Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay, Immunalysis Fentanyl Urine Calibrators

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k161216 B. Purpose for Submission: New device C. Measurand: Fentanyl D. Type of Test: Qualitative E. Applicant: Immunalysis Corporation F. Proprietary and Established Names: Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay Immunalysis SEFRIA Fentanyl Urine Calibrators G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | DJG | Class II | 21 CFR 862.3650, Opiate test system | Toxicology (91) | | DLJ | Class II | 21 CFR 862.3200, Clinical toxicology calibrator | Toxicology (91) | H. Intended Use: 1. Intended use(s): Refer to Indications for Use below. {1} 2. Indication(s) for use: 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 Enzyme Immunoassay provides only a preliminary analytical test result. A more specific alternate chemical method 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. 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. 3. Special conditions for use statement(s): For prescription use only. For in vitro diagnostic use only. 4. Special instrument requirements: The Beckman Coulter AU400e Chemistry Analyzer was used to generate the performance data in this submission. Instruments must be capable of maintaining a constant reaction temperature, pipetting samples and reagents, mixing reagents, timing reactions accurately, and measuring enzyme rates precisely at 570nm. I. Device Description: The Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay contains two reagents, which are provided as ready-to-use: - Antibody/Enzyme Donor (EA) – This reagent contains rabbit antibodies to fentanyl, and a partial sequence to galactosidase in PIPES buffer with Sodium Azide as a preservative. - Substrate/Enzyme Acceptor (ED) – This reagent contains the complementary sequence of β-galactosidase labeled with fentanyl in malic acid buffer with Sodium Azide as a preservative. All of the Immunalysis Fentanyl Urine Calibrators are sold as individual bottles and are liquid and ready to use. The negative calibrator is a processed, drug-free synthetic urine matrix with sodium azide as a preservative. Each calibrator (1, 2, and 4 ng/mL) contains a 2 {2} known concentration of Fentanyl spiked into the negative calibrator matrix. # J. Substantial Equivalence Information: 1. Predicate device name(s): Siemens Healthcare Diagnostics, Inc. Emit II Plus Buprenorphine Assay 2. Predicate $510(\mathrm{k})$ number(s): k150606 3. Comparison with predicate: | Similarities - Reagent | | | | --- | --- | --- | | Item | Predicate Device Siemens Healthcare Diagnostics. Emit II Plus Buprenorphine Assay k150606 | Candidate Device Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay | | Test System | Homogenous enzyme immunoassay | Same | | User Environment | Clinical laboratories | Same | | Sample Matrix | Urine | Same | | Mass Spectrometry Confirmation | Required to confirm preliminary positive analytical results | Same | | Storage | 2 – 8°C until expiration date | Same | | Materials | Antibody coated tube and reagents | Antibody/substrate reagents and enzyme labeled conjugate | | Differences - Reagent | | | | --- | --- | --- | | Item | Predicate Device Siemens Healthcare Diagnostics. Emit II Plus Buprenorphine Assay k150606 | Candidate Device Immunalysis SEFRIA™ Fentanyl Urine Enzyme Immunoassay | | Intended Use | For the qualitative and semi-quantitative determination of Buprenorphine in human urine at a cutoff of 5 ng/mL | For the qualitative determination of Fentanyl in human urine at a cutoff of 1 ng/mL | | Detection | Absorbance change measured spectrophotometrically at | Absorbance change measured spectrophotometrically at | {3} | | 340 nm | 570 nm | | --- | --- | --- | | Measurand Analytes | Buprenorphine | Fentanyl | | Cutoff Levels | 5 ng/mL | 1 ng/mL | | Antibody | Mouse monoclonal antibody to Buprenorphine | Enzyme Acceptor protein and rabbit antibodies to Fentanyl | | Reagents Form | R1 and R2-Liquid-Ready to Use | EA and ED: Liquid-Ready to Use | | Calibrators | | | | --- | --- | --- | | Item | Predicate Device Siemens Healthcare Diagnostics. Emit II Plus Specialty Drug Calibrator/Control Levels 1 - 4 k150606 | Candidate Device Immunalysis Immunalysis Fentanyl Urine Calibrators | | Analytes | Buprenorphine | Fentanyl | | Calibrator Levels | One negative and four levels (2.5, 5, 15, 25 ng/mL) | One negative and three levels (1, 2, and 4 ng/mL) | | Calibrator Form | Liquid | Same | | Storage | 2 – 8°C until expiration date | Same | # K. Standard/Guidance Document Referenced (if applicable): CLSI EP5-A3: "Evaluation of Precision of Quantitative Measurement Procedures: Approved Guideline-Third Edition" CLSI EP07-A2: "Interference Testing in Clinical Chemistry: Approved Guideline - Second Edition" ISO 14971 Second edition 2007-03-01, "Medical devices - application of risk management to medical devices" # L. Test Principle: The SEFRIA technology is based on artificial fragments of the $E.$ coli enzyme $\beta$ -galactosidase. A mutant enzyme, termed Enzyme Acceptor (EA), is created by deletion of 28 amino acids in the amino-terminal region of the sequence for $\beta$ -galactosidase. EA is inactive, but can combine Enzyme Donors (ED's), containing the deleted sequence, to form active $\beta$ -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 $\beta$ -D-galactopyranoside (CPRG). The ED peptides are attached to a derivative of fentanyl that does not interfere with the {4} formation of active $\beta$-galactosidase. However, when fentanyl antibodies bind to the ED-fentanyl conjugate complementation is blocked, thus preventing the formation of active $\beta$-galactosidase. 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 $\beta$-galactosidase activity is determined spectrophotometrically at $570~\mathrm{nm}$ by the conversion of CPRG (orange) to chlorophenol red (red) and galactose. ## M. Performance Characteristics: ### 1. Analytical performance: #### a. Precision/Reproducibility: The sponsor performed precision studies for 20 days, 2 runs per day in duplicate $(\mathrm{N} = 80)$ on drug-free negative urine samples spiked with Fentanyl to concentrations of $\pm 25\%$, $\pm 50\%$, $\pm 75\%$, and $\pm 100\%$ of the cutoff. Fentanyl concentrations in spiked samples were confirmed by mass spectrometry. Results were analyzed on a Beckman Coulter / Olympus AU400e Chemistry Analyzer. The data are summarized in the following tables: **Qualitative analysis (1.0 ng/mL cutoff)** | Concentration (ng/mL) | % of cutoff | Result | | --- | --- | --- | | 0 | -100 | 80 Neg / 0 Pos | | 0.25 | -75 | 80 Neg / 0 Pos | | 0.5 | -50 | 80 Neg / 0 Pos | | 0.75 | -25 | 80 Neg / 0 Pos | | 1.0 | Cutoff | 32 Neg / 48 Pos | | 1.25 | +25 | 80 Pos / 0 Neg | | 1.5 | +50 | 80 Pos / 0 Neg | | 1.75 | +75 | 80 Pos / 0 Neg | | 2.0 | +100 | 80 Pos / 0 Neg | #### b. Linearity/assay reportable range: Not applicable, this device is intended for qualitative use only #### c. Traceability, Stability, Expected values (controls, calibrators, or methods): ##### Traceability The analytes in the calibrators are traceable to a commercially available standard solution. The standard is certified material with the concentration verified by GC-MS or LC/MS-MS. {5} 6 # Value Assignment/Expected Values The calibrators are prepared by spiking known concentrations of Fentanyl into a negative calibrator matrix. Concentrations are confirmed by GC/MS or LC/MS/MS. # Stability Real time, closed vial and on-board stability studies for calibrators were conducted. Stability protocols and acceptance criteria were reviewed and found to be acceptable, and support that, when stored at 2 – 8 °C, calibrators are stable for one year. The results support that, once opened, calibrators are stable for 60 days when stored at 2 – 8 °C. d. Detection limit: Not applicable. e. Analytical specificity: Cross reactivity from structurally related compounds was evaluated by testing drug-free urine samples spiked with these compounds. Each potential cross-reacting compound was spiked and evaluated independently, and each spiked sample was tested in singlicate. Compounds were tested at a high concentration, and if cross-reactivity was observed, were diluted to identify the lowest concentration that produced a positive result. These concentrations were used to determine the percent cross-reactivity. The compounds and concentrations tested, and the approximate cross-reactivity are provided in the table below. | Compound | Concentration Tested (ng/mL) | Result | Cross-Reactivity (%) | | --- | --- | --- | --- | | Fentanyl | 1 | Positive | 100 | | Butryl Fentanyl | 0.8 | Positive | 125 | | Acetyl Fentanyl | 1 | Positive | 100 | | Despropionyl Fentanyl | 40 | Positive | 2.5 | | Sufentanil | 175 | Negative | <0.5714 | | Norfentanyl | 20,000 | Positive | 0.005 | | 6-Acetyl Morphine | 100,000 | Negative | <0.0010 | | Codeine | 100,000 | Negative | <0.0010 | | Methadone | 100,000 | Negative | <0.0010 | | Hydrocodone | 100,000 | Negative | <0.0010 | | Hydromorphone | 100,000 | Negative | <0.0010 | | Oxycodone | 100,000 | Negative | <0.0010 | | Morphine | 100,000 | Negative | <0.0010 | | Morphine-3-glucuronide | 100,000 | Negative | <0.0010 | {6} | Compound | Concentration Tested (ng/mL) | Result | Cross-Reactivity (%) | | --- | --- | --- | --- | | Ethylmorphine | | | | | EDDP | 100,000 | Negative | <0.0010 | | EMDP | 100,000 | Negative | <0.0010 | | Levorphanol | 100,000 | Negative | <0.0010 | | Oxymorphone | 100,000 | Negative | <0.0010 | | Tramadol | 100,000 | Negative | <0.0010 | | Nalorphine | 100,000 | Negative | <0.0010 | | Naloxone | 100,000 | Negative | <0.0010 | | Naltrexone | 100,000 | Negative | <0.0010 | | Normorphine | 100,000 | Negative | <0.0010 | | 6-Acetyl Codeine | 100,000 | Negative | <0.0010 | | Dihydrocodeine | 100,000 | Negative | <0.0010 | | Diacetyl Morphine | 100,000 | Negative | <0.0010 | | Pentazocine | 75,000 | Positive | 0.0013 | | Meperidine | 100,000 | Negative | <0.0010 | | Morphine-6-glucuronide | 100,000 | Negative | <0.0010 | | Buprenorphine | 100,000 | Negative | <0.0010 | | Norcodeine | 100,000 | Negative | <0.0010 | | Propoxyphene | 100,000 | Negative | <0.0010 | | Labetalol | 15,000 | Positive | 0.0067 | | Nordiazepam | 100,000 | Negative | <0.0010 | | Trimethoprim | 100,000 | Negative | <0.0010 | | Fluoxetine | 60,000 | Positive | 0.0017 | | Amitryptyline | 75,000 | Positive | 0.0013 | | Doxepin | 100,000 | Positive | 0.001 | | Nortriptyline | 100,000 | Positive | 0.001 | | Protryptyline | 100,000 | Negative | <0.0010 | | Trimipramine | 100,000 | Negative | <0.0010 | | Buproprion | 100,000 | Negative | <0.0010 | | Trazodone | 10,000 | Positive | 0.01 | | Clomipramine | 45,000 | Positive | 0.0022 | | Desipramine | 100,000 | Positive | 0.001 | | Imipramine | 100,000 | Positive | 0.001 | | Haloperidol | 1,250 | Positive | 0.08 | | Pipamperone | 1,500 | Positive | 0.0667 | | Risperidone | 2,500 | Positive | 0.04 | | Chlorpromazine | 75,000 | Positive | 0.0013 | | Meta-chlorphenyl piperazine | 100,000 | Positive | 0.001 | | Venlafaxine | 100,000 | Negative | <0.0010 | | PCP | 100,000 | Positive | 0.001 | | Diphenhydramine | 100,000 | Positive | 0.001 | {7} | Compound | Concentration Tested (ng/mL) | Result | Cross-Reactivity (%) | | --- | --- | --- | --- | | Methamphetamine | 70,000 | Positive | 0.0014 | | Benzylpiperazine | 50,000 | Positive | 0.002 | | Fenfluramine | 60,000 | Positive | 0.0017 | | Cyclobenzaprine | 100,000 | Positive | 0.001 | Potential interference from endogenous substances was evaluated by spiking these substances into drug free urine containing fentanyl at $\pm 50\%$ of the 1.0 cutoff (0.5 ng/mL and 1.5 ng/mL). The following endogenous substances, at the concentrations listed below, did not interfere with the assay: | Compound | Concentration Tested | | --- | --- | | Acetone | 1.0 g/dL | | Ascorbic Acid | 0.56 g/dL | | Bilirubin | 2.0 mg/dL | | Creatinine | 0.5 g/dL | | Ethanol | 1.0 g/dL | | Galactose | 10 mg /dL | | γ-Globulin | 0.5 g/dL | | Glucose | 2.0 g/dL | | Hemoglobin | 0.5 g/dL | | Human Serum Albumin | 0.5 g/dL | | Oxalic Acid | 0.1 g/dL | | Riboflavin | 7.5 mg/dL | | Sodium Azide | 1% w/v | | Sodium Chloride | 6.0 g/dL | | Sodium Fluoride | 1% w/v | | Urea | 2.0 g/dL | Boric acid at a concentration of $1\%$ w/v was evaluated by spiking the potential interferent into drug free urine containing Fentanyl at $\pm 50\%$ of the cutoff (0.5 ng/mL and 1.5 ng/mL). Effect of pH: The sponsor evaluated the effect of pH using drug free urine containing Fentanyl at $\pm 50\%$ of the cutoff (0.5 ng/mL and 1.5 ng/mL). The pH was adjusted using hydrochloric acid or sodium hydroxide. pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 did not interfere with the test. Effect of specific gravity: The sponsor evaluated the effect of specific gravity on the test results using drug free urine containing Fentanyl at $\pm 50\%$ of the cutoff (0.5 ng/mL and $1.5~\mathrm{ng / mL}$ ) that were adjusted using salt or albumin. Specific Gravity values of 1.000, 1.002, 1.005, 1.010, 1.015, 1.020, 1.025 and 1.030 did not interfere with the test. {8} Potential interference from non-structurally related compounds were evaluated by spiking these compounds into drug free urine containing fentanyl at $\pm 50\%$ of the 1.0 cutoff (0.5 ng/mL and 1.5ng/mL). The following non-structurally related substances at the concentrations listed below did not interfere with the assay: | Compound | Concentration Tested (ng/mL) | | --- | --- | | 11-hydroxy-delta-9-THC | 75,000 | | 11-nor-9 carboxy THC | 100,000 | | 1S, 2R(+)-Ephedrine | 100,000 | | 7-Aminoclonazepam | 100,000 | | Aminoflunitrazepam | 100,000 | | 7-Aminonitrazepam | 100,000 | | Acetaminophen | 500,000 | | Amobarbital | 100,000 | | Barbital | 100,000 | | Benzoylecgonine | 100,000 | | Bromazepam | 100,000 | | Butabarbital | 100,000 | | Caffeine | 100,000 | | Cannabidiol | 100,000 | | Cannabinol | 75,000 | | Carbamazepine | 100,000 | | Carisoprodol | 100,000 | | Chlordiazepoxide | 100,000 | | cis-Tramadol | 100,000 | | Clobazam | 100,000 | | Clonazepam | 100,000 | | Cotenine | 100,000 | | Delta-9-THC | 100,000 | | Demoxepam | 100,000 | | Ecgonine | 100,000 | | Ecgonine methyl ester | 100,000 | | Ethylbeta-D glucuronide | 100,000 | | Flunitrazepam | 100,000 | | Heroin | 100,000 | | Hexobarbital | 100,000 | | Ibuprofen | 100,000 | | Ketamine | 100,000 | | Lamotrigine | 100,000 | | Lidocaine | 100,000 | | Lorazepam Glucuronide | 50,000 | {9} | Compound | Concentration Tested (ng/mL) | | --- | --- | | LSD | 100,000 | | Mephobarbital | 100,000 | | Methaquolone | 100,000 | | Naproxen | 100,000 | | Nitrazepam | 100,000 | | Normorphine | 100,000 | | Norpseudoephedrine | 100,000 | | Oxazepam | 100,000 | | Pentobarbital | 100,000 | | Phenobarbital | 100,000 | | Phenylephedrine | 100,000 | | Salicylic Acid | 100,000 | | Secobarbital | 100,000 | | Temazepam | 100,000 | | Phenytoin | 100,000 | | PMA | 100,000 | | Propranolol | 100,000 | | (+)-MDA | 75,000 | | 4-Bromo-2,5,Dimethoxyphenethylamine | 75,000 | | Desalkyflurazepam | 75,000 | | Dextromethorphan | 75,000 | | Diazepam | 75,000 | | Flurazepam | 75,000 | | Lorazepam | 75,000 | | Lormetazepam | 75,000 | | Maprotiline | 75,000 | | Medezapam | 75,000 | | Meprobamate | 75,000 | | Methyphenidate | 75,000 | | Midazolam | 75,000 | | N Desmethyltapentadol | 75,000 | | Oxazepam glucuronide | 75,000 | | Phentermine | 75,000 | | Phenylpropanolamine | 75,000 | | R,R(-)-Pseudoephedrine- | 75,000 | | Ranitidine | 75,000 | | Ritalinic Acid | 75,000 | | Sertraline | 75,000 | 10 {10} | Compound | Concentration Tested (ng/mL) | | --- | --- | | Theophylline | 75,000 | | Thioridazine | 75,000 | | Zolpidem Tartrate | 75,000 | | Cocaine | 40,000 | | MDEA | 50,000 | | MDMA | 50,000 | | S-(+) Amphetamine | 50,000 | | Triazolam | 50,000 | | Trifluoromethylphenyl-piperazine | 40,000 | f. Assay cut-off: Analytical performance of the device around the claimed cutoff is described in the precision section M.1.a. above. 2. Comparison studies: a. Method comparison with predicate device: A method comparison study was performed using unaltered, clinical urine samples obtained from clinical testing laboratories. A total of 80 samples were analyzed in singlicate on a Beckman Coulter AU400e Chemistry Analyzer and the result was compared to that obtained by liquid chromatography/mass spectroscopy (LC/MS). The results of the assay performance compared to LC/MS are summarized below: | Candidate Device Result | Fentanyl Concentration by LC/MS (ng/mL) | | | | | --- | --- | --- | --- | --- | | | < 0.5 <50% | 0.5-0.9 ≥50% <100% | 1.0-1.5 ≥100% ≤150% | > 1.5 >150% | | POS | 0 | 1* | 9 | 31 | | NEG | 30 | 9 | 0 | 0 | *Discordant sample: One sample was determined as positive by the candidate device, whereas this sample was determined to be negative by the confirmatory LC/MS method (0.9 ng/mL). The sponsor conducted a supplemental method comparison study, which included twenty (20) subjects who were prescribed fentanyl and twenty (20) subjects who were suspected fentanyl abuse users. The urine samples from all study subjects were determined to be positive for fentanyl using the candidate device. Subject urine concentrations, as determined by LC/MS, ranged from 1.0 to 367 ng/mL for the {11} prescription fentanyl users and from 0.12 to 385 ng/mL for the suspected fentanyl abuse users. Two (2) of the twenty (20) subjects in the suspected fentanyl abuser subpopulation were determined as false positive*. *Discordant samples: The first sample was confirmed negative for fentanyl by LC/MS (fentanyl concentration: 0.12 ng/mL). Further analysis identified despropionyl fentanyl at a concentration consistent with a positive result for the candidate device, as determined by their cross-reactivity study (despropionyl fentanyl concentration: 46.6 ng/mL) The second sample was confirmed negative for fentanyl by LC/MS (fentanyl concentration: 0.17 ng/mL). Further analysis identified acetyl fentanyl at a concentration consistent with a positive result for the candidate device, as determined by their cross-reactivity study (acetyl fentanyl concentration: 1.03 ng/mL) b. Matrix comparison: Not applicable. This device is intended to be used with urine samples only. 3. Clinical studies: a. Clinical Sensitivity: Not applicable. b. Clinical specificity: Not applicable. c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. 4. Clinical cut-off: Not applicable. 5. Expected values/Reference range: Not applicable. N. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. 12 {12} O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 13