(69 days)
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No
The description details a standard in vitro diagnostic assay based on kinetic interaction of microparticles, with no mention of AI or ML algorithms for data analysis or interpretation.
No.
This device is an in vitro diagnostic test used to measure free phenytoin levels, which informs therapy but does not directly provide therapeutic treatment.
Yes
The "Intended Use / Indications for Use" section explicitly states that the device is "an in vitro test for the quantitative determination of free phenytoin in human serum and plasma" and that this determination "is used in monitoring levels of free phenytoin to ensure appropriate therapy," which is a diagnostic purpose to guide treatment.
No
The device description explicitly states it is an "in vitro test" for use on "cobas c systems" and involves "kinetic interaction of microparticles in a solution (KIMS), photometrically detected by turbidity measurements". This indicates the device is a reagent and method used with specific hardware analyzers, not a standalone software application.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use/Indications for Use: The description explicitly states it is an "in vitro test for the quantitative determination of free phenytoin in human serum and plasma". This clearly indicates it is used to test samples taken from the human body outside of the body.
- Device Description: The description further elaborates on the process of testing human serum and plasma samples using a specific method (KIMS) to measure free phenytoin levels.
- Nature of the Test: The test involves analyzing biological samples (serum and plasma) to provide information about a substance (free phenytoin) within those samples. This is the core function of an in vitro diagnostic device.
The information provided aligns perfectly with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
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.
Product codes (comma separated list FDA assigned to the subject device)
MOJ
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.
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
Mentions image processing
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Mentions AI, DNN, or ML
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Input Imaging Modality
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Anatomical Site
Not Found
Indicated Patient Age Range
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Intended User / Care Setting
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Description of the training set, sample size, data source, and annotation protocol
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Description of the test set, sample size, data source, and annotation protocol
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Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Precision (Repeatability and Intermediate Precision)
Results for repeatability and intermediate precision were obtained on the cobas c 503 analyzer in accordance with CLSI EP05-A3 requirements.
Sample size: n = 84 for repeatability. Intermediate precision utilized 2 aliquots per run, 2 runs per day, over 21 days.
Key results: All acceptance criteria were met.
Repeatability CV (Control 1a): 2.2%
Repeatability CV (Control 2b): 2.4%
Repeatability CV (Human Serum 1-5): 2.1% - 2.9%
Intermediate Precision CV (Control 1a): 2.9%
Intermediate Precision CV (Control 2b): 2.8%
Intermediate Precision CV (Human Serum 1-5): 2.8% - 3.5%
Analytical Sensitivity (Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ))
LoB: Determined with one analyte-free sample measured with three reagent lots in 6 runs, 10-fold determination per run, over 6 days, on one cobas c 503 analyzer, according to CLSI EP17-A2.
Key results: LoB = 0.100 µg/mL (0.396 µmol/L).
LoD: Determined with 5 serum samples with low analyte concentrations (spiked and ultrafiltrated) measured on three reagent lots with 2-fold determination per run on one cobas c 503 analyzer, over 6 days, according to CLSI EP17-A2.
Key results: LoD = 0.200 µg/mL (0.792 µmol/L).
LoQ: Determined with 6 serum samples (spiked and ultrafiltrated) measured with three reagent lots on one cobas c 503 in 1 run per day over 5 days, 5 replicates per run for each LoQ sample, according to CLSI EP17-A2.
Key results: LoQ = 0.400 µg/mL (1.58 µmol/L).
Linearity/Assay Reportable Range
Study type: Linearity assessment according to CLSI EP06-A-Ed2.
Description: A dilution series was prepared from a spiked human ultrafiltrated serum pool and a negative ultrafiltrated serum pool, spanning > 9 levels. Samples were assayed on one cobas c 503 analyzer in 1 run using 3 reagent lots and 4 replicates per sample. Same process for K3-EDTA plasma.
Key results: Linearity was confirmed for the measuring range of 0.400-4.00 µg/mL (1.58-15.8 µmol/L).
Dilution
Study type: Post Dilution Check experiments.
Description: Three ultrafiltrates with phenytoin concentrations above the measuring range (5.00 µg/mL, 6.00 µg/mL, 7.00 µg/mL) were measured via automatic rerun function and manual dilution on the cobas c 503.
Key results: The 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
Study type: Evaluation of potential interference from endogenous substances (hemoglobin, lipemia (Intralipid), conjugated and unconjugated bilirubin, Immunoglobulin G (IgG), albumin, rheumatoid factor, total protein, triglycerides, HAMA).
Description: Endogenous interferents were tested for analytical interference (added to ultrafiltrate) and for phenytoin release and binding effects (present in sample before ultrafiltration).
Key results: All predefined acceptance criteria were met. Claims for no interference were established for specific concentrations of Hemolysis (H index of 1000), Icterus (I index of 60 for conjugated bilirubin), Triglycerides (700 mg/dL), Albumin (60 g/L), Total protein (2-12 g/dL), and Rheumatoid factors (1200 IU/mL) for analytical interference. For phenytoin release/binding effects, no significant effect was observed up to H index of 1000 for Hemolysis, L index of 1000 for Lipemia, and 60 g/L for Immunoglobulin G.
Analytical Specificity/Cross-Reactivity
Study type: Cross-reactivity study.
Description: Conducted with the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 analyzer. Two ultrafiltrated human serum pools spiked with phenytoin (1.00 µg/mL and 2.50 µg/mL) were used for each potential cross-reacting compound. Phenytoin concentration determined in at least 5-fold determination and compared to reference.
Key results: All acceptance criteria for cross reactivity were met. Specific cross-reactivity percentages are provided for Fosphenytoin (≤ 50.0 %), m-HPPH (≤ 10.0 %), p-HPPH (≤ 5.0 %), and 5(p-methylphenyl)-5-phenylhydantoin (≤ 5.0 %).
Exogenous Interferences - Drugs
Study type: Exogenous interference study.
Description: Evaluated commonly used and special pharmaceuticals.
Key results: No increase in free phenytoin concentrations was observed at the tested concentrations for several listed drugs. Increased concentrations of free phenytoin were correctly observed in the presence of drugs that bind to human albumin in serum and release phenytoin (e.g., butabarbital, carbamazepine, ibuprofen, valproic acid). For phenobarbital and mephenytoin, analytical interference was noted at higher drug concentrations in addition to competition for albumin binding.
Sample Matrix Comparison
Study type: Matrix comparison.
Description: Effect on quantitation of free phenytoin values in the presence of anticoagulants determined by comparing values from samples collected in serum, Li-Heparin, K2-EDTA, and K3-EDTA plasma tubes. Samples were spiked and ultrafiltrated.
Sample size: ≥50 samples.
Key results: All predefined acceptance criteria were met, supporting that serum, Li-Heparin, K2-EDTA, and K3-EDTA plasma are acceptable sample types. Correlation coefficients (Pearson r) were 0.988 for Serum vs. Li-Heparin plasma, 0.992 for Serum vs. K2-EDTA plasma, and 0.992 for Serum vs. K3-EDTA plasma.
Method Comparison to Phenytoin - Free Phenytoin Application on COBAS INTEGRA 400 plus
Study type: Method comparison.
Description: Compared the ONLINE TDM Phenytoin - Free Phenytoin application on the cobas c 503 (candidate device) with the Phenytoin - Free Phenytoin Application on the COBAS INTEGRA 400 plus (reference device).
Sample size: 138 native human ultrafiltrated serum samples (≤10% spiked).
Key results: Sample concentrations were between 0.400 µg/mL (1.58 µmol/L) and 3.78 µg/mL (15.0 µmol/L). Passing/Bablok regression: y = 1.035x - 0.0165 µg/mL, τ = 0.972. Deming Regression: y = 1.019x + 0.0107 µg/mL, r = 0.998.
Stability
Key results: The stability data supports Roche Diagnostic's claims as reported in the package labeling.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Sensitivity (Analytical):
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) = 0.400 µg/mL (1.58 µmol/L)
Precision:
Repeatability CV: 2.1% - 2.9%
Intermediate precision CV: 2.8% - 3.5%
Correlation Coefficient for Method Comparison to Predicate (Deming Regression): r = 0.998
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 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.
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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
1
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
2
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)
CONTINUE ON A SEPARATE PAGE IF NEEDED.
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DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.
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"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."
3
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 Road |
| P.O. Box 50416 | |
| Indianapolis, IN 46250-0457 | |
| Contact | Leslie Patterson |
| Phone: (317) 225-8563 | |
| Email: 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: 9610126 |
| Roche Diagnostics GmBH Penzberg, Germany: 9610529 | |
| Roche 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
5
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 - Free
Phenytoin application | Predicate Device:
COBAS-FP Free Phenytoin |
|------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use /
Indications for
Use | 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 COBAS-FP Reagents for Free
Phenytoin are an in vitro test for the
quantitative determination of free
phenytoin in serum and heparinized
plasma on the COBAS FARA II chemistry
system. |
| Assay Method | Kinetic Interaction of Microparticles in a
Solution (KIMS) | Fluorescence Polarization Immunoassay
(FPIA) |
| Sample
Type/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 |
| Measuring
Range | 0.400-4.00 µg/mL | 0.09-4 µg/mL |
| Lower Limits
of
Measurement | 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) = 0.400
µg/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 |
| Intermediate
| precision | 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
| Endogenous
| Substance | 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
$(µg/mL)$ | Correlation
Coefficient
(Pearson r) | Concentration of
Samples
$(µg/mL, serum)$ |
|--------------------------------|-------|------------------------|-------------------------------------------|-------------------------------------------------|
| Serum vs. Li-Heparin
plasma | 1.018 | -0.0149 | 0.988 | 0.592 – 3.84 |
| Serum vs. K2-EDTA
plasma | 0.923 | -0.0328 | 0.992 | 0.592 – 3.83 |
| Serum vs. K3-EDTA
plasma | 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.