The Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay is an enzyme immunoassay with a cutoff of 1.0 ng/mL. The assay is intended for use in laboratories for the qualitative analysis of Fentanyl in human urine with automated clinical chemistry analyzers. This assay is calibrated against Fentanyl. This in vitro diagnostic device is for prescription use only.

The Immunalysis SEFRIA Fentanyl Urine Enzyne Immunoassay provides only a preliminary analytical test result. A more specific alternate chemical must be used in order to obtain a confirmed analytical result. Gas Chromatography / Mass Spectrometry (GC-MS) or Liquid Chromatography / Mass Spectrometry (LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

Immunalysis Fentanyl Urine Calibrators:

The Immunalysis Fentanyl Urine Calibrators are used as calibrators in the Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay for the qualitative determination of Fentanyl in urine on automated clinical chemistry analyzers.

Device Description

The assay consists of antibody/ substrate reagent and enzyme conjugate reagent. The antibody/ substrate reagent includes EA protein and rabbit antibodies to Fentanyl in PIPES buffer with sodium azide as a preservative. The enzyme conjugate reagent includes ED peptide labeled with Fentanyl and CPRG substrate in malic acid buffer with sodium azide as a preservative. Calibrators and controls are included as part of the test system and provided separately. The Fentanyl calibrators consist of a Level 1 calibrator at 1 ng/mL, a Level 2 calibrator at 2 ng/mL, and a Level 3 calibrator at 4 ng/mL. The control set contains a LOW control at 0.5 ng/mL and a HIGH control at 1.5 ng/mL.

Automated clinical chemistry analyzers capable of maintaining a constant temperature, pipetting samples and reagents, mixing reagents, timing the reaction accurately and measuring enzymatic rates at 570nm can be used to perform the assay.

The SEFRIA™ technology is based on artificial fragments of the E. coli enzyme ß-galactosidase. A mutant enzyme, termed Enzyme Acceptor (EA), is created by deletion of 28 amino acids in the amino-terminal region of the sequence. EA is inactive, but can combine with peptides, termed Enzyme Donors (ED's), containing the deleted sequence, to form active B-galactosidase. This process is termed complementation, and the active enzyme formed as a result can be measured by hydrolysis of a chromogenic substrate such as chlorophenolred ß-D-galactopyranoside (CPRG). The ED peptides can be modified by attachment of a derivative of fentanyl, which does not interfere with the formation of active ß-galactosidase. However antibodies to fentanyl bind to the ED-fentanyl conjugate, and block complementation. The assay is based on the competition of fentanyl in a urine sample with the ED-fentanyl conjugate for the fixed amount of antibody binding sites. In the absence of the free drug in the sample, the antibody binds the ED-fentanyl conjugate, resulting in inhibition of enzyme formation. As the fentanyl concentration in the sample increases, ED-fentanyl becomes available for complementation, creating a dose response relationship between fentanyl concentration in the urine and enzyme formation. The Bgalactosidase activity is determined spectrophotometrically at 570 nm by the conversion of CPRG (orange) to chlorophenol red (red) and galactose.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study information for the Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay, based on the provided text:

Important Note: This document describes an in vitro diagnostic (IVD) device, not an AI/ML powered device for image analysis. Therefore, some of the requested information (like experts for ground truth, adjudication methods, MRMC studies, effect size of human readers with AI assistance, and standalone algorithm performance) is not applicable or not present in the context of this type of device. I will address only the information that can be extracted from the provided text for this specific device type.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device are mainly demonstrated through various performance studies, showing that the device accurately identifies fentanyl in urine at defined concentrations and is not significantly affected by interferences. Since specific numerical acceptance criteria (e.g., "must achieve >95% accuracy") are not explicitly stated in a consolidated table, I've inferred them from the study results presented as "verification" of performance.

Acceptance Criteria & Reported Device Performance for Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay

Acceptance Criterion (Inferred from study objectives)	Reported Device Performance
Precision/Cutoff Characterization/Reproducibility	- At -100% to -25% of cutoff (0-0.75 ng/mL): 100% Negative (80/80)
(Ability to consistently and accurately classify samples around the cutoff)	- At cutoff (1.0 ng/mL): 32 Negative / 48 Positive (Note: a mix is expected at the cutoff)
	- At +25% to +100% of cutoff (1.25-2.0 ng/mL): 100% Positive (80/80)
Specificity and Cross-Reactivity	- Fentanyl, Butyryl Fentanyl, Acetyl Fentanyl were positive at low concentrations (1 ng/mL, 0.8 ng/mL, 1 ng/mL respectively).
(Ability to exclusively determine fentanyl and minimize false positives from related compounds)	- Other structurally related compounds (e.g., Sufentanil, Norfentanyl, various other drugs) showed very low or no cross-reactivity at significantly higher concentrations, indicating good specificity.
Interference (Structurally Unrelated Compounds)	- All tested structurally unrelated compounds (e.g., Acetaminophen, Caffeine, Ibuprofen) at high concentrations (75,000 to 500,000 ng/mL) did not interfere, correctly yielding Negative results at 0.5 ng/mL Fentanyl and Positive results at 1.5 ng/mL Fentanyl.
(Assay performance unaffected by externally ingested compounds)
Interference (Endogenous Compounds)	- All tested endogenous compounds (e.g., Acetone, Bilirubin, Glucose, Hemoglobin) at physiologically relevant concentrations did not interfere, correctly yielding Negative results at 0.5 ng/mL Fentanyl and Positive results at 1.5 ng/mL Fentanyl.
(Assay performance unaffected by internally existing physiological conditions)
Boric Acid Interference	- Boric acid (1% w/v) did not interfere, correctly yielding Negative results at 0.5 ng/mL Fentanyl and Positive results at 1.5 ng/mL Fentanyl.
(Assay performance unaffected by boric acid)
pH Interference	- No positive or negative interference observed with urine pH values ranging from 3.0 to 11.0.
(Assay performance unaffected by varying urine pH)
Specific Gravity Interference	- No positive or negative interference observed with urine specific gravity values ranging from 1.000 to 1.030.
(Assay performance unaffected by varying urine specific gravity)
Method Comparison (with LC-MS/MS as confirmatory method)	- Overall Agreement for Positive results: 100% (40/40)
(Concordance with a gold standard confirmatory method)	- Overall Agreement for Negative results: 98% (39/40)
	- One discordant result: Device gave a Positive for a sample confirmed by LC/MS at 0.9 ng/mL (which is below the 1.0 ng/mL cutoff, but still close).

2. Sample Size Used for the Test Set and Data Provenance

Precision/Cutoff Characterization/Reproducibility Study:
- Sample Size: 80 drug-free urine samples, spiked with fentanyl at 9 different concentrations, each tested in replicates of 4 over 10 days (total determinations = 80 per concentration point).
- Data Provenance: Drug-free urine was spiked with fentanyl. The provenance of the original drug-free urine is not specified (e.g., country of origin). The study is prospective in nature as samples were intentionally prepared for the test.
Specificity and Cross-Reactivity Study:
- Sample Size: Not explicitly stated, samples were spiked with various compounds to represent different concentrations.
- Data Provenance: Not explicitly stated, samples were spiked into drug-free urine.
Interference (Structurally Unrelated & Endogenous Compounds), Boric Acid, pH, and Specific Gravity Interference Studies:
- Sample Size: Not explicitly detailed per compound, but samples were spiked into drug-free urine containing fentanyl at ±50% of the cutoff.
- Data Provenance: Samples were spiked into drug-free urine.
Method Comparison Study:
- Sample Size: 80 de-identified, unaltered leftover clinical urine samples.
- Data Provenance: Obtained from clinical testing laboratories. Specific country of origin is not mentioned, but it implies a retrospective collection of existing clinical samples.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

This device is an in vitro diagnostic immunoassay, not an imaging AI device requiring expert interpretation for ground truth.

Ground Truth Establishment for Analytical Studies (Precision, Specificity, Interference): The "ground truth" was established by precisely spiking known concentrations of fentanyl or other compounds into drug-free urine, and/or confirming concentrations via mass spectrometry (LC-MS/MS). No human experts are used for this.
Ground Truth Establishment for Method Comparison Study: The ground truth was established by a confirmatory method, Liquid Chromatography / Mass Spectrometry (LC/MS or LC-MS/MS), which is considered the gold standard for drug quantification. No human experts are mentioned for establishing this ground truth.

4. Adjudication Method for the Test Set

Not applicable for this type of IVD device. Adjudication methods like 2+1 or 3+1 typically apply to human readers interpreting complex data (e.g., medical images) where discrepancies need to be resolved. For an immunoassay, the result is quantitative (absorbance) and then interpreted qualitatively based on a cutoff. Confirmatory testing (e.g., LC-MS/MS) serves as the definitive assessment, not a human adjudication process.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done

No, this is not applicable. MRMC studies evaluate the performance of human readers, sometimes with and without AI assistance, on a set of cases. This device is an automated laboratory test, not an AI-powered reader for medical images or other data that requires human interpretation.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the studies evaluate the performance of the device itself (the immunoassay) in a standalone fashion. Human involvement is limited to preparing samples, loading them onto the automated clinical chemistry analyzer, and reading the qualitative result (positive/negative) that the instrument provides based on its internal reaction and cutoff. There is no "human-in-the-loop" performance as would be understood in an AI-assisted diagnostic context for interpretation.

7. The Type of Ground Truth Used

Analytical Studies (Precision, Specificity, Interference): Ground truth was established by known concentrations of spiked analytes in drug-free urine, often confirmed by Mass Spectrometry (MS).
Method Comparison Study: Ground truth was established by Liquid Chromatography / Mass Spectrometry (LC/MS or LC-MS/MS), which is the preferred confirmatory method mentioned in the "Indications for Use" and is considered the gold standard.

8. The Sample Size for the Training Set

This is not an AI/ML device that requires a "training set" in the traditional sense. The device is a chemical immunoassay based on enzymatic reactions and antibody binding. Its "development" involves chemical formulation and optimization, not machine learning model training.

9. How the Ground Truth for the Training Set was Established

As noted above, there is no "training set" for this immunoassay device in the context of AI/ML. The development and optimization of the immunoassay reagents (antibodies, enzymes, etc.) would have relied on biochemical principles and extensive internal testing with known Fentanyl concentrations. The calibrators themselves have their values assigned based on mass spectrometry, as described in section 10 of the performance characteristics.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

June 14, 2017

IMMUNALYSIS CORPORATION MICHELLE BODIEN, ASSOCIATE DIRECTOR, REGULATORY AFFAIRS 829 TOWNE CENTER DRIVE POMONA, CA 91767

Re: K161216

Trade/Device Name: Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay and Immunalysis Fentanyl Urine Calibrators Regulation Number: 21 CFR 862.3650 Regulation Name: Opiate test system Regulatory Class: II Product Code: DJG, DLJ Dated: May 30, 2017 Received: June 1, 2017

Dear Ms. Bodien:

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. 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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Kellie B. Kelm -S

for Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K161216

Device Name

Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay, Immunalysis Fentanyl Urine Calibrators

Indications for Use (Describe)

Immunalysis Fentanyl Urine Calibrators:

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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510(k) SUMMARY

A. General Information
1.	Applicant Name:	Immunalysis Corporation
		829 Towne Center Drive
		Pomona, CA 91767
2.	Company Contact:	Michelle Bodien
		Associate Director, Regulatory Affairs
		Phone: (858) 805-2283
		Email: michelle.bodien@alere.com
3.	Date prepared:	June 13, 2017
B. Device Identification
1.	Trade Name:	Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay
		Immunalysis Fentanyl Urine Calibrators
2.	Common Name:	Fentanyl Urine Enzyme Immunoassay
		Fentanyl Urine Calibrators
C. Regulatory Information
1.	Device Classification:	II
2.	Regulation Number:	21 CFR 862.3650 Enzyme Immunoassay,Opiates
		21 CFR 862.3200 Calibrators, Drug Specific
3.	Panel:	Toxicology (91)
4.	Product Code:	DJGDLJ
5.	Predicate Device:	Emit® II Plus Buprenorphine Assay
6.	Predicate Company:	Siemens Healthcare Diagnostics Inc.
7.	Predicate K Number:	K150606

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D. Device Description

E. Intended Use

1. The Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay is an in vitro diagnostic test for the qualitative analysis of Fentanyl in human urine with automated clinical chemistry analyzers. This test is an enzyme immunoassay with a cutoff of 1.0 ng/mL. The assay is intended for use in laboratories and for prescription use only. This assay is calibrated against Fentanyl.
  The Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay provides only a preliminary analytical test result. A more specific alternate chemical must be used in order to obtain a confirmed analytical result. Gas Chromatography/ Mass Spectrometry (GC-MS) or Liquid Chromatography / Mass Spectrometry (LC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

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2. Immunalysis Fentanyl Urine Calibrators

The Immunalysis Fentanyl Urine Calibrators are used as calibrators in the Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay for the qualitative determination of Fentanyl in urine on automated clinical chemistry analyzers.

Attribute	Predicate Device K150606Emit® II Plus Buprenorphine Assay	Candidate DeviceImmunalysis Fentanyl Urine EIA
Similarities
Test System	Homogeneous enzyme immunoassay	Same
Sample Matrix	Urine	Same
User Environment	For use in laboratories	Same
Mass Spectrometry Confirmation	Required for preliminary positive analytical results	Same
Storage	2 – 8° C	Same
Materials	Antibody/substrate reagents and enzyme labeled conjugate	Same
Calibrator Form	Liquid	Same
Differences
Intended Use	For the qualitative and semi-quantitative determination of the presence of Buprenorphine in human urine at a cutoff of 5 ng/mL	For the qualitative determination of the presence of Fentanyl in human urine at a cutoff of 1 ng/mL
Detection	Absorbance change measured spectrophotometrically at 340 nm	Absorbance change measured spectrophotometrically at 570 nm
Measured Analytes	Buprenorphine	Fentanyl
Cutoff Levels	5 ng/mL of Buprenorphine	1 ng/mL of Fentanyl
Antibody	Mouse monoclonal antibody to buprenorphine	Rabbit antibodies to Fentanyl
Reagents Form	R1 and R2: Liquid – Ready to use	EA and ED: Liquid – Ready to use
Calibrator Levels	One negative and four levels (0, 2.5, 5, 15 and 25 ng/mL)	One negative and three levels (0, 1, 2 and 4 ng/mL

F. Comparison With Predicate

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G. Performance Characteristics:
The following laboratory performance studies were performed to determine substantial equivalence of the Immunalysis SEFRIATM Fentanyl Urine Enzyme Immunoassay to the predicate. All studies utilized the Beckman Coulter AU 400e instrument.
1. Precision/ Cutoff Characterization/ Reproducibility Study was performed for 10 days, 2 runs per day in replicates of 4 on drug free urine (N=80) spiked with fentanyl to concentrations of ±25%, ±50%, ±75%, and ±100% of the cutoff (1.0 ng/mL). The spiked concentrations were confirmed by mass spectrometry (MS). The study verified that the cutoff serves as a boundary between a negative and positive interpretation of a qualitative result.

Table 1 - Precision Results
Concentration (ng/mL)	% of cutoff	# of determinations	Total Result
0	-100%	80	80 Negative
0.25	-75%	80	80 Negative
0.5	-50%	80	80 Negative
0.75	-25%	80	80 Negative
1.0	Cutoff	80	32 Negative/48 Positive
1.25	+25%	80	80 Positive
1.5	+50%	80	80 Positive
1.75	+75%	80	80 Positive
2.0	+100%	80	80 Positive

The following is a summary table precision results for the 1.0 ng/mL cutoff test data results.

1. Specificity and Cross-Reactivity Structurally similar compounds were spiked into drug free urine at levels that will yield a result that is equivalent to the cutoff. The study verified assay performance relative to the ability of the device to exclusively determine certain drugs.

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Table 3 - Structurally Related Compounds
Compound	Concentration Tested (ng/mL)	Result	Cross-Reactivity (%)
Fentanyl	1	Positive	100.0000
Butyryl Fentanyl	0.8	Positive	125.0000
Acetyl Fentanyl	1	Positive	100.0000
Despropionyl Fentanyl	40	Positive	2.5000
Sufentanil	175	Negative	<0.5714
Haloperidol	1,250	Positive	0.0800
Pipamperone	1,500	Positive	0.0667
Risperidone	2,500	Positive	0.0400
Norfentanyl	20,000	Positive	0.0050
Trazodone	10,000	Positive	0.0100
Labetalol	15,000	Positive	0.0067
Clomipramine	45,000	Positive	0.0022
Benzylpiperazine	50,000	Positive	0.0020
Fluoxetine	60,000	Positive	0.0017
Fenfluramine	60,000	Positive	0.0017
Methamphetamine	70,000	Positive	0.0014
Amitryptyline	75,000	Positive	0.0013
Chlorpromazine	75,000	Positive	0.0013
PCP	100,000	Positive	0.0010
Pentazocine	75,000	Positive	0.0013
6-Acetyl Codeine	100,000	Negative	<0.0010
6-Acetyl Morphine	100,000	Negative	<0.0010
Buprenorphine	100,000	Negative	<0.0010
Buproprion	100,000	Negative	<0.0010
Codeine	100,000	Negative	<0.0010
Cyclobenzaprine	100,000	Positive	0.0010
Desipramine	100,000	Positive	0.0010
Diacetyl Morphine	100,000	Negative	<0.0010
Dihydrocodeine	100,000	Negative	<0.0010
Diphenhydramine	100,000	Positive	0.0010
Doxepin	100,000	Positive	0.0010
EDDP	100,000	Negative	<0.0010
EMDP	100,000	Negative	<0.0010
Hydrocodone	100,000	Negative	<0.0010
Hydromorphone	100,000	Negative	<0.0010
Imipramine	100,000	Positive	0.0010
Levorphanol	100,000	Negative	<0.0010
Meta-chlorphenylpiperazine	100,000	Positive	0.0010
Meperidine	100,000	Negative	<0.0010
Table 3 - Structurally Related Compounds
Compound	Concentration Tested (ng/mL)	Result	Cross-Reactivity (%)
Methadone	100,000	Negative	<0.0010
Morphine	100,000	Negative	<0.0010
Morphine-3-glucuronide	100,000	Negative	<0.0010
Morphine-6-glucuronide	100,000	Negative	<0.0010
Nalorphine	100,000	Negative	<0.0010
Naloxone	100,000	Negative	<0.0010
Naltrexone	100,000	Negative	<0.0010
Norcodeine	100,000	Negative	<0.0010
Nordiazepam	100,000	Negative	<0.0010
Normorphine	100,000	Negative	<0.0010
Nortriptyline	100,000	Positive	0.0010
Oxycodone	100,000	Negative	<0.0010
Oxymorphone	100,000	Negative	<0.0010
Propoxyphene	100,000	Negative	<0.0010
Protryptyline	100,000	Negative	<0.0010
Tramadol	100,000	Negative	<0.0010
Trimethoprim	100,000	Negative	<0.0010
Trimipramine	100,000	Negative	<0.0010
Venlafaxine	100,000	Negative	<0.0010

The following is a summary table of results:

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1. Interference Structurally unrelated compounds were evaluated by spiking the potential interferent into drug free urine containing fentanyl at ±50% of the cutoff. All potential interferents analyzed verified that assay performance is unaffected by externally ingested compounds. The results of this study are indicated in Table 5 below.

Table 5 - Structurally Unrelated Compounds
Compound	ConcentrationTested (ng/mL)	-50% Cutoff(0.5 ng/mL)	+50% Cutoff(1.5 ng/mL)
11-hydroxy-delta-9-THC	75,000	Negative	Positive
11-nor-9 carboxy THC	100,000	Negative	Positive
1S, 2R(+)-Ephedrine	100,000	Negative	Positive
7-Aminoclonazepam	100,000	Negative	Positive
7-Aminoflunitrazepam	100,000	Negative	Positive
7-Aminonitrazepam	100,000	Negative	Positive
Acetaminophen	500,000	Negative	Positive
Amobarbital	100,000	Negative	Positive
Barbital	100,000	Negative	Positive
Benzoylecgonine	100,000	Negative	Positive
Bromazepam	100,000	Negative	Positive
Butabarbital	100,000	Negative	Positive
Caffeine	100,000	Negative	Positive
Cannabidiol	100,000	Negative	Positive
Cannabinol	75,000	Negative	Positive
Carbamazepine	100,000	Negative	Positive
Table 5 - Structurally Unrelated Compounds
	Concentration	-50% Cutoff	+50% Cutoff
Compound	Tested (ng/mL)	(0.5 ng/mL)	(1.5 ng/mL)
Carisoprodol	100,000	Negative	Positive
Chlordiazepoxide	100,000	Negative	Positive
cis-Tramadol	100,000	Negative	Positive
Clobazam	100,000	Negative	Positive
Clonazepam	100,000	Negative	Positive
Cotenine	100,000	Negative	Positive
Delta-9-THC	100,000	Negative	Positive
Demoxepam	100,000	Negative	Positive
Ecgonine	100,000	Negative	Positive
Ecgonine methyl ester	100,000	Negative	Positive
Ethyl beta-D-glucuronide	100,000	Negative	Positive
Flunitrazepam	100,000	Negative	Positive
Heroin	100,000	Negative	Positive
Hexobarbital	100,000	Negative	Positive
Ibuprofen	100,000	Negative	Positive
Ketamine	100,000	Negative	Positive
Lamotrignine	100,000	Negative	Positive
Lidocaine	100,000	Negative	Positive
Lorazepam Glucuronide	50,000	Negative	Positive
LSD	100,000	Negative	Positive
Mephobarbital	100,000	Negative	Positive
Methaquolone	100,000	Negative	Positive
Naproxen	100,000	Negative	Positive
Nitrazepam	100,000	Negative	Positive
Normorphine	100,000	Negative	Positive
Norpseudoephedrine	100,000	Negative	Positive
Oxazepam	100,000	Negative	Positive
Pentobarbital	100,000	Negative	Positive
Phenobarbital	100,000	Negative	Positive
Phenylephedrine	100,000	Negative	Positive
Salicylic Acid	100,000	Negative	Positive
Secobarbital	100,000	Negative	Positive
Temazepam	100,000	Negative	Positive
Phenytoin	100,000	Negative	Positive
PMA	100,000	Negative	Positive
Propranolol	100,000	Negative	Positive
(+)-MDA	75,000	Negative	Positive
4-Bromo-2,5,Dimethoxyphenethylamine	75,000	Negative	Positive
Desalkyflurazepam	75,000	Negative	Positive
Dextromethorphan	75,000	Negative	Positive
Diazepam	75,000	Negative	Positive
Flurazepam	75,000	Negative	Positive
Lorazepam	75,000	Negative	Positive
Lormetazepam	75,000	Negative	Positive
Maprotiline	75,000	Negative	Positive
Medezapam	75,000	Negative	Positive
Table 5 - Structurally Unrelated Compounds
Compound	ConcentrationTested (ng/mL)	-50% Cutoff(0.5 ng/mL)	+50% Cutoff(1.5 ng/mL)
Meprobamate	75,000	Negative	Positive
Methyphenidate	75,000	Negative	Positive
Midazolam	75,000	Negative	Positive
N-Desmethyltapentadol	75,000	Negative	Positive
Oxazepam glucuronide	75,000	Negative	Positive
Phentermine	75,000	Negative	Positive
Phenylpropanolamine	75,000	Negative	Positive
R,R(-)-Pseudoephedrine	75,000	Negative	Positive
Ranitidine	75,000	Negative	Positive
Ritalinic Acid	75,000	Negative	Positive
Sertraline	75,000	Negative	Positive
Theophylline	75,000	Negative	Positive
Thioridazine	75,000	Negative	Positive
Zolpidem Tartrate	75,000	Negative	Positive
Cocaine	40,000	Negative	Positive
MDEA	50,000	Negative	Positive
MDMA	50,000	Negative	Positive
S-(+) Amphetamine	50,000	Negative	Positive
Triazolam	50,000	Negative	Positive
Trifluoromethylphenyl-piperazine	40,000	Negative	Positive

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1. Interference Endogenous compounds were evaluated by spiking the potential interferent into drug free urine containing fentanyl at ±50% of the cutoff. All potential interferents analyzed verified that assay performance is unaffected by internally existing physiological conditions. The results of this study are indicated in Table 6 below:

Table 6 - Endogenous Compounds
Compound	Concentration Tested (ng/mL)	-50% Cutoff (0.5 ng/mL)	+50% Cutoff (1.5 ng/mL)
Acetone	1.0 g/dL	Negative	Positive
Ascorbic Acid	0.56 g/dL	Negative	Positive
Bilirubin	2.0 mg/dL	Negative	Positive
Creatinine	0.5 g/dL	Negative	Positive
Ethanol	1.0 g/dL	Negative	Positive
Galactose	10 mg/dL	Negative	Positive
$\gamma$-Globulin	0.5 g/dL	Negative	Positive
Glucose	2.0 g/dL	Negative	Positive
Hemoglobin	0.5 g/dL	Negative	Positive
Human Serum Albumin	0.5 g/dL	Negative	Positive
Oxalic Acid	0.1 g/dL	Negative	Positive
Riboflavin	7.5 mg/dL	Negative	Positive
Sodium Azide	1% w/v	Negative	Positive
Sodium Chloride	6.0 g/dL	Negative	Positive
Sodium Fluoride	1% w/v	Negative	Positive
Urea	2.0 g/dL	Negative	Positive

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1. Boric Acid Interference - Boric acid at a concentration of 1% w/v was evaluated by spiking the potential interferent into drug free urine containing fentanyl at ±50% of the cutoff. The results of this study are indicated in Table 7 below.

Table 7 - Boric Acid
Compound	Concentration	-50% Cutoff	+50% Cutoff
	Tested (ng/mL)	(0.5 ng/mL)	(1.5 ng/mL)
Boric Acid	1% w/v	Negative	Positive

pH Interference To evaluate potential interference from the effect of urine pH, device 6. performance was tested using a range of urine pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0). All test samples were prepared in drug free urine containing fentanyl at ±50% of the cutoff. No positive or negative interference was observed at urine pH values ranging from 3.0 to 11.0 for each test mode. The results of this study are indicated in Table 8 below.

Table 8 - Effect of pH
pH Value	-50% Cutoff(0.5 ng/mL)	+50% Cutoff(1.5 ng/mL)
3.0	Negative	Positive
4.0	Negative	Positive
5.0	Negative	Positive
6.0	Negative	Positive
7.0	Negative	Positive
8.0	Negative	Positive
9.0	Negative	Positive
10.0	Negative	Positive
11.0	Negative	Positive

1. Specific Gravity Interference - To evaluate potential interference from the specific gravity of urine, device performance was tested using a range of physiologically relevant urine specific gravity values (1.000, 1.005, 1.010, 1.015, 1.020, 1.025 and 1.030). All test samples were prepared in drug free urine containing fentanyl at ±50% of the cutoff. No positive or negative interference was observed at urine specific gravity values ranging from 1.000 to 1.030 for each test mode. The results of this study are indicated in Table 9 below.

Table 9 - Effect of Specific Gravity
Specific GravityValue	-50% Cutoff(0.5 ng/mL)	+50% Cutoff(1.5 ng/mL)
1.000	Negative	Positive
1.002	Negative	Positive
1.005	Negative	Positive
1.010	Negative	Positive
1.015	Negative	Positive
1.020	Negative	Positive
1.025	Negative	Positive
1.030	Negative	Positive

1. Linearity/ Recovery Not applicable, this device is intended for qualitative use only

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1. Method Comparison Eighty deidentified, unaltered leftover clinical urine samples obtained from clinical testing laboratories were analyzed for fentanyl at an assay cutoff of 1.0 ng/mL with the Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay compared to results by mass spectrometry (LC-MS/MS). The instruments used were a Beckman Coulter AU 400e and an Agilent 6430 Liquid Chromatography Tandem Mass Spectrometer.

Table 12 -Performance Verified by LC/MS
SEFRIATMFentanyl UrineEIA Result	< 0.5ng/mL	0.5 ~ 0.9ng/mL	1.0 ~ 1.5ng/mL	> 1.5ng/mL	Agreement(%)
Positive	0	1	9	31	100%(40/40)
Negative	30	9	0	0	98%(39/40)

The following is a summary table of qualitative discordant results for the 1.0 ng/mL cutoff:

Table 13 - Discordant Result Summary
Sample ID	In-House ID	1ng CutoffTest Device	LC/MS Confirmation
Negative 36	21378	Positive	0.9 ng/mL

10. Immunalysis Fentanyl Urine Calibrators

Traceability all components of the calibrators have been traced to a commercially available a. fentanyl solution.
Value Assignment Calibrators are manufactured and tested by mass spectrometry. The b. negative calibrator is a processed, drug free urine matrix. The negative calibrator is compared to a reference negative standard to ensure that it is free of analyte. The non-zero calibrators are prepared by spiking a known concentration of fentanyl in the negative calibrator matrix. If any of the analytes are not within the acceptable range, then the calibrator is adjusted and retested. Values are assigned to the calibrators once the mass spectrometry results are within the acceptable ranges.

H. Conclusion

The information provided in this pre-market notification demonstrates that the Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay is substantially equivalent to the legally marketed predicate device for its intended use.

Regulation Number and Section

§ 862.3650 Opiate test system.

(a)
Identification. An opiate test system is a device intended to measure any of the addictive narcotic pain-relieving opiate drugs in blood, serum, urine, gastric contents, and saliva. An opiate is any natural or synthetic drug that has morphine-like pharmocological actions. The opiates include drugs such as morphine, morphine glucoronide, heroin, codeine, nalorphine, and meperedine. Measurements obtained by this device are used in the diagnosis and treatment of opiate use or overdose and in monitoring the levels of opiate administration to ensure appropriate therapy.(b)
Classification. Class II (special controls). An opiate test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (e.g., programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).