Predicate Devices

Orasure Benzodiazepine Intercept MICRO-PLATE EIA

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard immunoassay and LC/MS/MS techniques, with no mention of AI or ML algorithms for data analysis or interpretation.

Therapeutic

No.
The device is an in vitro diagnostic device used for the qualitative detection of benzodiazepines in hair, not for therapeutic purposes.

Diagnostic

Yes
The "Intended Use / Indications for Use" section explicitly states, "The Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair."

SaMD (Software as a Medical Device)

No

The device description clearly outlines a multi-component system including a pre-analytical hair treatment procedure, a screening immunoassay with various reagents and a microplate reader, and a confirmation assay utilizing LC/MS/MS hardware. This is not a software-only device.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use/Indications for Use: The document explicitly states "The Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair." This directly identifies it as an IVD.
Nature of the Test: The device performs a test on a biological sample (hair) in vitro (outside the body) to detect the presence of a substance (benzodiazepines) for diagnostic purposes (in this case, for use in workplace settings).
Device Description: The description details the components and process of an immunoassay, which is a common type of in vitro diagnostic test.
Performance Studies: The document describes performance studies (precision, linearity, specificity, accuracy, method comparison) which are typical evaluations for IVD devices to demonstrate their analytical performance.

The information provided clearly aligns 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 Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair. The assay is intended for use in workplace settings for the qualitative analysis of human head and body hair. The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.
Psychemedics plans to perform this test at one site. Psychemedics has not performed an evaluation of reproducibility at different laboratories.
The Psychemedics Microplate EIA For Benzodiazepines in Hair provides only a preliminary analytical test result. A more specific alternate chemical method must be used to obtain a confirmed analytical result. Liquid Chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS) using deuterated internal standards in multiple reaction monitoring (MRM) mode is the confirmatory method used by Psychemedics Corporation.

Product codes

JXM

Device Description

The immunoassay consists of two parts; a pre-analytical hair treatment procedure (to convert the solid matrix of hair to a measurable liquid matrix) and the screening assay, the Psychemedics Microplate EIA for Benzodiazepines. The screening portion of the test system consists of microplate wells coated with Oxazepam conjugated to bovine serum albumin (BSA), the prepared hair sample, a cutoff calibrator added to the sample at a concentration of 1 ng Oxazepam/10 mg hair, monoclonal mouse anti-Oxazepam antibody, goat anti-mouse secondary antibody conjugated to HRP (horseradish peroxidase), substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify (and stop the reaction), and wash buffer for washing the plates. Absorbance in the wells is read with a microplate reader.
The confirmation assay consists of a AB Sciex API 3200 LC/MS/MS (Serial numbers AA24661109 and AA28841310) linked to two Shimazu LC-20AD Micro pumps and a Leap Technologies PAL autosampler.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

human head and body hair

Indicated Patient Age Range

Not Found

Intended User / Care Setting

workplace settings

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

Precision of the immunoassay screening method:

Intra-Assay Precision: Nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) in replicates of 15 within a single run.
Inter-Assay Precision: Nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) over the course of five days, in replicates of 15 per day (total 75 samples per concentration).

Precision of the LC-MS/MS method:

A study performed to evaluate the precision of triplicate analyses of authentic (not spiked) benzodiazepine-positive samples. All six benzodiazepines measured with the confirmation method were included. Each replicate measurement was made from a separate hair sample taken through the entire extraction process prior to analysis.

Linearity of the LC-MS/MS method:

Evaluated by spiking each of the benzodiazepine analytes (Alprazolam, Lorazepam, Diazepam, Nordiazepam, and Temazepam) into a negative hair digest matrix over the desired linear range of 0.05 to 20.0 ng/10 mg hair. Eight concentrations within this range plus a zero concentration sample were evaluated.

Detection Limit of the LC-MS/MS method:

A study performed to determine the Lower limit of Quantitation (LLOO) of the LC-MS/MS assay.

Specificity of the immunoassay screening method:

Cross-reactivity with structurally related compounds: Evaluated by spiking various concentrations of benzodiazepines into drug-free hair samples and comparing the result to the cutoff calibrator.
Interference from structurally unrelated compounds: 40 potentially interfering compounds were tested at a concentration of 100 ng/10 mg hair on samples spiked with oxazepam at the cutoff and at ± 50% of the cutoff.

Specificity of the LC-MS/MS method:

Cross-reactivity with structurally related compounds: Evaluated by measuring contrived samples containing Clonazepam, Estazolam, Flurazepam, Meprobromate, Midazolam, Nitrazepam, Prazepam, Triazolam, and Zolpidem.
Interference from structurally unrelated compounds: Evaluated by measuring contrived samples containing 31 compounds (e.g., Cotinine, Nicotine, Caffeine, Ibuprofen, Morphine, Cocaine).

Accuracy of the immunoassay screening method:

Method comparison studies were performed by comparing the results from 382 hair samples on the immunoassay screening method to the confirmatory LC/MS/MS method. The 382 samples included 318 head hair samples and 64 body hair samples.
The samples included various hair colors (159 black, 197 brown, 8 blond, 4 red, 14 gray) and ethnicities (175 Caucasian, 40 African-American, 125 Hispanic, 41 Asian).

Key results:

Immunoassay Precision: Reported in tables showing Negative/Positive rates for various concentrations relative to the cutoff for both intra-assay and inter-assay.
LC-MS/MS Precision: Detailed tables show mean, SD, and %CV for triplicate analyses of Alprazolam, Lorazepam, Diazepam, Nordiazepam, Oxazepam, and Temazepam in ng/10 mg hair for multiple samples. %CV values ranged from 0.91% to 13.12%.
LC-MS/MS Linearity: All conditions were met, and the linear range of 0.05 to 20.0 ng/10 mg hair was claimed.
LC-MS/MS Detection Limit: LLOO of 0.05 ng/10 mg hair for all six drugs was demonstrated.
Immunoassay Cross-reactivity for structurally related compounds: Listed for 27 compounds, ranging from 550% (Clobazam, Diazepam, Nimetazepam) to 1.500 ng).
- Positive results: 5 for 1.500 ng.
- Negative results: 233 for 1.500 ng.
- Discordant results were detailed, showing immunoassay positive results with LC/MS/MS concentrations below the cutoff or near cutoff (e.g., 0.092 ng Alprazolam, 0.493 ng Lorazepam, 0.308 ng Temazepam).

Key Metrics

Intra-Assay Precision: 15 replicates per concentration.
- Negative: 15/0 (Negative/Positive)
- 25%: 15/0
- 50%: 15/0
- 75%: 15/0
- Cutoff: 7/8
- 125%: 0/15
- 150%: 0/15
- 175%: 0/15
- 200%: 0/15
Inter-Assay Precision: 75 replicates per concentration (15 per day over 5 days).
- Negative: 75/0 (Positive/Negative)
- 25%: 75/0
- 50%: 75/0
- 75%: 75/0
- Cutoff: 43/32
- 125%: 0/75
- 150%: 0/75
- 175%: 0/75
- 200%: 0/75
LC-MS/MS Precision: %CV ranged from 0.91% to 13.12%.
LC-MS/MS Linearity: %CV for each level 0.995, mean of 5 replicates within +/- 15% of predicted value.
LC-MS/MS LLOO: 0.05 ng/10 mg hair for all six drugs.
Immunoassay Cross-reactivity: Reported as percent cross-reactivity and expected concentration equivalent to cutoff calibrator. Range from 550% (0.18 ng/10 mg hair) down to 300 ng/10 mg hair).

Predicate Device(s)

Orasure Benzodiazepine Intercept MICRO-PLATE EIA

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 862.3170 Benzodiazepine test system.

(a)
Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and urine. The benzodiazepine compounds include chlordiazepoxide, diazepam, oxazepam, chlorzepate, flurazepam, and nitrazepam. Measurements obtained by this device are used in the diagnosis and treatment of benzodiazepine use or overdose and in monitoring levels of benzodiazepines to ensure appropriate therapy.(b)
Classification. Class II (special controls). A benzodiazepine 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

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Image /page/0/Picture/1 description: The image shows the logo for the United States Department of Health and Human Services. The logo features a stylized caduceus, a symbol often associated with medicine and healthcare, with three intertwined snakes forming a wing-like shape. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the caduceus. The logo is presented in black and white.

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

September 15, 2017

PSYCHEMEDICS CORPORATION VIRGINIA HILL VICE PRESIDENT, RESEARCH AND DEVELOPMENT 5832 UPLANDER WAY CULVER CITY, CA 90230

Re: K163590

Trade/Device Name: Psychemedics Microplate EIA For Benzodiazepines in Hair Regulation Number: 21 CFR 862.3170 Regulation Name: Benzodiazepine test system Regulatory Class: II Product Code: JXM Dated: August 15, 2017 Received: August 15, 2017

Dear Ms. Hill:

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

1

electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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/Resourcesfor You/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,

Courtney H. Lias -S

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) K163590

Device Name

Psychemedics Microplate EIA for Benzodiazepines in Hair

Indications for Use (Describe)

The Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair. The assay is intended for use in workplace settings for the qualitative analysis of human head and body hair. The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.

Psychemedics plans to perform this test at one site. Psychemedics has not performed an evaluation of reproducibility at different laboratories.

The Psychemedics Microplate EIA For Benzodiazepines in Hair provides only a preliminary analytical test result. A more specific alternate chemical method must be used to obtain a confirmed analytical result. Liquid Chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS) using deuterated internal standards in multiple reaction monitoring (MRM) mode is the confirmatory method used by Psychemedics Corporation. This confirmatory method uses a cutoff of 0.2 ng of the identified benzodiazepine/10 mg hair.

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

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR § 807.92.

The assigned 510(k) number is: K163590

| Submitted By: | Psychemedics Corporation
5832 Uplander Way
Culver City, CA 90230
TEL: 310 216 7776
FAX: 310 216 6662 |
|-------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Submission Contact: | Virginia Hill |
| Date Prepared: | September 15, 2017 |
| Device Trade Name: | Psychemedics Microplate EIA for Benzodiazepines in Hair |
| Common/Usual Name: | Benzodiazepine Enzyme Immunoassay |
| Predicate Device: | Orasure Benzodiazepine Intercept MICRO-PLATE EIA |
| Product Code: | JXM |
| Device Classification and Name: | 21 CFR§862.3170, Benzodiazepine Test System, Class II |
| Intended Use: | The Psychemedics Microplate EIA For Benzodiazepines in Hair is
an in vitro diagnostic device for the qualitative detection of
benzodiazepines in hair. The assay is intended for use in workplace
settings for the qualitative analysis of human head and body hair.
The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.
Psychemedics plans to perform this test at one site. Psychemedics
has not performed an evaluation of reproducibility at different
laboratories.
The Psychemedics Microplate EIA For Benzodiazepines in Hair
provides only a preliminary analytical test result. A more specific
alternate chemical method must be used to obtain a confirmed
analytical result. Liquid Chromatography/Mass spectrometry/Mass
spectrometry (LC/MS/MS) using deuterated internal standards in
multiple reaction monitoring (MRM) mode is the confirmatory |
| | Psychemedics Corporation |
| Assay Description: | The immunoassay consists of two parts; a pre-analytical hair
treatment procedure (to convert the solid matrix of hair to a
measurable liquid matrix) and the screening assay, the
Psychemedics Microplate EIA for Benzodiazepines. The
screening portion of the test system consists of microplate wells
coated with Oxazepam conjugated to bovine serum albumin
(BSA), the prepared hair sample, a cutoff calibrator added to the
sample at a concentration of 1 ng Oxazepam/10 mg hair,
monoclonal mouse anti-Oxazepam antibody, goat anti-mouse
secondary antibody conjugated to HRP (horseradish peroxidase),
substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify
(and stop the reaction), and wash buffer for washing the plates.
Absorbance in the wells is read with a microplate reader. |
| | The confirmation assay consists of a AB Sciex API 3200
LC/MS/MS (Serial numbers AA24661109 and AA28841310)
linked to two Shimazu LC-20AD Micro pumps and a Leap
Technologies PAL autosampler. |
| Sample Collection
and Stability: | Psychemedics provides sample collection kits, which are
recommended but not required for sample collection. A sample of
hair should be cut as close as possible to the skin. The hair is
placed in a V-shaped aluminum foil sample holder with the root
end of the hair protruding beyond the slanted edge of the foil. The
aluminum foil is crimped around the sample, securing the hair
specimen firmly into place within the foil. The hair sample,
crimped within the foil, is placed in a sample acquisition card
envelope and the envelope is sealed with a tamper-evident seal.
Hair specimens are kept at ambient temperature in a secure
location until they are shipped without refrigeration to the
laboratory. Stability of benzodiazepines in hair samples stored at
room temperature has been shown to last for over one year.
Benzodiazepines in samples shipped coast-to-coast twice were
stable. |
| Materials required: | Hair sample collection kit Liquid Handling Equipment,
Microplate washer and reader AB Sciex API 3200 LC/MS/
MS (Serial numbers AA24661109 and AA28841310). |
| | BSA-Oxazepam coated Microplate
Cutoff Calibrator (.8 ng Oxazepam/37 uL) added to 8 mg hair
Controls (minus 50% of cutoff, and plus 100% of cutoff) |

4

5

Primary antibody against Oxazepam and other benzodiazepines HRP-labeled secondary antibody directed against the primary antibody species Substrate TMB (tetramethylbenzidine) Acidic stop solution (2 N HCl)

Comparison with Predicate:

Item	Proposed Device	Orasure OTI Benzodiazepine Assay
Indications/
Intended use	The Psychemedics Microplate
EIA for Benzodiazepines is an
enzyme immunoassay (EIA) for
the preliminary qualitative
detection of benzodiazepines in
human head and body hair using
an Oxazepam calibrator at 1 ng
/10 mg hair cutoff for identifying
benzodiazepine use. The
Psychemedics Microplate EIA
Benzodiazepine assay provides
only a preliminary analytical test
result. A more specific
alternative chemical method
must be used in order to obtain a
confirmed analytical result.
Liquid Chromatography / Mass
spectrometry /Mass
spectrometry (LC/MS/MS) using
deuterated internal standards in
multiple reaction monitoring
(MRM) mode is the
confirmatory method used by
Psychemedics Corporation.	The OTI Benzodiazepine test is
intended for use by clinical
laboratories in the qualitative
determination of benzodiazepines
in oral fluid using a 1 ng/mL
(Nordiazepam) cutoff. For in vitro
diagnostic use. This assay
provides only a preliminary
analytical test result. A more
specific alternative chemical
method must be used in order to
obtain a confirmed analytical
result. Gas Chromatography/Mass
Spectrometry/Mass Spectrometry
(GC/MS) is the preferred
confirmatory method.
Product Code	JXM	JXM
Measurand	Benzodiazepines in Hair	Benzodiazepines in Oral Fluid
Test System	Psychemedics Microplate EIA
for Benzodiazepines in Head and
Body Hair	Orasure OTI Benzodiazepine test
Sample Matrix	Human Hair	Human oral fluid
Method of Measurement	Microplate reader, read at 450
nm	Microplate reader, read at 450 nm
Cutoff	1 ng Oxazepam/10 mg hair	1 ng/mL Nordiazepam
Type of Test	Enzyme Immunoassay	Enzyme Immunoassay
Extraction Method	Patented Digestion method	Not applicable
Confirmation Method	LC/MS/MS	GC/MS/MS

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Summary of Performance Characteristics

Precision of the immunoassay screening method

Intra-Assay Precision was demonstrated by measuring nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) in replicates of 15 within a single run. Results are summarized below.

| Concentration relative
to cutoff (1 ng/10 mg

hair)	No. Samples	Negative / Positive
Negative	15	15/0
25%	15	15/0
50%	15	15/0
75%	15	15/0
Cutoff	15	7/8
125%	15	0/15
150%	15	0/15
175%	15	0/15
200%	15	0/15

Inter-Assay Precision of the assay was evaluated by measuring nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) over the course of five days, in replicates of 15 per day. Results are summarized below.

| Concentration relative
to cutoff (1 ng/10 mg

hair)	No. Samples	Positive / Negative
Negative	75	75/0
25%	75	75/0
50%	75	75/0
75%	75	75/0
Cutoff	75	43/32
125%	75	0/75
150%	75	0/75
175%	75	0/75
200%	75	0/75

Precision of the LC-MS/MS method

A study was performed to evaluate the precision of triplicate analyses of authentic (not spiked) benzodiazepine-positive samples. All six benzodiazepines measured with the confirmation method were included in the study. Each replicate measurement was made from a separate hair sample that was taken through the entire extraction process prior to analysis. Results for the six benzodiazepines measured by the LC-MS/MS assay are summarized below.

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Alprazolam (ng/10 mg hair)
Sample #	1	2	3	4	5
Rep 1	6.477	0.199	2.941	7.408	3.802
Rep 2	6.713	0.202	2.886	7.702	4.746
Rep 3	5.711	0.243	3.159	7.269	4.189
Mean	6.300	0.215	2.995	7.460	4.246
SD	0.524	0.025	0.144	0.221	0.475
%CV	8.31	11.45	4.82	2.96	11.18
Lorazepam (ng/10 mg hair)
Sample #	1	2	3	4
Rep 1	1.174	0.555	0.536	0.341
Rep 2	1.222	0.643	0.57	0.375
Rep 3	1.102	0.591	0.59	0.333
Mean	1.166	0.596	0.565	0.350
SD	0.060	0.044	0.027	0.022
%CV	5.18	7.42	4.83	6.38
Diazepam (ng/10 mg hair)
Sample #	1	2	3	4	5
Rep 1	0.229	8.365	1.417	4.642	0.77
Rep 2	0.235	8.443	1.572	3.75	0.903
Rep 3	0.205	8.519	1.215	3.929	0.818
Mean	0.223	8.442	1.401	4.107	0.830
SD	0.016	0.077	0.179	0.472	0.067
%CV	7.12	0.91	12.77	11.49	8.11
Nordiazepam (ng/10 mg hair)
Sample #	1	2	3	4
Rep 1	9.227	3.377	0.884	0.25
Rep 2	9.928	3.725	0.923	0.226
Rep 3	10.258	3.202	0.954	0.252
Mean	9.804	3.435	0.920	0.243
SD	0.527	0.266	0.035	0.014
%CV	5.37	7.75	3.81	5.96
Oxazepam (ng/10 mg hair)
Sample #	1	2	3	4
Rep 1	0.176	0.262	0.161	0.073
Rep 2	0.179	0.267	0.149	0.071
Rep 3	0.146	0.274	0.143	0.066
Mean	0.167	0.268	0.151	0.070
SD	0.018	0.006	0.009	0.004
%CV	10.82	2.25	6.07	5.15

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Temazepam (ng/10 mg hair)
Sample

| 1 | 2 | 3 | 4 | 5 |

| Rep 1 | 1.342 | 0.22 | 0.141 | 0.133 | 0.136 |
| Rep 2 | 1.327 | 0.22 | 0.147 | 0.129 | 0.139 |
| Rep 3 | 1.291 | 0.233 | 0.114 | 0.136 | 0.132 |
| Mean | 1.320 | 0.224 | 0.134 | 0.133 | 0.136 |
| SD | 0.026 | 0.008 | 0.018 | 0.004 | 0.004 |
| %CV | 1.99 | 3.35 | 13.12 | 2.65 | 2.59 |

Linearity of the LC-MS/MS method

The linearity of the confirmation method was evaluated by spiking each of the benzodiazepine analytes (Alprazolam, Lorazepam, Diazepam, Nordiazepam, and Temazepam) into a negative hair digest matrix over the desired linear range of 0.05 to 20.0 ng/10 mg hair. Eight concentrations within this range plus a zero concentration sample were evaluated. In order for the linear range to be considered validated, the following conditions needed to be met: the %CV for each level tested must be 10% or less, the concentrations tested must quantitate within ± 15% of the target value, the correlation coefficient must be >0.995, and the mean of the five replicates for each level must be within ± 15% of the predicted value based upon the linear regression line. All of these conditions were met, and the linear range of 0.05 to 20.0 ng/10 mg hair was claimed.

Detection Limit of the LC-MS/MS method

A study was performed to determine the Lower limit of Quantitation (LLOO) of the LC-MS/MS assay with acceptance criteria for analyte and blank response, and acceptable chromatography:

. The analyte response at the LLOO should be at least five times the response compared to blank response.
Analyte peak (response) should be identifiable, discrete, and reproducible, and the ● concentrations measured should have precision that does not exceed 20% of the CV and accuracy within 20% of the nominal concentration.

The acceptance criteria were satisfied, demonstrating an LLOO of 0.05 ng/10 mg hair for all six drugs.

Specificity of the immunoassay screening method

Cross-reactivity with structurally related compounds

The cross-reactivity characteristics of the screening immunoassay were evaluated by spiking various concentrations of benzodiazepines into drug-free hair samples and comparing the result to the cutoff calibrator. The table below lists the percent cross-reactivity and the approximate concentration of each compound required to produce a response approximately equivalent to the cutoff concentration of the assay.

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| Compound Cross-
reactivity | Expected
Concentration in
ng/10 mg hair
equivalent to cutoff
calibrator |
|--------------------------|---- | Clobazam | 550 | Diazepam | 550 | Nimetazepam | 550 | Estazolam | 400 | Temazepam | 312 | Alprazolam | 277 | Flunitrazepam | 222 | Nordiazepam | 220 | Nitrazepam | 220 | N-Desmethylflunitrazepam | 172 | Lormetazepam | 133 | Oxazepam | 100 | Midazolam | 42 | Clonazepam | 33 | Triazolam | 31 | alpha-Hydroxyalprazolam | 22 | Lorazepam | 13 | Bromazepam | 8.3 | Chlordiazepoxide | 4 | 7-Aminoflunitrazepam | 3.1 | Hydroxymethylflurazepam | 2 | Alpha-Hydroxytriazolam | 1.7 | desalkylflurazepam | 1 | 7-Aminonitrazepam | 0.6 | 7-aminoclonazepam | 300 | Flurazepam | 300 | Prazepam | 300 | Zolpidem Tartrate | 300 | Percent
-----------------------------|-------------------------------------------------------------------------------------|
| 0.18 |
| 0.18 |
| 0.18 |
| 0.25 |
| 0.32 |
| 0.36 |
| 0.45 |
| 0.45 |
| 0.45 |
| 0.58 |
| 0.75 |
| 1 |
| 2.4 |
| 3 |
| 3.2 |
| 4.5 |
| 7.5 |
| 12 |
| 25 |
| 32 |
| 50 |
| 60 |
| 100 |
| 170 |
|
|
|
|

Interference from structurally unrelated compounds

The following potentially interfering compounds were tested using the screening immunoassay on samples spiked with oxazepam at the cutoff and at ± 50% of the cutoff to assess possible positive or negative interference. All potential interferents listed below were tested at a concentration of 100 ng/10 mg hair. No positive or negative interference was observed in this study.

10

11-nor-9-Carboxy-delta-9-THC	Dextromethorphan	Naproxen
1S, 2R Ephedrine	Doxepin	Nicotine
5-hydroxyindole-3-acetic acid	Doxylamine succinate	Normetanephrine (+/-)
8(-)-11-nor-9-Carboxy-delta-9
THC	Ecgononine	Nortriptyline
Acetaminophen	Erythromycin	O-Desmethyvenlafaxine
Amitriptyline	Ethosuximide	Oxycodone
Amobarbital	Ethylmorphine	Penicillin G
Amoxicillin	Fentanyl	Pentazocine
Anhydroecgonine methyl ester	Haloperidol	Phendimetrazine
Atropine	Hexobarbital	Phendimetrazine bitartrate
Benzocaine	Hydrocodone	Phenmetrazine
Benzocaine	Hydromorphone	Phenobarbital
Buprenorphine	Ibuprofen	Procaine
Bupropion	Imipramine	Promethazine
Butabarbital	Lidocaine	Propanolol
Caffeine	LSD	Propoxyphene
Cannabinol	Meperidine	Protriptyline
Chlorpheniramine maleate	Mepivacaine	R-(-)Phenylephrine
Cis-tramadol HCl	Metanephrine (+/-)	R,R(-)-Pseudoephedrine
Codeine	Methadone (+/-)	Secobarbital
Cotinine	Methanol	Streptomycin
Synthetic cannabinoids CP 47,
497 (+/-)	Methaqualone	Synthetic cannabinoid AM2201
Delta 8-THC	Methyl phenidate	Synthetic cannabinoid HU-211
Desipramine	Morphine	Synthetic cannabinoids JWH-019,
081, 122, 200, and 250
Desmethyldoxepin	Nalorphine	Thioridazine
Desipramine	Naloxone	Trimipramine
Despropionyl fentanyl	Naltrexone	Vanilmandelic acid(+/-)

Specificity of the LC-MS/MS method

Cross-reactivity with structurally related compounds

The cross-reactivity characteristics of the LC-MS/MS confirmation assay were evaluated by measuring contrived samples containing the following structurally related compounds: Clonazepam, Estazolam, Flurazepam, Meprobromate, Midazolam, Nitrazepam, Prazepam, Triazolam, and Zolpidem. No cross-reactivity was observed for these compounds.

Interference from structurally unrelated compounds

The specificity of the LC-MS/MS confirmation assay with structurally unrelated compounds was evaluated by measuring contrived samples containing the following compounds: Cotinine,

11

Nicotine, Caffeine, Ibuprofen, Naproxen, Phentermine, Pseudoephedrine, Morphine, Hydrocodone, Oxycodone, Codeine, Cocaine, Phencyclidine, Methamphetamine, Methadone, Phenobarbital, Phenylephrine, Carbamazepine, Gabapentin, Salicylic Acid, Valproic Acid, Oxcarbazepine, Propoxyphene, Acetaminophen, Norfloxacin, Psilocybin, Nimesulide, Etifoxine, Chlorcyclizine, and Teriflunomide. No interference was observed for these compounds.

Accuracy of the immunoassay screening method

Method comparison studies were performed by comparing the results from 382 hair samples on the immunoassay screening method to the confirmatory LC/MS/MS method. The 382 samples included 318 head hair samples and 64 body hair samples.

The color of the hair samples included 159 black samples, 197 brown (from light brown to dark brown), 8 blond, 4 red, and 14 gray. The ethnicities represented included 175 Caucasian subjects, 40 African-American subjects, 125 Hispanic subjects, and 41 Asian subjects.

| Immunoassay
Screening
Test Result | Negative by
the predicate
device or less
than half the
cutoff
concentration
by
LC/MS/MS
analysis | Near Cutoff
Negative
(Between
50% below
the cutoff and
the cutoff
concentration) | Near Cutoff
Positive
(Between the
cutoff and
50% above
the cutoff
concentration) | High Positive
(greater than
50% above
the cutoff
concentration) |
|-----------------------------------------|--------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------|
| | (1.500 ng
oxazepam
equivalents/10
mg hair) |
| Positive | 5* | 19** | 17 | 107 |
| Negative | 233 | 1 | 0 | 0 |

Discordant Results

| Immunoassay
Result | LC/MS/MS
Result | Sum of
concentrations
based on cross-
reactivities |
|-----------------------|--------------------------------------------|-------------------------------------------------------------|
| POS* | 0.092 ng Alprazolam | 0.255 |
| POS* | 0.093 ng Alprazolam | 0.258 |
| POS* | 0.196 ng Nordiazepam | 0.431 |
| POS* | 0.156 ng Alprazolam | 0.432 |
| POS* | 0.111 ng Nordiazepam, 0.217
ng Oxazepam | 0.461 |
| POS** | 0.191 ng Alprazolam | 0.529 |
| POS** | 0.201 ng Alprazolam | 0.557 |
| POS** | 0.493 ng Lorazepam | 0.584 |
| POS** | 0.24 ng Alprazolam | 0.665 |

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| Immunoassay
Result | LC/MS/MS
Result | Sum of
concentrations
based on cross-
reactivities |
|-----------------------|----------------------------------------------|-------------------------------------------------------------|
| POS** | 0.121 ng Diazepam | 0.666 |
| POS** | 0.073 ng Diazepam, 0.133 ng
Nordiazepam | 0.694 |
| POS** | 0.266 ng Alprazolam | 0.737 |
| POS** | 0.267 ng Alprazolam | 0.74 |
| POS** | 0.267 ng Alprazolam | 0.74 |
| POS** | 0.272 ng Alprazolam | 0.753 |
| POS** | 0.347 ng Nordiazepam | 0.763 |
| POS** | 0.283 ng Alprazolam | 0.784 |
| POS** | 0.293 ng Nordiazepam, 0.158
ng Oxazepam | 0.803 |
| POS** | 0.099 ng Diazepam, 0.131 ng
Nordiazepam | 0.833 |
| POS** | 0.069 ng Alprazolam, 0.303 ng
Nordiazepam | 0.858 |
| POS** | 0.266 ng Alprazolam, 0.023 ng
Diazepam | 0.863 |
| POS** | 0.322 ng Alprazolam | 0.892 |
| POS** | 0.427 ng Nordiazepam | 0.939 |
| POS** | 0.308 ng Temazepam | 0.961 |

Conclusion: The Psychemedics Microplate EIA for Benzodiazepines in Hair is substantially equivalent to the predicate, based on performance studies including precision, specificity, interference, and accuracy.