Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a standard enzyme immunoassay based on chemical reactions and spectrophotometric measurement, with no mention of AI or ML algorithms for data analysis or interpretation.

Therapeutic

No

This device is an in vitro diagnostic assay used to determine the presence of tricyclic antidepressants, not for treatment.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device is "intended for the qualitative and/or semiquantitative determination of the presence of tricyclic antidepressants (TCAs) in human serum, plasma, or urine of patients at a cutoff concentration of 300 ng/mL in patients suspected of drug overdose," and "For In Vitro Diagnostic Use Only." These statements indicate its use in diagnosing or aiding in the diagnosis of a patient's condition.

SaMD (Software as a Medical Device)

No

The device description clearly states it is a homogeneous enzyme immunoassay using ready-to-use liquid reagents (Reagent A and Reagent E) and involves spectrophotometric measurement at 340 nm, indicating it is a hardware-based in vitro diagnostic device, not software only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicit Statement: The document explicitly states "For In Vitro Diagnostic Use Only."
Intended Use: The intended use is for the qualitative and/or semiquantitative determination of tricyclic antidepressants in human serum, plasma, or urine. This involves testing samples taken from the human body, but performed outside the body (in vitro).
Device Description: The device is a homogeneous enzyme immunoassay using reagents to analyze biological samples. This is a typical method for in vitro diagnostic tests.
Performance Studies: The document details performance studies conducted on human serum, plasma, and urine samples, which is standard for validating an IVD.
Predicate Device: The mention of a predicate device (K983268; Tricyclic Serum Tox EIA Assay) further indicates that this device is being compared to an existing IVD.

All these factors align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semiquantitative determination of the presence of tricyclic antidepressants (TCAs) in human serum, plasma, or urine of patients at a cutoff concentration of 300 ng/mL in patients suspected of drug overdose.

The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical test result. A more specific alternative chemical must be used to obtain a confirmed analytical result. Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) is the preferred confirmatory method.

Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

For In Vitro Diagnostic Use Only.

Product codes

LFH

Device Description

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay using ready to-use liquid reagents. Specific tricyclic antibodies were used to detect most tricyclic antidepressants in serum, plasma, or urine. The test is based on the competition of an enzyme, glucose-6-phosphate dehydrogenase (G6PDH), labeled-drug and the drug from the sample for a fixed amount of specific antibody binding sites. In the absence of the drug from the sample, the specific antibody binds the enzyme-labeled drug and the enzyme activity is inhibited. This phenomenon creates a direct relationship between drug concentration in the sample and the enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.

The DRI™ Tricyclics Serum Tox assay is supplied as a two liquid reagent kit (Reagent A and Reagent E). Thev are bottled separately within the same kit, see details below:

Antibody/Substrate Reagent (Reagent A): Contains polyclonal anti-tricyclics . antibodies (sheep), glucose-6- phosphate (G6P), and nicotinamide adenine dinucleotide (NAD) in Tris buffer with sodium azide as a preservative.
Enzyme Conjugate Reagent (Reagent E): Contains glucose-6-phosphate ● dehydrogenase (G6PDH) labeled with nortriptyline in Tris buffer with sodium azide as a preservative.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Trained professionals

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance:
The performance was evaluated on Architect c4000 clinical chemistry analyzer.

a) Precision
Precision studies were performed in accordance with CLSI Guideline EP05-A3.
Samples were prepared by spiking Nortriptyline in both drug-free serum and drug-free urine at the cutoff, 25%, 50%, 75%, and 100% above and below the cutoff.

Repeatability:
The study was conducted for 20 days with 2 runs per day, 2 replicates per run in both qualitative and semi-quantitative modes with 1 lot of reagent, calibrators, and controls. There were 80 replicates for each level of precision samples. Serum and urine samples were run separately.
- Serum:
  - 300 ng/mL cutoff (100% of cutoff):
    - Qualitative: 10/70 (Negative/Positive)
    - Semi-Quantitative: 19/61 (Negative/Positive)
  - For values -100% to -25% of cutoff (0-225 ng/mL), all 80 replicates were found Negative in both qualitative and semi-quantitative modes.
  - For values +25% to +100% of cutoff (375-600 ng/mL), all 80 replicates were found Positive in both qualitative and semi-quantitative modes.
- Urine:
  - 300 ng/mL cutoff (100% of cutoff):
    - Qualitative: 68/12 (Negative/Positive)
    - Semi-Quantitative: 73/7 (Negative/Positive)
  - For values -100% to -25% of cutoff (0-225 ng/mL), all 80 replicates were found Negative in both qualitative and semi-quantitative modes.
  - For values +25% to +100% of cutoff (375-600 ng/mL), all 80 replicates were found Positive in both qualitative and semi-quantitative modes.
Reproducibility:
The study was conducted for 5 days with 1 run per day, 5 replicates per run in both qualitative and semi-quantitative modes with 2 lots of reagents on 3 different instruments. Serum and urine samples were run separately.
- Serum:
  - 300 ng/mL cutoff (100% of cutoff):
    - Qualitative: 30/45 (Negative/Positive)
    - Semi-quantitative: 29/46 (Negative/Positive)
  - For values -100% to -25% of cutoff (0-225 ng/mL), all 75 replicates were found Negative in both qualitative and semi-quantitative modes.
  - For values +25% to +100% of cutoff (375-600 ng/mL), all 75 replicates were found Positive in both qualitative and semi-quantitative modes.
- Urine:
  - 300 ng/mL cutoff (100% of cutoff):
    - Qualitative: 61/14 (Negative/Positive)
    - Semi-quantitative: 66/9 (Negative/Positive)
  - For values -100% to -25% of cutoff (0-225 ng/mL), all 75 replicates were found Negative in both qualitative and semi-quantitative modes.
  - For values +25% to +100% of cutoff (375-600 ng/mL), all 75 replicates were found Positive in both qualitative and semi-quantitative modes.

b) Spike Recovery
The spike recovery study was performed using 20 replicates. Samples were prepared by spiking Nortriptyline in both drug-free serum and drug-free urine at 225 ng/mL, 300 ng/mL, 375 ng/mL. The study demonstrated the accuracy of spike recovery at low control (225 ng/mL) and high control (375 ng/mL) in both qualitative and semiquantitative mode. Serum and urine samples were run separately.

Serum: All 20 replicates at low control (225 ng/mL) were Negative. All 20 replicates at high control (375 ng/mL) were Positive. No overlap observed.
Urine: All 20 replicates at low control (225 ng/mL) were Negative. All 20 replicates at high control (375 ng/mL) were Positive. No overlap observed.

c) Dilution Linearity
The Dilution Linearity study is performed using the CLSI guideline CLSI EP06-A.
Drug-free serum or drug-free urine were spiked using Nortriptyline and diluted with drug-free serum or drug-free urine, respectively to generate 9 levels between 0 and 1000 ng/mL. Each sample was run in replicates of 5 in semi-quantitative mode and the average was used to determine percent recovery compared to the expected target value. Serum and urine samples were run separately.

Serum: Recovery ranged from 96% to 115% for spiked concentrations from 125 ng/mL to 1000 ng/mL.
Urine: Recovery ranged from 94% to 118% for spiked concentrations from 125 ng/mL to 1000 ng/mL.

d) Method Comparison and Accuracy
The method comparison study was performed in accordance with CLSI guideline CLSI EP09c-A3.
One (1) replicate of each of the 61 negative patient samples for serum and 50 negative samples for urine and 56 positive patient samples for serum and 50 positive samples for urine were analyzed in both qualitative and semi-quantitative modes. The results were compared to LC-MS/MS lab testing.

Serum (Qualitative Mode):
- 61 negative patient samples, 56 positive patient samples.
- % Negative Sample Agreement: 98% (60/61)
- % Positive Sample Agreement: 100% (56/56)
- % Total Sample Agreement: 99% (116/117)
- Discordant Samples: 2 samples were positive by immunoassay but negative by LC-MS/MS, or positive by LC-MS/MS and negative by immunoassay.
Serum (Semi-Quantitative Mode):
- 61 negative patient samples, 56 positive patient samples.
- % Negative Sample Agreement: 97% (59/61)
- % Positive Sample Agreement: 100% (56/56)
- % Total Sample Agreement: 98% (115/117)
- Discordant Samples: 2 samples were positive by immunoassay but negative by LC-MS/MS, or positive by LC-MS/MS and negative by immunoassay.
Urine (Qualitative Mode):
- 50 negative patient samples, 50 positive patient samples.
- % Negative Sample Agreement: 96% (48/50)
- % Positive Sample Agreement: 98% (49/50)
- % Total Sample Agreement: 97% (97/100)
- Discordant Samples: 3 samples showed discordance between immunoassay and LC-MS/MS results.
Urine (Semi-Quantitative Mode):
- 50 negative patient samples, 50 positive patient samples.
- % Negative Sample Agreement: 96% (48/50)
- % Positive Sample Agreement: 98% (49/50)
- % Total Sample Agreement: 97% (97/100)
- Discordant Samples: 3 samples showed discordance between immunoassay and LC-MS/MS results.

e) Matrix equivalency
Matrix Equivalency is performed in accordance with CLSI Guideline CLSI-EP35.
Two (2) replicates of 50 patient samples of K2 EDTA Plasma, K3 EDTA plasma, Lithium Heparin Plasma, Sodium Citrate Plasma, Potassium Oxalate Plasma and 45 patient samples of Sodium Heparin Plasma were run.

Results: All tested matrices (K2 EDTA Plasma, K3 EDTA plasma, Lithium Heparin Plasma, Sodium Citrate Plasma, Potassium Oxalate Plasma, Sodium Heparin Plasma) showed 100% agreement with serum matrix across both qualitative and semi-quantitative modes for both positive and negative samples.

f) Specificity

Cross-Reactivity to structurally related compounds: Evaluated by adding known amounts of compounds into drug-free serum and urine.
- Serum: Various TCA drugs and metabolites (e.g., Amitriptyline, Clomipramine, Desipramine, Imipramine, Nortriptyline) showed cross-reactivity ranging from 0.2% to 157.9%, with many at 100% or above at tested concentrations. Other structurally related compounds (e.g., Chlorpromazine, Cyclobenzaprine) showed cross-reactivity from

Regulation Number and Section

§ 862.3910 Tricyclic antidepressant drugs test system.

(a)
Identification. A tricyclic antidepressant drugs test system is a device intended to measure any of the tricyclic antidepressant drugs in serum. The tricyclic antidepressant drugs include imipramine, desipramine, amitriptyline, nortriptyline, protriptyline, and doxepin. Measurements obtained by this device are used in the diagnosis and treatment of chronic depression to ensure appropriate therapy.(b)
Classification. Class II (special controls). A tricyclic antidepressant drugs 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).

Summary

0

Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health and Human Services logo on the left and the FDA text logo on the right. The FDA text logo is in blue and includes the acronym "FDA" followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in a stacked format.

December 21, 2022

Microgenics Corporation Pranjali Shinde Senior Regulatory Affairs Specialist 46500 Kato Road Fremont, CA 94538

Re: K213875

Trade/Device Name: DRI™ Tricyclics Serum Tox Assay Regulation Number: 21 CFR 862.3910 Regulation Name: Tricyclic antidepressant drugs test system Regulatory Class: Class II Product Code: LFH Dated: August 19, 2022 Received: August 23, 2022

Dear Pranjali Shinde:

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 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR

1

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.

Paula --Digitally signed by Caposino - Date: 2022.12.21 Paula Caposino -S 11:51:25 -05'00'

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 Ouality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K213875

Device Name DRI™ Tricyclics Serum Tox Assay

Indications for Use (Describe)

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semiquantitative determination of the presence of tricyclic antidepressants (TCAs) in human serum, plasma, or urine of patients at a cutoff concentration of 300 ng/mL in patients suspected of drug overdose.

The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical test result. A more specific alternative chemical must be used to obtain a confirmed analytical result. Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) is the preferred confirmatory method.

Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

For In Vitro Diagnostic Use Only.

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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3

510(k) Summary

510(k) Number: K213875

This 510(k) Summary of Safety and Effectiveness is being submitted in accordance with the requirements of Safe Medical Device Act of 1990 and 21 CFR 807.92.

A. Device Information

| Sponsor: | Microgenics Corporation
Thermo Fisher Scientific
46500 Kato Road
Fremont, CA 94538
Phone: 510-979-5000
FAX: 510-979-5002 |
|----------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Correspondent Contact
Information: | Pranjali Shinde
Microgenics Corporation
Thermo Fisher Scientific
46500 Kato Road
Fremont, CA-94538
Email: Pranjali.Shinde@Thermofisher.com
Phone: 213-344-9952
Fax: 510-979-5002 |
| Device Common Name: | Tricyclics Serum Tox Assay |
| Trade or Proprietary Name | DRI™ Tricyclics Serum Tox Assay |
| Brand Name | DRI™ Tricyclics Serum Tox Assay |
| Candidate Device Product
Code, Classification,
Classification Name & Panel | LFH, Class II,
21 CFR 862. 3910 – Tricyclic antidepressant drugs test
system, 91 - Toxicology |

Predicate Device Information

Predicate Device:	Tricyclic Serum Tox EIA Assay
Predicate Device Manufacturer:	Microgenics Corporation
Predicate Device Common Name	Tricyclic Serum Tox Assay
Predicate Device Premarket Notification #:	K983268
Predicate Device Product Code, Classification, Classification Name & Panel	LFH, Class II,
21 CFR 862. 3910 – Tricyclic antidepressant drugs test
system, 91 - Toxicology

B. Date Summary Prepared

November 14, 2022

4

C. Description of Device

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay using ready to-use liquid reagents. Specific tricyclic antibodies were used to detect most tricyclic antidepressants in serum, plasma, or urine. The test is based on the competition of an enzyme, glucose-6-phosphate dehydrogenase (G6PDH), labeled-drug and the drug from the sample for a fixed amount of specific antibody binding sites. In the absence of the drug from the sample, the specific antibody binds the enzyme-labeled drug and the enzyme activity is inhibited. This phenomenon creates a direct relationship between drug concentration in the sample and the enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.

The DRI™ Tricyclics Serum Tox assay is supplied as a two liquid reagent kit (Reagent A and Reagent E). Thev are bottled separately within the same kit, see details below:

Antibody/Substrate Reagent (Reagent A): Contains polyclonal anti-tricyclics . antibodies (sheep), glucose-6- phosphate (G6P), and nicotinamide adenine dinucleotide (NAD) in Tris buffer with sodium azide as a preservative.
Enzyme Conjugate Reagent (Reagent E): Contains glucose-6-phosphate ● dehydrogenase (G6PDH) labeled with nortriptyline in Tris buffer with sodium azide as a preservative.

CI. Intended Use

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay intended for the qualitative and/or semiquantitative determination of the presence of tricyclic antidepressants (TCAs) in human serum, plasma, or urine of patients at a cutoff concentration of 300 ng/mL in patients suspected of drug overdose.

The semi-quantitative mode is for the purpose of enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) or permitting laboratories to establish quality control procedures.

The assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) is the preferred confirmatory method.

Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

For In Vitro Diagnostic Use Only.

5

E. Comparison to Predicate Device

| Characteristics | Candidate Device:
DRI™ Tricyclics Serum Tox Assay
(K213875) | Predicate Device:
Tricyclic Serum Tox EIA Assay
(K983268) |
|----------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------|
| Intended Use | The DRI™ Tricyclics Serum Tox
Assay is a homogeneous enzyme
immunoassay intended for the
qualitative and/or
semiquantitative determination of
the presence of tricyclic
antidepressants (TCAs) in human
serum, plasma, or urine of patients
at a cutoff concentration of 300
ng/mL in patients suspected of
drug overdose.

The semi-quantitative mode is for
the purpose of enabling
laboratories to determine an
appropriate dilution of the
specimen for confirmation by a
confirmatory method such as
Liquid Chromatography/Tandem
Mass Spectrometry (LC-MS/MS)
or permitting laboratories to
establish quality control
procedures.

The assay provides only a
preliminary analytical test result.
A more specific alternative
chemical method must be used to
obtain a confirmed analytical
result. Liquid
Chromatography/Tandem Mass
Spectrometry (LC-MS/MS) is the
preferred confirmatory method.

6

F. Test Principle

The DRI™ Tricyclics Serum Tox Assay is a homogeneous enzyme immunoassay using ready-to use liquid reagents. The assay uses specific tricyclic antibodies, which can detect most tricyclic antidepressants in serum, plasma, or urine. The DRI technology is based on the competition of an enzyme glucose-6-phosphate dehydrogenase (G6PDH) labeled-drug and the drug from the serum, plasma, or urine sample for a fixed amount of specific antibody binding sites. In the presence of free drug from the sample, the free drug occupies the antibody binding sites, allowing the drug-labeled G6PDH to interact with the substrate, resulting in enzyme activity. In the absence of drug from the sample, the specific antibody binds to the drug labeled with G6PDH and the enzyme activity is inhibited. This phenomenon creates a direct relationship between the drug concentration in the serum, plasma, or urine and the enzyme activity. The enzyme G6PDH activity is determined spectrophotometrically at 340 nm by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH.

7

G. Summary of Supporting Data

Analytical Performance:

The performance was evaluated on Architect c4000 clinical chemistry analyzer.

a) Precision
Precision studies were performed in accordance with CLSI Guideline EP05-A3.

Samples were prepared by spiking Nortriptyline in both drug-free serum and drug-free urine at the cutoff, 25%, 50%, 75%, and 100% above and below the cutoff.

Repeatability:

The study was conducted for 20 days with 2 runs per day, 2 replicates per run in both qualitative and semi-quantitative modes with 1 lot of reagent, calibrators, and controls. There were 80 replicates for each level of precision samples. Serum and urine samples were run separately. The results of the precision study (Repeatability) are presented in the table below.

Table 1: Precision study data (Repeatability) in qualitative and semi-quantitative mode for 300 ng/mL cutoff - Serum

| Spiked
Concentration
(ng/mL) | % of
Cutoff | # of
Determinants | Total Precision (n=80) | |
|------------------------------------|----------------|----------------------|-----------------------------------------------------------|-----------------------------------------------------------------|
| | | | Qualitative
Immunoassay Results
(Negative/Positive) | Semi-Quantitative
Immunoassay Results
(Negative/Positive) |
| 0 | -100 | 80 | 80/0 | 80/0 |
| 75 | -75 | 80 | 80/0 | 80/0 |
| 150 | -50 | 80 | 80/0 | 80/0 |
| 225 | -25 | 80 | 80/0 | 80/0 |
| 300 | 100 | 80 | 10/70 | 19/61 |
| 375 | +25 | 80 | 0/80 | 0/80 |
| 450 | +50 | 80 | 0/80 | 0/80 |
| 525 | +75 | 80 | 0/80 | 0/80 |
| 600 | +100 | 80 | 0/80 | 0/80 |

8

| Spiked
Concentration
(ng/mL) | % of
Cutoff | # of
Determinants | Total Precision (n=80) | |
|------------------------------------|----------------|----------------------|-----------------------------------------------------------|-----------------------------------------------------------------|
| | | | Qualitative
Immunoassay Results
(Negative/Positive) | Semi-Quantitative
Immunoassay Results
(Negative/Positive) |
| 0 | -100 | 80 | 80/0 | 80/0 |
| 75 | -75 | 80 | 80/0 | 80/0 |
| 150 | -50 | 80 | 80/0 | 80/0 |
| 225 | -25 | 80 | 80/0 | 80/0 |
| 300 | 100 | 80 | 68/12 | 73/7 |
| 375 | +25 | 80 | 0/80 | 0/80 |
| 450 | +50 | 80 | 0/80 | 0/80 |
| 525 | +75 | 80 | 0/80 | 0/80 |
| 600 | +100 | 80 | 0/80 | 0/80 |

Table 2: Precision study data (Repeatability) in qualitative and semi-quantitative mode for 300 ng/mL cutoff = Urine

Reproducibility:

The study was conducted for 5 days with 1 run per day, 5 replicates per run in both qualitative and semi-quantitative modes with 2 lots of reagents on 3 different instruments. Serum and urine samples were run separately. The results of the precision study (Reproducibility) are presented in the table below.

Table 3: Precision study data (Reproducibility) in qualitative and semi-quantitative mode for 300 ng/mL cutoff - Serum

| Spiked
Concentration
(ng/mL) | % of
Cutoff | # of
Determinants | Total Precision (n=75) | |
|------------------------------------|----------------|----------------------|-----------------------------------------------------------|-----------------------------------------------------------------|
| | | | Qualitative
Immunoassay Results
(Negative/Positive) | Semi-quantitative
Immunoassay Results
(Negative/Positive) |
| 0 | -100 | 75 | 75/0 | 75/0 |
| 75 | -75 | 75 | 75/0 | 75/0 |
| 150 | -50 | 75 | 75/0 | 75/0 |
| 225 | -25 | 75 | 75/0 | 75/0 |
| 300 | 100 | 75 | 30/45 | 29/46 |
| 375 | +25 | 75 | 0/75 | 0/75 |
| 450 | +50 | 75 | 0/75 | 0/75 |
| 525 | +75 | 75 | 0/75 | 0/75 |
| 600 | +100 | 75 | 0/75 | 0/75 |

9

Table 4: Precision study data (Reproducibility) in qualitative and semi-quantitative mode for 300 ng/mL cutoff - Urine

| Spiked
Concentration
(ng/mL) | % of
Cutoff | # of
Determinants | Total Precision (n=75) | |
|------------------------------------|----------------|----------------------|-----------------------------------------------------------|-----------------------------------------------------------------|
| | | | Qualitative
Immunoassay Results
(Negative/Positive) | Semi-quantitative
Immunoassay Results
(Negative/Positive) |
| 0 | -100 | 75 | 75/0 | 75/0 |
| 75 | -75 | 75 | 75/0 | 75/0 |
| 150 | -50 | 75 | 75/0 | 75/0 |
| 225 | -25 | 75 | 75/0 | 75/0 |
| 300 | 100 | 75 | 61/14 | 66/9 |
| 375 | +25 | 75 | 0/75 | 0/75 |
| 450 | +50 | 75 | 0/75 | 0/75 |
| 525 | +75 | 75 | 0/75 | 0/75 |
| 600 | +100 | 75 | 0/75 | 0/75 |

10

b) Spike Recovery
The spike recovery study was performed using 20 replicates. Samples were prepared by spiking Nortriptyline in both drug-free serum and drug-free urine at 225 ng/mL, 300 ng/mL, 375 ng/mL. The study demonstrated the accuracy of spike recovery at low control (225 ng/mL) and high control (375 ng/mL) in both qualitative and semiquantitative mode. Serum and urine samples were run separately. The results of the study are presented in the table below.

	Table 5: Spike Recovery in qualitative and semi-quantitative mode for 300 ng/mL cutoff -
Serum

| Replicates | (-25% of cutoff) (n=20) (+25% of cutoff)(n=20) |
|-----------------|--------- | 1 | Negative | 2 | Negative | 3 | Negative | 4 | Negative | 5 | Negative | 6 | Negative | 7 | Negative | 8 | Negative | 9 | Negative | 10 | Negative | 11 | Negative | 12 | Negative | 13 | Negative | 14 | Negative | 15 | Negative | 16 | Negative | 17 | Negative | 18 | Negative | 19 | Negative | 20 | Negative | Overlap | No | Relative to C/O | All 20 below C/O Low Control: 225 ng/mL
| High Control: 375 ng/mL
------------------------------------------|---------------------------------------------------|
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| Positive |
| No |
| All 20 above C/O |

Table 6: Recovery in qualitative and semi-quantitative mode for 300 ng/mL cutoff -Urine

| Replicates | Low Control: 225 ng/mL
-25% of cutoff (n=20) | High Control: 375 ng/mL
+25% of cutoff (n=20) |
|------------|-------------------------------------------------|--------------------------------------------------|
| 1 | Negative | Positive |
| 2 | Negative | Positive |
| 3 | Negative | Positive |
| 4 | Negative | Positive |

11

| Replicates | Low Control: 225 ng/mL
-25% of cutoff (n=20) | High Control: 375 ng/mL
+25% of cutoff (n=20) |
|-----------------|-------------------------------------------------|--------------------------------------------------|
| 5 | Negative | Positive |
| 6 | Negative | Positive |
| 7 | Negative | Positive |
| 8 | Negative | Positive |
| 9 | Negative | Positive |
| 10 | Negative | Positive |
| 11 | Negative | Positive |
| 12 | Negative | Positive |
| 13 | Negative | Positive |
| 14 | Negative | Positive |
| 15 | Negative | Positive |
| 16 | Negative | Positive |
| 17 | Negative | Positive |
| 18 | Negative | Positive |
| 19 | Negative | Positive |
| 20 | Negative | Positive |
| Overlap | No | No |
| Relative to C/O | All 20 below C/O | All 20 above C/O |

12

c) Dilution Linearity

The Dilution Linearity study is performed using the CLSI guideline CLSI EP06-A.

To demonstrate the dilution linearity for purposes of sample dilution and quality control of the entire assay range, drug-free serum or drug-free urine were spiked using Nortriptyline and diluted with drug-free serum or drug-free urine, respectively to generate 9 levels between 0 and 1000 ng/mL. Each sample was run in replicates of 5 in semi-quantitative mode and the average was used to determine percent recovery compared to the expected target value. Serum and urine samples were run separately. The study results are presented in the table below.

| Level

| Expected Nortriptyline

Values (ng/mL) | Serum | |
|------------|------------------------------------------|-------------------------|--------------|
| | | Observed Values (ng/mL) | Recovery (%) |
| 1 | 0 | 7 | N/A |
| 2 | 125 | 121 | 97 |
| 3 | 250 | 242 | 97 |
| 4 | 375 | 385 | 103 |
| 5 | 500 | 479 | 96 |
| 6 | 625 | 720 | 115 |
| 7 | 750 | 839 | 112 |
| 8 | 875 | 913 | 104 |
| 9 | 1000 | 1046 | 105 |

Table 7: Dilution linearity data - Serum

| Level

| Expected Nortriptyline

Values (ng/mL) | Urine | |
|------------|------------------------------------------|-------------------------|--------------|
| | | Observed Values (ng/mL) | Recovery (%) |
| 1 | 0 | 8 | N/A |
| 2 | 125 | 148 | 118 |
| 3 | 250 | 271 | 108 |
| 4 | 375 | 393 | 105 |
| 5 | 500 | 470 | 94 |
| 6 | 625 | 620 | 99 |
| 7 | 750 | 741 | 99 |
| 8 | 875 | 838 | 96 |
| 9 | 1000 | 946 | 95 |

Table 8: Dilution linearity data – Urine

13

d) Method Comparison and Accuracy
The method comparison study was performed in accordance with CLSI guideline CLSI EP09c-A3.

One (1) replicate of each of the 61 negative patient samples for serum and 50 negative samples for urine and 56 positive patient samples for serum and 50 positive samples for urine were analyzed in both qualitative and semi-quantitative modes. Serum and urine samples were run separately. The results were compared to LC-MS/MS lab testing.

Serum and urine samples were run separately. The results of the study are presented in the tables below.

Table 9: Stratified data comparing immunoassay in qualitative mode with LC-MS/MS –
Serum
				Near Cutoff
DRI
TCA ST
Assay
Results	Negative
by LC-
MS/MS	450
ng/mL)
Positive	0	11	0	31	25
Negative	1	35	24	0	0
% Negative Sample Agreement		98% or (60/61)
% Positive Sample Agreement		100% or (56/56)
% Total Sample Agreement		99% or (116/117)

1 Refer to Table 11 for discordant samples of serum.

Table 10: Stratified data comparing immunoassay in semi-quantitative mode with LC MS/MS -

Serum
DRI
TCA ST
Assay
Results	Negative
by LC-
MS/MS	450
ng/mL)
Positive	0	21	0	31	25
Negative	1	34	24	0	0
% Negative Sample Agreement			97% or (59/61)

14

% Positive Sample Agreement	100% or (56/56)
% Total Sample Agreement	98% or (115/117)

1 Refer to Table 11 for discordant samples of serum.

| Sample ID | Qualitative
(mA/min)
Result | Semi-Quantitative
Observed concentration
(ng/mL) | Final LC-MS/MS
Value
(ng/mL) |
|------------|-----------------------------------|--------------------------------------------------------|------------------------------------|
| APP9489-22 | Negative | 354 (Positive) | 110 |
| APP9474-23 | Positive | 418 (Positive) | 120 |

1Table 11: Discordant Samples - Serum
---------------------------------------

2 Sample APP9489-2 contains Amitriptyline at 8.08 ng/mL, Imipramine at 18.35 ng/mL, Desipramine at 46.35 ng/mL, and Nortriptyline at 17 ng/mL by LC-MS/MS and cross-reacts at 100%, 158%, 120%, and 100% by immunoassay, respectively.

3 Sample APP9474-2 contains Amitriptyline at 9.08 ng/mL, Imipramine at 19.9 ng/mL, Desipramine at 49.8 ng/mL, and Nortriptyline at 19.65 ng/mL by LC-MS/MS and cross-reacts at 100%, 158%, 120%, and 100% by immunoassay, respectively.

Note: In addition, both samples APP9489-2 and APP9474-2 were confirmed by LC-MS/MS to contain Carbamazepine and Carbamazepine Epoxide with concentrations (4275 ng/mL & 5285 ng/mL) and (595 ng/mL & 955 ng/mL), respectively.

Urine
DRI
TCA ST
Assay
Results	Negative
by LC-
MS/MS	450
ng/mL)
Positive	0	0	24	7	42
Negative	22	11	15	14	0
% Negative Sample Agreement		96% or (48/50)
% Positive Sample Agreement		98% or (49/50)
% Total Sample Agreement		97% or (97/100)

Table 12: Stratified data comparing immunoassay in qualitative mode with LC-MS/MS -I Irine

4 Refer to Table 14 for discordant samples of urine.

15

| DRI
TCA ST
Assay
Results | Negative
by LC-
MS/MS | 450
ng/mL) |
|-----------------------------------|-----------------------------|-------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------|
| Positive | 0 | 0 | 24 | 7 | 42 |
| Negative | 22 | 11 | 15 | 14 | 0 |
| % Negative Sample Agreement | | 96% or (48/50) | | | |
| % Positive Sample Agreement | | 98% or (49/50) | | | |
| % Total Sample Agreement | | 97% or (97/100) | | | |

Table 13: Stratified data comparing immunoassay in semi-quantitative mode with LC-MS/MS - Urine

4 Refer to Table 14 for discordant samples of urine.

4Table 14: Discordant Samples - Urine

| Sample ID | Qualitative (mA/min)
Result | Semi-Quantitative
Observed concentration
(ng/mL) | LC-MS/MS Value
(ng/mL) |
|------------|--------------------------------|--------------------------------------------------------|---------------------------|
| APP6058-25 | Positive | 347 (Positive) | 285 |
| APP6060-26 | Positive | 375 (Positive) | 230 |
| APP6056-27 | Negative | 267 (Negative) | 372 |

5 Sample APP6058-2 contains Amitriptyline at 146 ng/mL and Nortriptyline at 139 ng/mL by LC-MS/MS and cross-reacts at 100% and 100% by immunoassay, respectively.

6 Sample APP6060-2 contains Amitriptyline at 114 ng/mL, Nordoxepin at 3.05 ng/mL, and Nortriptyline at 115 ng/mL by LC-MS/MS and cross-reacts at 100%, 17.1% and 100% by immunoassay, respectively.

7 Sample APP6056-2 contains Amitriptyline at 186 ng/ mL and Nortriptyline at 186 ng/mL by LC-MS/MS and cross-reacts at 100% and 100% by immunoassay, respectively.

16

e) Matrix equivalency
Matrix Equivalency is performed in accordance with CLSI Guideline CLSI-EP35.

Two (2) replicates of 50 patient samples of K2 EDTA Plasma, K3 EDTA plasma, Lithium Heparin Plasma, Sodium Citrate Plasma, Potassium Oxalate Plasma and 45 patient samples of Sodium Heparin Plasma were run in both qualitative and semiquantitative modes to demonstrate Nortriptyline concentrations obtained in different test plasma matrices with different anticoagulants are equivalent to those measured in the primary or control matrix, serum. The results of the matrix equivalency study are presented in the table below.

		Qualitative		Semi-quantitative
Serum Matrix		Positive	Negative	Positive	Negative
K2 EDTA Plasma	Positive	25	0	25	0
	Negative	0	25	0	25
K3 EDTA plasma	Positive	25	0	25	0
	Negative	0	25	0	25
Lithium Heparin Plasma	Positive	25	0	25	0
	Negative	0	25	0	25
Sodium Citrate Plasma	Positive	25	0	25	0
	Negative	0	25	0	25
Potassium Oxalate Plasma	Positive	25	0	25	0
	Negative	0	25	0	25
Sodium Heparin Plasma	Positive	25	0	25	0
	Negative	0	20	0	20

17

f) Specificity

Cross-Reactivity to structurally related compounds:

The cross-reactivity of Tricyclic compounds and other structurally related compounds were evaluated by adding known amount of each compound into drug-free serum and drug-free urine at concentrations indicated. Serum and urine samples were run separately.

	Serum
Structurally Related Compounds
(TCA drugs and Metabolites)	Tested
Concentrations
(ng/mL)	Positive/
Negative	Cross-
Reactivity (%)
2-Hydroxy Imipramine	1,300	Positive	23.1
7-Hydroxy Quetiapine	100,000	Negative	7.8 are lethal. Varied urine pH is to support in case of sample adulteration.

Specific gravity for urine only:

The specific gravity of multiple negative urine samples from different donors were tested on available clinical chemistry analyzer to select ten (10) negative urine samples with specific gravity range from 1.004 to 1.030.

The selected negative urine samples with a specific gravity range from 1.004 to 1.030 were spiked at control levels of 225 ng/mL and 375ng/mL using intermediate drug stock at 10,000 ng/mL.

| Specific Gravity | Low Control
-25% of cutoff
(225 ng/mL) | High Control
+25% of cutoff
(375 ng/mL) |
|------------------|----------------------------------------------|-----------------------------------------------|
| 1.004 | Negative | Positive |
| 1.006 | Negative | Positive |
| 1.008 | Negative | Positive |
| 1.010 | Negative | Positive |
| 1.011 | Negative | Positive |
| 1.012 | Negative | Positive |
| 1.013 | Negative | Positive |
| 1.016 | Negative | Positive |
| 1.022 | Negative | Positive |
| 1.030 | Negative | Positive |

Specific Gravity	Number of Patients
1.001-1.010	10
1.011-1.020	25
1.021-1.030	15
>1.030	5

26

H. Conclusion

The information supports a determination of substantial equivalence between DRI™ Tricyclics Serum Tox Assay (K213875) and the predicate device Tricyclic Serum Tox EIA Assay (K983268).