(69 days)
The ONLINE TDM Phenytoin - Free Phenytoin application is an in vitro test for the quantitative determination of free phenytoin in human serum and plasma on cobas c systems. The determination of free phenytoin is used in monitoring levels of free phenytoin to ensure appropriate therapy.
The ONLINE TDM Phenytoin - Free Phenytoin application is an in vitro test for the quantitative determination of free phenytoin in human serum and plasma on cobas c systems. The determination of free phenytoin is used in monitoring levels of free phenytoin to ensure appropriate therapy.
Prior to measurement using the ONLINE TDM Phenytoin - Free Phenytoin application, the sample is processed by ultrafiltration to remove the bound phenytoin generating a result for free phenytoin.
The ONLINE TDM Phenytoin - Free Phenytoin application is based on the kinetic interaction of microparticles in a solution (KIMS). Phenytoin antibody is covalently coupled to microparticles and the drug derivative is linked to a macromolecule. The kinetic interaction of microparticles in solutions, photometrically detected by turbidity measurements is induced by binding of drugconjugate to the antibody on the microparticles and is inhibited by the presence of phenytoin in the sample. A competitive reaction takes place between the drug conjugate and phenytoin in the serum sample for binding to the phenytoin antibody on the microparticles. The resulting turbidity is indirectly proportional to the amount of drug present in the sample.
Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:
Device Name: ONLINE TDM Phenytoin - Free Phenytoin application
1. Table of Acceptance Criteria and Reported Device Performance
| Performance Characteristic | Acceptance Criteria (Stated or Implied) | Reported Device Performance |
|---|---|---|
| Precision | Implicit: Acceptable repeatability and intermediate precision, likely defined by a maximum allowable Coefficient of Variation (CV%) or Standard Deviation (SD) as per CLSI EP05-A3. (No specific numerical criteria for CV% or SD are explicitly stated as "acceptance criteria" in the text, but the claim "All acceptance criteria were met" implies adherence to predefined thresholds.) | Repeatability (CV%): - 2.2% - 2.9% for Control 1, Control 2, and Human Serums 1-5 Intermediate Precision (CV%): - 2.8% - 3.5% for Control 1, Control 2, and Human Serums 1-5 |
| Analytical Sensitivity | Implicit: LoB, LoD, and LoQ determined according to CLSI EP17-A2, and likely within a range consistent with the intended clinical use. (No specific numerical criteria are explicitly stated for these limits as "acceptance criteria"). | LoB: 0.100 µg/mL (0.396 µmol/L) LoD: 0.200 µg/mL (0.792 µmol/L) LoQ: 0.400 µg/mL (1.58 µmol/L) |
| Linearity/Reportable Range | Linearity confirmed for the measuring range of 0.400-4.00 µg/mL (1.58-15.8 µmol/L) per CLSI EP06-A-Ed2. | Confirmed for the measuring range of 0.400-4.00 µg/mL (1.58-15.8 µmol/L). |
| Dilution | Automatic rerun function (1:2 dilution) for samples above measuring range should demonstrate acceptable deviation. (Exact % deviation acceptance criteria not stated, but "All acceptance criteria were met" implies compliance). | Demonstrated % deviation results of -7.3% to -11.6% when using the automatic rerun function for samples above the measuring range. |
| Endogenous Interferences | No significant interference at specified concentrations for various endogenous substances (e.g., Hemolysis, Icterus, Triglycerides, Albumin, Total protein, Rheumatoid factors, Immunoglobulin G). (Specific acceptance criteria for "no interference up to" certain levels are explicitly stated in the claims summary, e.g., H index of 1000 for Hemolysis). | All predefined acceptance criteria were met. Specific claims are: - Hemolysis: No interference up to H index of 1000 - Icterus: I index of 60 for conjugated bilirubin - Triglycerides: 700 mg/dL - Albumin: 60 g/L - Total protein: between 2-12 g/dL - Rheumatoid factors: 1200 IU/mL - Lipemia: L index of 1000 - Immunoglobulin G: 60 g/L – No significant phenytoin release for conjugated bilirubin up to 18 mg/dL, unconjugated bilirubin up to 9 mg/dL, triglycerides up to 183 mg/dL, and rheumatoid factors up to 284 IU/mL. |
| Analytical Specificity/Cross-Reactivity | Acceptable cross-reactivity for tested compounds (e.g., Fosphenytoin ≤ 50.0 %, m-HPPH ≤ 10.0 %). (Specific numerical criteria for % Cross Reactivity are explicitly stated in the table). | All acceptance criteria for cross-reactivity were met. Specific values reported: - Fosphenytoin: ≤ 50.0 % - m-HPPH: ≤ 10.0 % - p-HPPH: ≤ 5.0 % - 5(p-methylphenyl)-5-phenylhydantoin: ≤ 5.0 % |
| Exogenous Interferences (Drugs) | No increase in free phenytoin concentrations at tested concentrations for commonly used pharmaceuticals, or expected increases for drugs that compete for albumin binding (e.g., Valproic acid). For phenobarbital and mephenytoin, analytical interference starts above specific concentrations (e.g., >90 µg/mL for phenobarbital in ultrafiltrate). | No increase in free phenytoin concentrations observed for a long list of tested drugs. Increased concentrations observed for Butabarbital, Carbamazepine, Cefoxitin, Ethotoin, p-Hydroxyphenobarbital, Ibuprofen, Oxaprozine, Phenylbutazone, d-Propoxyphene, and Valproic acid when spiked into serum (due to competition for albumin binding). These did not show analytical interference in ultrafiltrate. Phenobarbital and Mephenytoin showed significant increase above 34.5 µg/mL and 40.0 µg/mL respectively in serum, and analytical interference above 90 µg/mL (phenobarbital) and 60 µg/mL (mephenytoin) in ultrafiltrate. |
| Sample Matrix Comparison | Acceptable correlation/agreement between serum and various plasma types (Li-Heparin, K2-EDTA, K3-EDTA plasma). (No specific numerical criteria for slope, intercept, or correlation coefficient are stated as "acceptance criteria"). | All predefined acceptance criteria were met. - Serum vs. Li-Heparin plasma: Slope 1.018, Intercept -0.0149, r 0.988 - Serum vs. K2-EDTA plasma: Slope 0.923, Intercept -0.0328, r 0.992 - Serum vs. K3-EDTA plasma: Slope 0.950, Intercept -0.0282, r 0.992 |
| Method Comparison | Acceptable agreement with the predicate device (Phenytoin - Free Phenytoin Application on COBAS INTEGRA 400 plus). (No specific numerical criteria for Passing/Bablok or Deming regression parameters are stated as "acceptance criteria"). | Passing/Bablok: y = 1.035x - 0.0165 µg/mL, τ = 0.972 Deming Regression: y = 1.019x + 0.0107 µg/mL, r = 0.998 |
| Stability | Implicit: Data supports Roche Diagnostic's claims as reported in the package labeling. (No specific criteria are detailed in this section.) | Stability data supports claims as reported in package labeling. |
2. Sample Size Used for the Test Set and Data Provenance
- Precision (Repeatability and Intermediate Precision):
- Repeatability: n = 84 (implied total measurements, as 2 aliquots per run, 2 runs per day, 21 days = 84 measurements for intermediate precision, and repeatability likely collected within this structure).
- Intermediate Precision: 2 aliquots per run, 2 runs per day, 21 days.
- Data Provenance: Not explicitly stated, implied to be laboratory-prepared controls and human serum samples.
- Analytical Sensitivity (LoB, LoD, LoQ):
- LoB: One analyte-free sample (ultrafiltrate of human phenytoin-free serum) measured with three reagent lots in 6 runs, each with 10-fold determination (total 180 measurements).
- LoD: 5 serum samples with low analyte concentrations (spiked with phenytoin and ultrafiltrated) measured on three reagent lots with 2-fold determination per run, over 6 runs (total 180 measurements).
- LoQ: 6 serum samples (spiked with phenytoin and ultrafiltrated) measured with three reagent lots, 5 replicates per run, over 5 days (total 450 measurements).
- Data Provenance: Human phenytoin-free serum, spiked human serum samples (ultrafiltrated).
- Linearity/Assay Reportable Range:
- Sample Size: A dilution series ( > 9 levels) prepared from a spiked human ultrafiltrated serum pool and a negative ultrafiltrated serum pool. Tested with 3 reagent lots and 4 replicates per sample. The process was repeated with K3-EDTA plasma.
- Data Provenance: Spiked human ultrafiltrated serum pool and negative ultrafiltrated serum pool, K3-EDTA plasma.
- Dilution:
- Sample Size: Three ultrafiltrates prepared with specific phenytoin concentrations (5.00, 6.00, 7.00 µg/mL).
- Data Provenance: Laboratory-prepared ultrafiltrates with spiked phenytoin.
- Endogenous Interferences:
- Sample Size: Not explicitly stated, but various endogenous substances were evaluated. The description suggests samples were prepared by adding interferents to ultrafiltrate or present in the sample before ultrafiltration.
- Data Provenance: Laboratory-prepared samples with added endogenous substances or naturally occurring levels.
- Analytical Specificity/Cross-Reactivity:
- Sample Size: Two ultrafiltrated human serum pools spiked with phenytoin (1.00 and 2.50 µg/mL) for each potential cross-reacting compound. Phenytoin concentration determined in at least 5-fold determination.
- Data Provenance: Ultrafiltrated human serum pools spiked with phenytoin and cross-reactants.
- Exogenous Interferences - Drugs:
- Sample Size: Not explicitly stated, but drugs were tested individually at specified concentrations. Some drugs were tested by spiking into serum, others into ultrafiltrate.
- Data Provenance: Laboratory-prepared samples with added drugs.
- Sample Matrix Comparison:
- Sample Size: ≥ 50 samples comparing serum, Li-Heparin, K2-EDTA, and K3-EDTA plasma.
- Data Provenance: Native human samples collected in different tube types, spiked and ultrafiltrated.
- Method Comparison:
- Sample Size: 138 native human ultrafiltrated serum samples (≤10% spiked).
- Data Provenance: Native human ultrafiltrated serum samples.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
This device is an in vitro diagnostic (IVD) test system for the quantitative determination of free phenytoin. The "ground truth" for such a device is typically established by reference methods or established analytical techniques, often involving highly skilled laboratory personnel operating calibrated equipment, rather than clinical experts (like radiologists).
The text does not mention the use of clinical experts (e.g., radiologists) or their qualifications to establish ground truth. The methodologies like CLSI guidelines (EP05-A3, EP17-A2, EP06-A-Ed2) imply adherence to established laboratory standards and practices for analytical validation, often performed by trained laboratory scientists or technicians.
4. Adjudication Method for the Test Set
This type of analytical device validation does not typically involve an "adjudication method" in the way it's understood for image-based diagnostics (e.g., 2+1, 3+1 consensus among radiologists). Results are quantitative and are compared directly to expected values, reference methods, or statistical acceptance criteria.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
No, an MRMC comparative effectiveness study was not done. MRMC studies are relevant for AI-powered diagnostic devices where human readers interpret images or data, and the study assesses how AI assistance impacts their performance. This submission is for an in vitro diagnostic (IVD) laboratory test system, not an image-interpretation AI device.
6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done
This device is a standalone in vitro diagnostic test system. Its performance characteristics (precision, sensitivity, linearity, interference, etc.) are evaluated directly as the device itself operates. There isn't a separate "human-in-the-loop performance" concept for this type of quantitative assay, as the device provides a direct numerical result. The reported performance is the standalone performance of the ONLINE TDM Phenytoin - Free Phenytoin application on cobas c systems.
7. The Type of Ground Truth Used
The ground truth for the analytical performance studies primarily relied on:
- Reference Materials: Calibrators (Preciset TDM I), Controls (TDM Control Set), and laboratory-prepared samples with known concentrations of phenytoin and/or interferents (e.g., spiked serum, ultrafiltrates).
- Established Methods: Comparison to a predicate device (Phenytoin - Free Phenytoin Application on COBAS INTEGRA 400 plus) in the method comparison study.
- Theoretical Standards: Adherence to CLSI (Clinical and Laboratory Standards Institute) guidelines (EP05-A3, EP17-A2, EP06-A-Ed2) for defining analytical limits and linearity.
There is no mention of pathology, outcomes data, or expert consensus in the clinical sense for establishing ground truth for this analytical performance.
8. The Sample Size for the Training Set
The document does not describe a "training set" as would be relevant for a machine learning or AI-based device. This device is a chemical assay (Kinetic Interaction of Microparticles in a Solution - KIMS). The development of the assay itself would involve optimization and calibration phases, but these are not referred to as statistical "training sets" in the context of general device validation.
9. How the Ground Truth for the Training Set Was Established
Since there is no "training set" described in the context of an AI/ML algorithm, this question is not applicable. The assay's performance is driven by its chemical and optical principles, not by learning from a labeled dataset. The "ground truth" during assay development would be established through careful analytical chemistry techniques to ensure the reagents and measurement system accurately quantify free phenytoin.
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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: the Department of Health & Human Services logo on the left and the FDA logo on the right. The FDA logo is in blue and includes the letters "FDA" followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in a stacked format.
March 30, 2023
Roche Diagnostics Dr. Leslie Patterson Regulatory Affairs Program Manager 9115 Hague Road Indianapolis, IN 46250
Re: K230161
Trade/Device Name: ONLINE TDM Phenytoin - Free Phenytoin application Regulation Number: 21 CFR 862.3350 Regulation Name: Diphenylhydantoin test system Regulatory Class: Class II Product Code: MOJ Dated: January 19, 2023 Received: January 20, 2023
Dear Dr. Leslie Patterson:
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 requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part
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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/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-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 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 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,
Image /page/1/Figure/6 description: The image shows a digital signature. The signature is for Paula V. Caposino -S. The date of the signature is 2023.03.30. The time of the signature is 18:04:32 -04'00'.
Paula Caposino, Ph.D. Acting Deputy Division Director 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
Form Approved: OMB No. 0910-0120 Expiration Date: 06/30/2023 See PRA Statement below.
Submission Number (if known)
Device Name
ONLINE TDM Phenytoin - Free Phenytoin application
Indications for Use (Describe)
ONLINE TDM Phenytoin - Free Phenytoin application is an in vitro test for the quantitative determination of free phenytoin in human serum and plasma on cobas c systems. The determination of free phenytoin is used in monitoring levels of free phenytoin 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 Phenytoin - Free Phenytoin application K230161 - 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 | Leslie PattersonPhone: (317) 225-8563Email: leslie.patterson@roche.com |
| Date Prepared | March 21, 2023 |
| Proprietary Name | ONLINE TDM Phenytoin - Free Phenytoin application |
| Common Name | Free Phenytoin application |
| Classification Name | Diphenylhydantoin test system |
| Product Codes, Regulation Numbers | MOJ, 862.3350 |
| Predicate Devices | Reagent Application for Free Phenytoin (K952555) |
| Establishment Registration | Roche Diagnostics GmbH Mannheim, Germany: 9610126Roche Diagnostics GmBH Penzberg, Germany: 9610529Roche Diagnostics Indianapolis, IN United States: 1823260 |
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1. DEVICE DESCRIPTION
The ONLINE TDM Phenytoin - Free Phenytoin application is an in vitro test for the quantitative determination of free phenytoin in human serum and plasma on cobas c systems. The determination of free phenytoin is used in monitoring levels of free phenytoin to ensure appropriate therapy.
Prior to measurement using the ONLINE TDM Phenytoin - Free Phenytoin application, the sample is processed by ultrafiltration to remove the bound phenytoin generating a result for free phenytoin.
The ONLINE TDM Phenytoin - Free Phenytoin application is based on the kinetic interaction of microparticles in a solution (KIMS). Phenytoin antibody is covalently coupled to microparticles and the drug derivative is linked to a macromolecule. The kinetic interaction of microparticles in solutions, photometrically detected by turbidity measurements is induced by binding of drugconjugate to the antibody on the microparticles and is inhibited by the presence of phenytoin in the sample. A competitive reaction takes place between the drug conjugate and phenytoin in the serum sample for binding to the phenytoin antibody on the microparticles. The resulting turbidity is indirectly proportional to the amount of drug present in the sample.
1.1. Reagents - working solutions
R1: Phenytoin conjugate, 1.0 µg/mL; piperazine-N,N'-bis (ethanesulfonic acid) (PIPES) buffer, pH 7.3; stabilizer; preservative
R2: Anti-phenytoin antibody (mouse monoclonal); latex microparticle, 0.003 % (w/w); 3-(Nmorpholino) propane sulfonic acid (MOPS) buffer, pH 7.4; stabilizer; preservative
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INDICATIONS FOR USE 2.
The ONLINE TDM Phenytoin - Free Phenytoin application is an in vitro test for the quantitative determination of free phenytoin in human serum and plasma on cobas c systems. The determination of free phenytoin is used in monitoring levels of free phenytoin to ensure appropriate therapy.
TECHNOLOGICAL CHARACTERISTICS 3.
The following table compares the ONLINE TDM Phenytoin - Free Phenytoin application on cobas c 503 with its predicate device, COBAS-FP Reagents for Free Phenytoin on COBAS FARA II chemistry system (K952555).
| Candidate Device:ONLINE TDM Phenytoin - FreePhenytoin application | Predicate Device:COBAS-FP Free Phenytoin | |
|---|---|---|
| Intended Use /Indications forUse | The ONLINE TDM Phenytoin - FreePhenytoin application is an in vitro test forthe quantitative determination of freephenytoin in human serum and plasma oncobas c systems. The determination offree phenytoin is used in monitoring levelsof free phenytoin to ensure appropriatetherapy. | The COBAS-FP Reagents for FreePhenytoin are an in vitro test for thequantitative determination of freephenytoin in serum and heparinizedplasma on the COBAS FARA II chemistrysystem. |
| Assay Method | Kinetic Interaction of Microparticles in aSolution (KIMS) | Fluorescence Polarization Immunoassay(FPIA) |
| SampleType/Matrix | Serum and Lithium heparin, K2-EDTA,K3-EDTA plasma | Serum and heparinized plasma |
| Calibrator | Preciset TDM I calibrators (diluted) | COBAS FP Free Phenytoin Calibrators |
| Controls | TDM Control Set (Level I and II,ultrafiltrated) | COBAS FP Free Phenytoin Controls |
| MeasuringRange | 0.400-4.00 µg/mL | 0.09-4 µg/mL |
| Lower LimitsofMeasurement | LoB (Limit of Blank) = 0.100 µg/mL(0.396 µmol/L)LoD (Limit of Detection) = 0.200 µg/mL(0.792 µmol/L)LoQ (Limit of Quantitation) = 0.400µg/mL (1.58 µmol/L) | Sensitivity (Analytical): 0.09 µg/mL |
Table 1: ONLINE TDM Phenytoin - Free Phenytoin application Technical Characteristics
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4. NON-CLINICAL PERFORMANCE EVALUATION
Performance characteristics were evaluated with ONLINE TDM Phenytoin - Free Phenytoin application 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 aliguots 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.
| Repeatability | Mean | SD | CV | ||
|---|---|---|---|---|---|
| µg/mL | µmol/L | µg/mL | µmol/L | % | |
| Control 1a) | 1.28 | 5.07 | 0.0277 | 0.110 | 2.2 |
| Control 2b) | 2.80 | 11.1 | 0.0670 | 0.265 | 2.4 |
| Human Serum 1 | 0.739 | 2.93 | 0.0198 | 0.0784 | 2.7 |
| Human Serum 2 | 1.19 | 4.71 | 0.0348 | 0.138 | 2.9 |
| Human Serum 3 | 1.74 | 6.89 | 0.0408 | 0.162 | 2.4 |
| Human Serum 4 | 2.50 | 9.90 | 0.0596 | 0.236 | 2.4 |
| Human Serum 5 | 3.52 | 13.9 | 0.0723 | 0.286 | 2.1 |
| Intermediateprecision | Mean | SD | CV | ||
|---|---|---|---|---|---|
| µg/mL | µmol/L | µg/mL | µmol/L | % | |
| Control 1a) | 1.28 | 5.07 | 0.0365 | 0.145 | 2.9 |
| Control 2b) | 2.80 | 11.1 | 0.0795 | 0.315 | 2.8 |
| Human Serum 1 | 0.739 | 2.93 | 0.0241 | 0.0954 | 3.3 |
| Human Serum 2 | 1.19 | 4.71 | 0.0379 | 0.150 | 3.2 |
| Human Serum 3 | 1.74 | 6.89 | 0.0600 | 0.238 | 3.5 |
| Human Serum 4 | 2.50 | 9.90 | 0.0797 | 0.316 | 3.2 |
| Human Serum 5 | 3.52 | 13.9 | 0.100 | 0.396 | 2.8 |
a) TDM Control Set Level I, ultrafiltrated, b) TDM Control Set Level II, ultrafiltrated
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Analytical Sensitivity 4.2.
Limit of Blank (LoB) 4.2.1.
For determination of LoB, one analyte-free sample (ultrafiltrate of human phenytoin-free serum) was measured with three reagent lots in 6 runs, each run with 10-fold determination, distributed over 6 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 LoB = 0.100 µg/mL (0.396 µmol/L).
Limit of Detection (LoD) 4.2.2.2
For determination of LoD, 5 serum samples with low analyte concentrations (spiked with phenytoin and ultrafiltrated) were measured on three reagent lots with 2-fold determination per run on one cobas c 503 analyzer. Six runs were distributed over 6 days. The LoD was determined according to CLSI EP17-A2. The LoD claim in the labeling will be set to LoD = 0.200 µg/mL (0.792 µmol/L).
Limit of Quantitation (LoQ) 4.2.3.
For determination of LoQ, 6 serum samples (spiked with phenytoin and ultrafiltrated) were measured with three reagent lots on one cobas c 503. These samples were tested in 1 run per day over 5 days, 5 replicates per run for each LoQ sample. The Limit of Quantitation (LoQ) was determined according to CLSI EP17-A2. For calculation according to the RMS model, the bias has not been considered. TErel corresponds to the intermediate precision of the LoQ samples. The LoQ claim in the labeling will be set to LoQ = 0.400 µg/mL (1.58 µmol/L).
Linearity/Assay Reportable Range 4.3.
The linearity of the ONLINE TDM Phenytoin - Free Phenytoin application was assessed according to CLSI EP06-A-Ed2.
A dilution series was prepared from a spiked human ultrafiltrated serum pool (sample High) and a negative ultrafiltrated serum pool (sample Blank). The dilution series spanning the measuring range was prepared to obtain > 9 levels. Samples were assayed on one cobas c 503 analyzer in 1 run using 3 reagent lots and 4 replicates per sample. The process was the same with K3-EDTA plasma. The linearity data is analyzed according to CLSI EP06-Ed2.
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Linearity was confirmed for the measuring range of 0.400-4.00 us/mL (1.58-15.8 umo//L).
4.4. Dilution
Post Dilution Check experiments were performed for samples above the measuring range and verify dilution of samples via the rerun function is a 1:2 dilution. Three ultrafiltrates were prepared with phenytoin concentrations above the measuring range: the volumes of the ultrafiltrate were adjusted to the weighed amount of phenytoin to achieve final concentrations of 5.00 µg/mL, 6.00 µg/mL and 7.00 µg/mL. The samples were measured with the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 via the automatic rerun function and after manual dilution. The ONLINE TDM Phenytoin - Free Phenytoin application demonstrated % deviation results of -7.3% to -11.6% when measuring samples above the measuring range and using the automatic rerun function.
Endogenous Interferences 4.5.
Endogenous substances (hemoglobin, lipemia (Intralipid), conjugated and unconjugated bilirubin, Immunoglobulin G (IgG), albumin, rheumatoid factor, total protein, triglycerides and HAMA) were evaluated for potential interference with the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 analyzer.
Endogenous interferents were tested for analytical interference (endogenous substances added to the ultrafiltrate).
All predefined acceptance criteria were met, and the proposed labeling claims for each endogenous substance can be found below:
| Endogenous | Claim |
|---|---|
| Substance | No interference up to |
| Hemolysis | H index of 1000 |
| (approximate hemoglobin concentration: 1000 mg/dL or 621 umol/L) | |
| Icterus | I index of 60 for conjugated bilirubin |
| (approximate conjugated bilirubin concentration: 1026 umol/L or 60 mg/dL) | |
| Triglycerides | 700 mg/dL (7.98 mmol/L) |
| Albumin | 60 g/L |
| Total protein | between concentrations of 2-12 g/dL |
| Rheumatoid factors | 1200 IU/mL |
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Endogenous substances were tested for phenytoin release and phenytoin binding effects
| EndogenousSubstance | Maximum Concentration without significant effect |
|---|---|
| Hemolysis | H index of 1000(approximate hemoglobin concentration: 1000 mg/dL or 621 µmol/L) |
| Lipemia | L index of 1000 (1000 mg/dL) |
| Immunoglobulin G | 60 g/L |
(endogenous substances were present in the sample before ultrafiltration).
Conjugated and unconjugated bilirubin, triglycerides and rheumatoid factors result in higher free phenytoin concentrations. No significant phenytoin release was observed for conjugated bilirubin up to 18 mg/dL, for unconjugated bilirubin up to 9 mg/dL, for triglycerides up to 183 mg/dL and for rheumatoid factors up to 284 IU/mL.
Phenytoin binds to albumin and total protein (the albumin fraction within total protein). Increased protein concentrations result in decreased free phenytoin concentrations.
4.6. Analytical Specificity/Cross-Reactivity
A cross-reactivity study was conducted with the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 analyzer to evaluate the potential cross-reacting compounds. For each potential cross-reacting compound two ultrafiltrated human serum pools were prepared by spiked with phenytoin to obtain approximately 1.00 and 2.50 µg/mL phenytoin. The phenytoin concentration was determined at least in 5-fold determination and compared to the reference aliquot. All acceptance criteria for cross reactivity were met.
| Compound | Concentration tested(µg/mL) | % Cross Reactivity |
|---|---|---|
| Fosphenytoin | 387 | ≤ 50.0 % |
| m-HPPH | 500 | ≤ 10.0 % |
| p-HPPH | 220 | ≤ 5.0 % |
| 5(p-methylphenyl)-5-phenylhydantoin | 500 | ≤ 5.0 % |
4.7. Exogenous Interferences - Drugs
An exogenous interference study was conducted to evaluate commonly used pharmaceuticals and in addition, special pharmaceuticals were tested with the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 analyzer. No increase in free phenytoin concentrations was observed at the concentrations tested.
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| Drug | Concentration tested mg/L (µg/mL) |
|---|---|
| Acetaminophen | 156 |
| N-Acetyl-L-cysteine | 150 |
| N-Acetylsalicylic acid | 30.0 |
| Amitriptyline | 0.48 |
| Amlodipine | 0.075 |
| Amobarbital | 36.0 |
| Amoxicillin | 54.0 |
| Ampicillin | 75.0 |
| L-Ascorbic acid | 52.5 |
| Atorvastatin | 0.750 |
| Budesonide | 0.00630 |
| Carbamazepine-10,11-epoxide | 140 |
| Chlordiazepoxide | 6.90 |
| Chlorpromazine | 3.30 |
| Citalopram | 5.43 |
| Cyclosporin A | 1.80 |
| Diazepam | 30.0 |
| Diphenhydramine | 50.0 |
| Doxycyclin | 18.0 |
| Ethosuximide | 1000 |
| Furosemide | 15.9 |
| Gabapentin | 26.7 |
| Gentamicin | 30.0 |
| Glutethimide | 36.0 |
| Heparin, sodium | 3300 IU/L |
| Hydantoin | 500 |
| 10-Hydroxycarbamazepine | 105 |
| Hydroxychloroquine | 0.624 |
| Imipramine | 0.315 |
| Levodopa | 7.50 |
| Levothyroxin | 0.429 |
| Lisinopril | 0.246 |
| Losartan | 1.164 |
| Mephobarbital (Methylphenobarbital) | 10.5 |
| Metformin | 12.0 |
| Methsuximide | 260 |
| Methyldopa | 22.5 |
| Metoprolol | 1.50 |
| Metronidazol | 123 |
| Oxcarbazepine | 100 |
| Pentobarbital | 126 |
| 2-Phenyl-2-ethyl-malonamide (PEMA) | 100 |
| Phenylsuximide | 500 |
| Prednisone | 0.0990 |
| Primidone | 120 |
| Promethazine | 0.297 |
| Rifampicin | 48.0 |
| Secobarbital | 15.9 |
| Sertraline | 0.927 |
| Simvastatin | 1.68 |
| Theophylline | 60.0 |
| Vancomycin | 120 |
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Increased concentrations of free phenytoin were correctly observed in the presence of butabarbital (secbutabarbital), carbamazepine, cefoxitin, ethotoin, p-hydroxyphenobarbital, ibuprofen, oxaprozine, phenylbutazone, d-propoxyphene and valproic acid; this effect may occur at therapeutic concentrations. These drugs bind to human albumin in serum and release phenytoin, thus increasing the concentration of free phenytoin. When these drugs were spiked into serum, significant effects were observed at concentrations above those indicated in the following table:
| Drug | No significant effect up to mg/L (µg/mL) |
|---|---|
| Butabarbital (Secbutabarbital) | 100 |
| Carbamazepine | 56.0 |
| Cefoxitin | 188 |
| Ethotoin | 15.0 |
| p-Hydroxyphenobarbital | 240 |
| Ibuprofen | 54.8 |
| Oxaprozine | 141 |
| Phenylbutazone | 32.1 |
| d-Propoxyphene | 40.0 |
| Valproic acid | 25.7 |
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The above drugs, which increase the free phenytoin concentration when present in serum, were also tested for analytical interference by spiking the ultrafiltrate. No analytical interference from these drugs was observed when present in ultrafiltrate, confirming that the increase of free phenytoin concentration observed when present in serum is a result from competition for albumin binding.
For phenobarbital and mephenytoin, when spiked into serum before ultrafiltration, significantly increased values for free phenytoin were observed above concentrations of 34.5 µg/mL and 40.0 ug/mL, respectively. When spiked into the ultrafiltrate, increased values for free phenytoin were found at concentrations above 90 µg/mL (356 µmol/L) for phenobarbital and above 60 µg/mL (238 umol/L) for mephenytoin, indicating analytical interference of these drugs with the assay in addition to the competition for albumin binding. For these drugs, analytical interference starts at higher drug concentrations compared to the competitive effect.
Sample Matrix Comparison 4.8.
The effect on quantitation of free phenytoin values in the presence of anticoagulants with the ONLINE TDM Phenytoin - Free Phenytoin application was determined on the cobas c 503 analyzer by comparing values obtained from samples collected in serum, Li-Heparin, K2-EDTA and K3-EDTA plasma tubes; after centrifugation the samples were spiked and ultrafiltrated. 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 K3-EDTA plasma are acceptable sample types.
| Anticoagulant | Slope | Intercept$(µg/mL)$ | CorrelationCoefficient(Pearson r) | Concentration ofSamples$(µg/mL, serum)$ |
|---|---|---|---|---|
| Serum vs. Li-Heparinplasma | 1.018 | -0.0149 | 0.988 | 0.592 – 3.84 |
| Serum vs. K2-EDTAplasma | 0.923 | -0.0328 | 0.992 | 0.592 – 3.83 |
| Serum vs. K3-EDTAplasma | 0.950 | -0.0282 | 0.992 | 0.592 - 3.84 |
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4.9. Method Comparison to Phenytoin - Free Phenytoin Application on COBAS INTEGRA 400 plus
A method comparison was performed with the Phenytoin - Free Phenytoin Application on the COBAS INTEGRA 400 plus and the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 analyzer (candidate device), using a total of 138 native human ultrafiltrated serum samples (≤10% spiked), 1 lot of reagent, 1 cobas c 503 analyzer, in 3 runs, in singlicate. The sample concentrations were between 0.400 µg/mL (1.58 µmol/L) and 3.78 µg/mL (15.0 umol/L). The results can be found below:
Passing/Bablok y = 1.035x - 0.0165 µg/mL τ = 0.972
Deming Regression y = 1.019x + 0.0107 μg/mL r = 0.998
4.10. Stability
The stability data supports Roche Diagnostic's claims as reported in the package labeling.
5. ADDITIONAL INFORMATION
The ONLINE TDM Phenytoin - Free Phenytoin application is intended to be used with the following calibrators and controls:
- . Preciset TDM I calibrators (diluted)
- . TDM Control Set (Level I and II, ultrafilatrated)
Preciset TDM I, product code DKB, is a Class II 510(k) Exempt device and therefore, is not included with this submission.
TDM Control Set, product code JJY, is a Class I 510(k) Exempt device and therefore, is not included with this submission.
Other devices required but not provided:
- . Serum Filters
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6. CONCLUSIONS
The analytical performance data for ONLINE TDM Phenytoin - Free Phenytoin application met the acceptance criteria and support the substantial equivalence of ONLINE TDM Phenytoin -Free Phenytoin application on cobas c 503 analyzer to the predicate.
§ 862.3350 Diphenylhydantoin test system.
(a)
Identification. A diphenylhydantoin test system is a device intended to measure diphenylhydantoin, an antiepileptic drug, in human specimens. Measurements obtained by this device are used in the diagnosis and treatment of diphenylhydantoin overdose and in monitoring levels of diphenylhydantoin to ensure appropriate therapy.(b)
Classification. Class II.