemical reactions (homogeneous enzyme-immunoassay) and instrumental calibration rather than iterative learning from a dataset.
9. How the Ground Truth for the Training Set Was Established:
As there is no training set for this type of IVD device (it's not an AI/ML product), this question is not applicable. The "ground truth" for the performance evaluation (test set) relied on validated analytical methods and known sample concentrations.
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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the seal of the Department of Health & Human Services. To the right of that is the FDA logo in blue, with the words "U.S. FOOD & DRUG" stacked on top of the word "ADMINISTRATION".
Roche Diagnostics Operations Elina Voronovsky Regulatory Affairs Manager 9115 Hague Road Indianapolis, Indiana 46250
Re: K233454
Trade/Device Name: ONLINE TDM Methotrexate Regulatory Class: Unclassified Product Code: LAO Dated: January 12, 2024 Received: January 12, 2024
Dear Elina Voronovsky:
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 (the 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/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.
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 820.30 and 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
Joseph A. Joseph A. Joseph A. Kotarek -S Kotarek -S Date: 2024.02.20
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
Submission Number (if known)
Device Name
ONLINE TDM Methotrexate
Indications for Use (Describe)
ONLINE TDM Methotrexate is an in vitro test for the quantitative determination of methotrexate in human serum and plasma on cobas c systems. The determination of methotrexate is used for monitoring levels of methotrexate to ensure appropriate therapy.
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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ONLINE TDM Methotrexate K233454 – 510(k) Summary
This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92
| Submitter Name | Roche Diagnostics |
|---|---|
| Address | 9115 Hague RoadP.O. Box 50416Indianapolis, IN 46250-0457 |
| Contact | Elina VoronovskyPhone: (317) 478-3317Email: elina.voronovsky.ev1@roche.com |
| Date Prepared | February 20, 2024 |
| Proprietary Name | ONLINE TDM Methotrexate |
| Common Name | ONLINE TDM Methotrexate |
| Classification Name | Enzyme Immunoassay, Methotrexate |
| Product Codes,Regulation Numbers | LAO, Unclassified |
| Predicate Devices | ARK Methotrexate Assay (K111904) |
| Establishment Registration | Roche Diagnostics GmbH Mannheim, Germany: 9610126Roche Diagnostics GmbH Penzberg, Germany: 9610529Roche Diagnostics Indianapolis, IN United States: 1823260 |
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DEVICE DESCRIPTION 1.
The ONLINE TDM Methotrexate assay is an in vitro test for the quantitative determination of methotrexate in human serum and plasma on cobas c systems. The determination of methotrexate is used for monitoring levels of methotrexate to ensure appropriate therapy.
The ONLINE TDM MTX assay is a two-reagent system used for the detection of methotrexate in serum and plasma. In this technology drug hapten attached to the enzyme glucose 6 phosphate dehydrogenase (G6PDH) serves as the binding partner to anti-methotrexate antibody. A competitive reaction to a limited amount of specific anti-methotrexate antibody takes place between the enzyme bound hapten and free methotrexate in the sample. Enzyme activity is reduced with bound antibody. Only active enzymes reduce NAD+ to NADH. The rate of NADH formation during the reaction correlates to the methotrexate concentration and is measured photometrically.
The ONLINE TDM MTX assay is a homogeneous enzyme-immunoassay.
Reagents - working solutions
R1: Anti-methotrexate antibody (rabbit monoclonal), 3 µg/mL; NAD, G6P, bovine serum albumin in water, pH 6.3; preservative
R3: Methotrexate hapten conjugated to G6PDH, 0.3 µg/mL; bovine serum albumin in buffer, pH 7.8; preservative
2. INDICATIONS FOR USE
ONLINE TDM Methotrexate is an in vitro test for the quantitative determination of methotrexate in human serum and plasma on cobas c systems. The determination of methotrexate is used for monitoring levels of methotrexate to ensure appropriate therapy.
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TECHNOLOGICAL CHARACTERISTICS 3.
The following table compares the ONLINE TDM Methotrexate assay on cobas c 503 with its predicate device, ARK Methotrexate Assay (K111904).
| Candidate Device:ONLINE TDMMethotrexate Assay | Predicate Device:ARK MethotrexateAssay (K111904) | |
|---|---|---|
| Intended Use /Indications for Use | The Roche ONLINE TDMMethotrexate Assay isintended as the In vitro testfor the quantitativedetermination ofmethotrexate in humanserum and plasma on cobasc systems. Thedetermination ofmethotrexate is used formonitoring levels ofmethotrexate to ensureappropriate therapy. | The ARK™ MethotrexateAssay is intended for thequantitative determinationof methotrexate in humanserum or plasma onautomated clinical chemistryanalyzers. |
| Assay Method | Same | Homogenous enzymeimmunoassay (EIA) |
| Detection Method | Same | Absorbance |
| Instrument Platform | Same | Automated clinicalchemistry analyzer |
| Sample Type/Matrix | Same | Serum or plasma |
| Calibrator | Roche MTX Calibrator | ARK MethotrexateCalibrator |
| Calibration Method | 1 calibrator diluted to 6levels by the instrument.The calibrator consists of asynthetic protein matrix and1.2 µmol/L. | Six-level set to calibrate theassay. The calibratorsconsist of a synthetic proteinmatrix and the levels are0.00 umol/L, 0.05 umol/L,0.15 umol/L, 0.25 umol/L,0.50 umol/L and 1.2 umol/L |
| Calibration Interval | - after reagent lot changeevery 2 weeksas required followingquality controlprocedures | - Whenever a new lotnumber of reagents isusedWhenever indicated byquality control resultsWhenever required bystandard laboratoryprotocols |
| Controls | Roche TDM Control | ARK Methotrexate Control |
| Candidate Device:ONLINE TDMMethotrexate Assay | Predicate Device:ARK MethotrexateAssay (K111904) | |
| Traceability/Standardization | Roche MTX Calibrator isprepared to contain knownquantities of methotrexate ina synthetic proteinaceousmatrix free of methotrexateand is traceable to USPreference standard. | ARK MethotrexateCalibrators and controls areprepared by volumetricdilution of high purity,certified Methotrexatesolution into a syntheticproteinaceous matrix free ofMethotrexate |
| Reagent Stability | Shelf life at 2-8 °C: Seeexpiration date on cobas cpack labelOn-board in use andrefrigerated on the analyzer:12 weeks | When not in use, reagentsmust be stored at 2-8°C(36-46°F), upright and withscrew caps tightly closed. Ifstored as directed, reagentsare stable until theexpiration date printed onthe label. |
| Measuring Range | Same | 0.04 – 1.20 µmol/LSpecimens containingmethotrexate in higherconcentrations are assayedby dilution of the specimen. |
| Lower Limits ofMeasurement | LoB = 0.0250 µmol/LLoD = 0.0350 µmol/LLoQ = 0.0400 µmol/L | LoB = 0.01 µmol/LLoD = 0.02 µmol/LLoQ = 0.04 µmol/L |
Table 1: ONLINE TDM Methotrexate Technical Characteristics
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4. NON-CLINICAL PERFORMANCE EVALUATION
Performance characteristics were evaluated with ONLINE TDM Methotrexate on cobas c 503 and are briefly summarized below.
All acceptance criteria were met.
Precision 4.1.
Repeatability and Intermediate Precision 4.1.1.
Precision was determined in accordance with the CLSI EP05-A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). Results for repeatability and intermediate precision were obtained on the cobas c 503 analyzer. The results are summarized below. All acceptance criteria were met.
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| Repeatability | Mean | SD | CV |
|---|---|---|---|
| µmol/L | µmol/L | % | |
| Control 1 | 0.0863 | 0.00376 | 4.4 |
| Control 2 | 0.485 | 0.00421 | 0.9 |
| Control 3 | 0.849 | 0.00567 | 0.7 |
| Human Serum 1 | 0.0872 | 0.00350 | 4.0 |
| Human Serum 2 | 0.526 | 0.00427 | 0.8 |
| Human Serum 3 | 0.889 | 0.00648 | 0.7 |
| Human Serum 4 | 4.85 | 0.0491 | 1.0 |
| Human Serum 5 | 44.2 | 1.76 | 4.0 |
| Human Serum 6 | 449 | 16.6 | 3.7 |
| Human Serum 7 | 1334 | 41.8 | 3.1 |
| Intermediateprecision | Mean | SD | CV |
|---|---|---|---|
| µmol/L | µmol/L | % | |
| Control 1 | 0.0737 | 0.00804 | 10.9 |
| Control 2 | 0.487 | 0.00561 | 1.2 |
| Control 3 | 0.841 | 0.00664 | 0.8 |
| Human Serum 1 | 0.0752 | 0.00845 | 11.2 |
| Human Serum 2 | 0.526 | 0.00683 | 1.3 |
| Human Serum 3 | 0.889 | 0.00971 | 1.1 |
| Human Serum 4 | 4.91 | 0.0970 | 2.0 |
| Human Serum 5 | 44.2 | 2.36 | 5.3 |
| Human Serum 6 | 449 | 28.5 | 6.3 |
| Human Serum 7 | 1316 | 67.3 | 5.1 |
Analytical Sensitivity 4.2.
Limit of Blank (LoB) 4.2.1.
For determination of LoB, one analyte-free sample was measured with three reagent lots in 6 runs, each run with 10-fold determination, distributed over 3 days, on one cobas c 503 analyzer. The LoB was determined according to CLSI EP17-A2. The LoB claim in the labeling will be set to ≤ 0.0250 µmol/L.
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Limit of Detection (LoD) 4.2.2.
For determination of LoD, 5 serum samples (spiked with methotrexate) were measured on three lots with 2-fold determination per run on one cobas c 503 analyzer. Six runs were distributed over 3 days. The LoD was determined according to CLSI EP17-A2. The LoD claim in the labeling will be set to < 0.0350 umo1/L.
Limit of Quantitation (LoQ) 4.2.3.
For determination of LoQ. 5 serum samples (spiked with methotrexate) were measured with three reagent lots on one cobas c 503. Six runs were distributed over 3 days. The Limit of Ouantitation (LoQ) was determined according to CLSI EP17-A2. The LoO claim in the labeling will be set to 0.0400 umol/L.
Linearity/Assay Reportable Range 4.3.
The linearity of the ONLINE TDM Methotrexate assay was assessed according to CLSI EP06-A-Ed2.
A dilution series was prepared from human serum spiked with methotrexate (sample High) and analyte-free human serum (sample Blank). The dilution series spanning the measuring range was prepared to obtain ≥ 9 levels. Samples were assayed on one cobas c 503 analyzer using 3 reagent lots and 4 replicates per sample. The linearity data was analyzed according to CLSI EP06-Ed2.
Linearity was confirmed for the measuring range of 0.0400 – 1.20 umol/L.
4.4. Dilution
Post Dilution Check experiments were performed for samples above the measuring range. Human serum samples were spiked with methotrexate and measured with the ONLINE TDM Methotrexate assay on the cobas c 503 via instrument and/or manual dilution, depending on the concentration level. The target value was determined with the LC-MS/MS.
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Endogenous Interferences 4.5.
Endogenous substances (conjugated and unconjugated bilirubin, hemoglobin, lipemia (Intralipid), Immunoglobulin G (IgG), albumin, rheumatoid factor, total protein,) were evaluated for potential interference with the ONLINE TDM Methotrexate assay on the cobas c 503 analyzer.
All predefined acceptance criteria were met, and the proposed labeling claims for each endogenous substance can be found below:
| EndogenousSubstance | ClaimNo interference up to |
|---|---|
| Icterus | I index of 60 for conjugated bilirubin and unconjugated bilirubin(approximate conjugated and unconjugated bilirubin concentration:1026 µmol/L or 60 mg/dL). |
| Hemolysis | H index of 1000(approximate hemoglobin concentration: 621 µmol/L or 1000 mg/dL) |
| Lipemia (Intralipid) | L index of 1000 |
| Albumin | 60 g/L |
| Immunoglobulin G | 60 g/L |
| Total protein | between concentrations of 2-12 g/dL |
| Rheumatoid factors | 1000 IU/mL |
Analytical Specificity/Cross-Reactivity 4.6.
DAMPA (4-[(2,4-Diaminopteridin-6-vl) methyl-methylamino] benzoic acid was tested for cross-reactivity with ONLINE TDM Methotrexate on the cobas c 503 analyzer. Two human serum sample pools were spiked with methotrexate to approximately 0.1 and 1.0 umol/L and then divided into 2 portions. One portion of each pool was spiked with 0.1 umol/L of DAMPA and the other portion of the pool with solvent used to dissolve DAMPA. which was used for the baseline reference methotrexate concentration. The methotrexate concentration was determined at least in 5-fold determination and compared to the reference aliquot.
DAMPA cross-reactivity was shown at 87.6 % at methotrexate level 0.1 umo1/L and at 64.6 % at methotrexate level 1.0 µmo1/L.
DAMPA can cross-react between 50 % and 150 %.
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Exogenous Interferences – Drugs 4.7.
An exogenous interference study was conducted to evaluate commonly used pharmaceuticals and in addition, special pharmaceuticals were tested with ONLINE TDM Methotrexate on the cobas c 503 analyzer. All acceptance criteria were met.
| Drug | Concentration tested (mg/L) |
|---|---|
| Acetaminophen | 156 |
| N-Acetylcysteine | 150 |
| Acetylsalicylic acid | 30 |
| Ampicillin-Na | 75 |
| Ascorbic acid | 52.5 |
| Cefoxitin | 750 |
| Cyclosporine | 1.8 |
| Doxycyclin | 18 |
| Heparin | 3300 IU/L |
| Ibuprofen | 219 |
| Levodopa | 7.5 |
| Methyldopa | 22.5 |
| Metronidazole | 123 |
| Phenylbutazone | 321 |
| Rifampicin | 48 |
| Theophylline | 60 |
| 5-Fluorouracil | 90 |
| 5-Methyl-THF | 459 |
| 6-Mercaptopurin | 1.48 |
| 6-Methyl-5,6,7,8-tetrahydropterin-dihydrochlorid | 254 |
| 7-Hydroxy methotrexate | 9.35 |
| Adriamycin | 580 |
| Carbamazepine | 45 |
| Chloramphenicol | 78 |
| Cisplatin | 15 |
| Cyclophosphamide | 549 |
| Cytosine | 111 |
| Digoxin | 0.039 |
| Dihydrofolic acid | 443 |
| Disopyramide | 16.8 |
| Erythromycin | 138 |
| Folic acid | 0.44 |
| Furosemide | 15.9 |
| Gabapentin | 26.7 |
| Hydrochlorothiazide | 1.13 |
| Isoproterenol hydrochloride | 0.0595 |
| Leucovorin | 1420 |
| Lidocaine | 15 |
| Naproxen | 360 |
| Phenobarbital | 690 |
| Phenytoin | 60.0 |
| Prednisolone | 1.2 |
| Prednisone | 0.099 |
| Pyrimethamine | 249 |
| Sulfamethoxazole | 405 |
| Tetrahydrofolic acid | 445 |
| Triamterene | 0.585 |
| Trimethoprim | 42.0 |
| Vancomycin | 120 |
| Vinblastine | 811 |
| Vincristine | 825 |
{11}------------------------------------------------
4.8. Sample Matrix Comparison
The effect on quantitation of methotrexate values in the presence of anticoagulants with the ONLINE TDM Methotrexate assay was determined on the cobas c 503 analyzer by comparing values obtained from samples collected in serum, Li-Heparin, K2-EDTA, K3-EDTA, and Na-Heparin plasma tubes. The study was performed using ≥ 50 samples, 1 lot of reagent and measured on 1 cobas c 503 analyzer. All predefined acceptance criteria were met, supporting the labeling claim that serum, Li-Heparin, K2-EDTA, and Na-Heparin are acceptable sample types.
| Anticoagulant | Slope | Intercept(µmol/L) | CorrelationCoefficient(Pearson r) | Concentration ofSamples(µmol/L) |
|---|---|---|---|---|
| Serum vs. Li-Heparin plasma | 1.005 | -0.00539 | 0.998 | 0.0642 – 1.19 |
{12}------------------------------------------------
| Serum vs. K2-EDTA plasma | 0.997 | 0.00309 | 0.999 | 0.0421 – 1.19 |
|---|---|---|---|---|
| Serum vs. K3-EDTA plasma | 1.003 | -0.00261 | 0.998 | 0.0642 – 1.19 |
| Serum vs. Na-Heparin plasma | 1.000 | -0.00300 | 0.998 | 0.0642 – 1.19 |
Method Comparison to Validated Comparator Method 4.9.
A method comparison of the ONLINE TDM Methotrexate on the cobas c 503 analyzer versus a validated comparator method on LC-MS/MS was completed. 105, native human plasma and serum samples, were tested on a cobas c 503 analyzer and LC-MS/MS, in singlet using 1 lot of reagent. The sample concentrations were between 0.0429 umol/L and 1.19 µmol/L. The results can be found below:
Deming Regression
y = 1.032x = 0.000831 μmol/L
r = 0.997
4.10. Stability
Stability studies were conducted to support Roche Diagnostic's claims as reported in the package labeling.
CONCLUSIONS 5.
The analytical performance data for ONLINE TDM Methotrexate assay met the acceptance criteria and support the substantial equivalence of ONLINE TDM Methotrexate assay on cobas c 503 analyzer to ARK Methotrexate Assay.
N/A