Psychemedics Homogeneous Enzyme Immunoassay (HEIA) for Opiates in Hair, Psychemedics Homogeneous Enzyme Immunoassay (HEIA) for Oxycodone in Hair

K201326 · Psychemedics Corporation · DJG · Jul 27, 2021 · Clinical Toxicology

Device Facts

• Record ID: K201326
• Device Name: Psychemedics Homogeneous Enzyme Immunoassay (HEIA) for Opiates in Hair, Psychemedics Homogeneous Enzyme Immunoassay (HEIA) for Oxycodone in Hair
• Applicant: Psychemedics Corporation
• Product Code: DJG · Clinical Toxicology
• Decision Date: Jul 27, 2021
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3650
• Device Class: Class 2

Indications for Use

The Psychemedics homogeneous enzyme immunoassay (HEIA) for opiates is an enzyme immunoassay for the preliminary qualitative detection of opiates in human head and body hair using a morphine calibrator at 2 ng morphine/10 mg hair for the purpose of identifying opiate use. This is an in vitro diagnostic device intended exclusively for Psychemedics use only and is not intended for sale to anyone. The Psychemedics homogeneous enzyme immunoassay 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) is the preferred confirmatory method. The Psychemedics homogeneous enzyme immunoassay (HEIA) for oxycodone is an enzyme immunoassay for the preliminary qualitative detection of oxycodone in human head and body hair using an oxycodone calibrator at 2 ng oxycodone/10 mg hair for the purpose of identifying opioid use. This is an in vitro diagnostic device intended exclusively for Psychemedics use only and is not intended for sale to anyone. The Psychemedics homogeneous enzyme immunoassay 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) is the preferred confirmatory method.

Device Story

Device performs preliminary qualitative screening for opiates or oxycodone in human hair; intended for use exclusively by Psychemedics laboratory. Process involves pre-analytical hair treatment (acidic phosphate buffer extraction) to convert solid hair matrix into liquid; followed by homogeneous enzyme immunoassay (HEIA) on automated clinical chemistry analyzer. Principle: competitive binding between drug in sample and drug-labeled recombinant glucose-6-phosphate dehydrogenase (G6PDH) for antibody binding sites; enzyme activity decreases as antibody binds labeled G6PDH; in presence of drug, enzyme activity increases proportionally. Active enzyme reduces NAD to NADH; absorbance change measured spectrophotometrically at 340 nm. Output is preliminary qualitative result; requires confirmatory testing via LC/MS/MS. Benefits include rapid screening of hair samples for drug use identification.

Clinical Evidence

No clinical data. Bench testing only. Performance evaluated via precision studies (intra-assay and inter-assay), analytical specificity (cross-reactivity and interference testing), and method comparison against LC-MS/MS. Cosmetic treatment interference studies (permanent wave, relaxer, dye, shampoo) showed no impact on assay results. Linearity and LLOQ established for LC-MS/MS confirmation method.

Technological Characteristics

Homogeneous enzyme immunoassay (HEIA) using mouse monoclonal antibodies and recombinant G6PDH-labeled drug conjugates. Measurement via automated clinical chemistry analyzer (Olympus AU 640) at 340 nm. Confirmation via AB Sciex 3200 LC/MS/MS with Perkin Elmer Series 200 pumps/autosampler. Sample matrix: human head/body hair. Extraction: acidic phosphate buffer at high temperature.

Indications for Use

Indicated for preliminary qualitative detection of opiates or oxycodone in human head and body hair to identify drug use. Intended for use by Psychemedics only; not for sale to others. Requires confirmatory testing via LC/MS/MS.

Regulatory Classification

Identification

An opiate test system is a device intended to measure any of the addictive narcotic pain-relieving opiate drugs in blood, serum, urine, gastric contents, and saliva. An opiate is any natural or synthetic drug that has morphine-like pharmocological actions. The opiates include drugs such as morphine, morphine glucoronide, heroin, codeine, nalorphine, and meperedine. Measurements obtained by this device are used in the diagnosis and treatment of opiate use or overdose and in monitoring the levels of opiate administration to ensure appropriate therapy.

Special Controls

*Classification.* Class II (special controls). An opiate test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (*e.g.,* programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).

Predicate Devices

• Psychemedics Microplate EIA for Opiates in Hair (k111926)
• Psychemedics Microplate EIA for Oxycodone in Hair (k123799)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K201326

B Applicant

Psychemedics Corporation

C Proprietary and Established Names

Psychemedics Homogeneous Enzyme Immunoassay for Opiates in Hair
Psychemedics Homogeneous Enzyme Immunoassay for Oxycodone in Hair

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| DJG | Class II | 21 CFR 862.3650 - Opiate Test System | TX - Clinical Toxicology |

## II Submission/Device Overview:

A Purpose for Submission:

New Device

B Measurand:

Opiates, Oxycodone

C Type of Test:

Qualitative screening test: homogeneous enzyme immunoassay
Quantitative confirmatory test: LC-MS/MS

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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## III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) for Use:

The Psychemedics homogeneous enzyme immunoassay (HEIA) for opiates is an enzyme immunoassay for the preliminary qualitative detection of opiates in human head and body hair using a morphine calibrator at 2 ng morphine/10 mg hair for the purpose of identifying opiate use. This is an in vitro diagnostic device intended exclusively for Psychemedics use only and is not intended for sale to anyone. The Psychemedics homogeneous enzyme immunoassay 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) is the preferred confirmatory method.

The Psychemedics homogeneous enzyme immunoassay (HEIA) for oxycodone is an enzyme immunoassay for the preliminary qualitative detection of oxycodone in human head and body hair using an oxycodone calibrator at 2 ng oxycodone/10 mg hair for the purpose of identifying opioid use. This is an in vitro diagnostic device intended exclusively for Psychemedics use only and is not intended for sale to anyone. The Psychemedics homogeneous enzyme immunoassay 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) is the preferred confirmatory method.

### C Special Conditions for Use Statement(s):

OTC - Over The Counter

The Psychemedics homogeneous enzyme immunoassay (HEIA) for opiates and homogeneous enzyme immunoassay (HEIA) for oxycodone combine a screening method (immunoassay) with a confirmation method (LC/MS/MS) in one test system.

The assay is to be performed only at Psychemedics Corporation.

### D Special Instrument Requirements:

Performance data for the Psychemedics homogeneous enzyme immunoassay (HEIA) for opiates in hair and Psychemedics homogeneous enzyme immunoassay (HEIA) for oxycodone in hair screening assays was collected on the Olympus AU 640 analyzer.

The confirmation assay consists of an AB Sciex 3200 LC/MS/MS linked to two Perkin Elmer Series 200 Micro pumps and a Perkin Elmer Series 200 autosampler.

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## IV Device/System Characteristics:

### A Device Description:

The homogeneous enzyme immunoassay (HEIA) test (screening assay) consists of two parts; a pre-analytical hair treatment procedure (to extract opiates from the solid hair matrix to form a measurable liquid matrix) and the screening assay, the Psychemedics Opiates HEIA and the Psychemedics Oxycodone HEIA.

The Psychemedics Opiates HEIA consists of reagents R1 (anti-opiates monoclonal antibody with substrate) and R2 (morphine labeled recombinant G6PDH). The Psychemedics Oxycodone HEIA consists of reagents R1 (anti-oxycodone monoclonal antibody with substrate) and R2 (oxycodone labeled recombinant G6PDH).

The confirmation assay consists of an AB Sciex 3200 LC/MS/MS linked to two Perkin Elmer Series 200 Micro pumps and a Perkin Elmer Series 200 autosampler.

### B Principle of Operation:

Hair specimens are extracted in a phosphate buffer solution at an acidic pH at high temperature. After the extraction, the sample is analyzed with opiates antibody and opiate-labeled G6PDH or oxycodone antibody and oxycodone-labeled G6PDH using a chemistry autoanalyzer. The cutoff calibrator and controls are also processed through the extraction and autoanalyzer steps.

The immunoassay is based on competition between the morphine-labeled G6PDH or oxycodone-labeled G6PDH and free drug from the extracted hair sample for a fixed amount of specific antibody binding sites. In the absence of free drug from the samples, the specific antibody binds the opiate- G6PDH or oxycodone-G6PDH and causes a decrease in enzyme activity. This phenomenon creates a direct relationship between the drug concentration in hair and enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by measuring the conversion of nicotinamide adenine dinucleotide (NAD) to NADH.

A change in milli-absorbance units (ΔmAU) greater than or equal to the ΔmAU of the 2 ng morphine/10 mg hair cutoff calibrator is indicative of the presence of opiates; a change in milli-absorbance units (ΔmAU) greater than or equal to the ΔmAU of the 2 ng oxycodone/10 mg hair cutoff calibrator is indicative of the presence of oxycodone.

For samples that are presumptive positive by the screening assay, a new aliquot of the hair sample is weighed, washed extensively to remove externally derived opioids, extracted by a different procedure and confirmed by LC/MS/MS for the presence of opioids or oxycodone.

### V Substantial Equivalence Information:

### A Predicate Device Name(s):

Psychemedics Microplate EIA for Opiates in Hair, Psychemedics Microplate EIA for Oxycodone in Hair

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B Predicate 510(k) Number(s):

K111926, K123799

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K201326 | K111926 |
| --- | --- | --- |
| Device Trade Name | Psychemedics Homogeneous Enzyme Immunoassay for Opiates in Hair | Psychemedics Microplate EIA for Opiates in Hair |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | Same | The Psychemedics Microplate EIA for Opiates is an enzyme immunoassay (EIA) for the preliminary qualitative detection of opiates in human head and body hair samples using a morphine calibrator at 2 ng/10 mg hair cutoff for the purpose of identifying opiate use. |
| Sample Matrix | Human Hair | Same |
| General Device Characteristic Differences | | |
| Method of Measurement | Automated Clinical Chemistry Analyzer at 340 nm | Microplate Reader at 450 nm |
| Antibody | Mouse monoclonal | Rabbit polyclonal |
| Device & Predicate Device(s): | K201326 | K123799 |
| --- | --- | --- |
| Device Trade Name | Psychemedics Homogeneous Enzyme Immunoassay for Oxycodone in Hair | Psychemedics Microplate EIA for Oxycodone in Hair |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | Same | The Psychemedics Microplate EIA for Oxycodone is an enzyme immunoassay (EIA) for the preliminary qualitative detection of oxycodone in human head and body hair samples using an oxycodone |

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| Device & Predicate Device(s): | K201326 | K123799 |
| --- | --- | --- |
| | | calibrator at 2 ng/10 mg hair cutoff for the purpose of identifying oxycodone use. |
| Sample Matrix | Same | Human hair |
| General Device Characteristic Differences | | |
| Method of Measurement | Automated Clinical Chemistry Analyzer at 340 nm | Microplate Reader at 450 nm |
| Antibody | Mouse monoclonal | Rabbit polyclonal |

## VI Standards/Guidance Documents Referenced:

CLSI EP06-A: Evaluation of Linearity of Quantitative Measurement Procedures - 2nd Edition

## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

1. Precision/Reproducibility:

### Opiates Screening Assay

Precision studies were performed by spiking negative hair with previously LC/MS/MS validated calibrator and control solutions to achieve concentrations of negative, the cutoff calibrator of 2 ng morphine/10 mg hair or 2 ng oxycodone/10 mg hair, and +/- 75%, +/-50% and +/-25% of the cutoff calibrator.

Intra-assay precision for both screening assays was performed on a single day in two replicates of four.

| Opiates HEIA Intra-assay Precision Results | | | |
| --- | --- | --- | --- |
| Concentration (ng/10 mg) | % of cutoff | # of determinations | Total Result |
| 0 | -100 | 8 | 0 Positive / 8 Negative |
| 0.5 | -75 | 8 | 0 Positive / 8 Negative |
| 1.0 | -50 | 8 | 0 Positive / 8 Negative |
| 1.5 | -25 | 8 | 0 Positive / 8 Negative |
| 2.0 | Cutoff | 8 | 4 Positive / 4 Negative |
| 2.5 | +25 | 8 | 8 Positive / 0 Negative |
| 3.0 | +50 | 8 | 8 Positive / 0 Negative |
| 3.5 | +75 | 8 | 8 Positive / 0 Negative |
| 4.0 | +100 | 8 | 8 Positive / 0 Negative |

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Inter-assay precision for both screening assays was performed for 10 days, 2 runs per day in replicates of 4 for a total of 80 results per sample.

| Opiates HEIA Inter-assay Precision Results | | | |
| --- | --- | --- | --- |
| Concentration (ng/10 mg) | % of cutoff | # of determinations | Total Result |
| 0 | -100 | 80 | 0 Positive / 80 Negative |
| 0.5 | -75 | 80 | 0 Positive / 80 Negative |
| 1.0 | -50 | 80 | 0 Positive / 80 Negative |
| 1.5 | -25 | 80 | 0 Positive / 80 Negative |
| 2.0 | Cutoff | 80 | 50 Positive / 30 Negative |
| 2.5 | +25 | 80 | 80 Positive / 0 Negative |
| 3.0 | +50 | 80 | 80 Positive / 0 Negative |
| 3.5 | +75 | 80 | 80 Positive / 0 Negative |
| 4.0 | +100 | 80 | 80 Positive / 0 Negative |

Inter-assay precision for both screening assays was performed for 10 days, 2 runs per day in replicates of 4 for a total of 80 results per sample.

| Opiates HEIA Inter-assay Precision Results | | | |
| --- | --- | --- | --- |
| Concentration (ng/10 mg) | % of cutoff | # of determinations | Total Result |
| 0 | -100 | 80 | 0 Positive / 80 Negative |
| 0.5 | -75 | 80 | 0 Positive / 80 Negative |
| 1.0 | -50 | 80 | 0 Positive / 80 Negative |
| 1.5 | -25 | 80 | 0 Positive / 80 Negative |
| 2.0 | Cutoff | 80 | 52 Positive / 28 Negative |
| 2.5 | +25 | 80 | 80 Positive / 0 Negative |
| 3.0 | +50 | 80 | 80 Positive / 0 Negative |
| 3.5 | +75 | 80 | 80 Positive / 0 Negative |
| 4.0 | +100 | 80 | 80 Positive / 0 Negative |

LC-MS/MS confirmation method
Intra-assay Precision around the Cutoff

| Morphine | 1.0 | 1.5 | 2.0 | 2.5 | 3.0 |
| --- | --- | --- | --- | --- | --- |
| Mean | 0.988 | 1.468 | 1.942 | 2.418 | 2.788 |
| SD | 0.046 | 0.016 | 0.094 | 0.073 | 0.049 |
| %CV | 4.694 | 1.119 | 4.822 | 3.031 | 1.746 |

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| Oxycodone | 1.0 | 1.5 | 2.0 | 2.5 | 3.0 |
| --- | --- | --- | --- | --- | --- |
| Mean | 0.975 | 1.496 | 1.912 | 2.458 | 2.88 |
| SD | 0.0475 | 0.0568 | 0.0823 | 0.0536 | 0.0938 |
| %CV | 4.867 | 3.799 | 4.303 | 2.180 | 3.257 |

Intra-assay Precision over the range of the Assay

| Morphine | 0.25 | 0.5 | 1 | 75 | 100 |
| --- | --- | --- | --- | --- | --- |
| Mean | 0.206 | 0.449 | 0.8546 | 62.52 | 82.94 |
| SD | 0.0044 | 0.0139 | 0.0278 | 0.5805 | 0.8264 |
| %CV | 2.15 | 3.10 | 3.25 | 0.93 | 1.00 |
| Oxycodone | 0.25 | 0.5 | 1 | 75 | 100 |
| Mean | 0.2404 | 0.48 | 1.0286 | 68.34 | 89.58 |
| SD | 0.0170 | 0.0239 | 0.0542 | 0.6066 | 3.0351 |
| %CV | 7.08 | 4.97 | 5.27 | 0.89 | 3.39 |

Inter-assay Precision around the cutoff

| Morphine | | | |
| --- | --- | --- | --- |
| Target concentration (ng/10 mg hair) | 1.00 | 2.00 | 3.00 |
| Mean | 0.98 | 1.94 | 2.89 |
| SD | 0.05 | 0.11 | 0.13 |
| %CV | 4.97 | 5.68 | 4.58 |
| Oxycodone | | | |
| --- | --- | --- | --- |
| Target concentration (ng/10 mg hair) | 1.00 | 2.00 | 3.00 |
| Mean | 1.05 | 2.11 | 3.09 |
| SD | 0.07 | 0.13 | 0.16 |
| %CV | 6.25 | 6.20 | 5.17 |

2. Linearity:

The screening immunoassays are qualitative tests. An evaluation of linearity is not applicable.

The linearity of the LC-MS/MS confirmation method for the calibrator drugs morphine and oxycodone was evaluated as follows. Morphine concentrations at 0.0, 0.25, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 10.0, 25.0, 50.0, 100.0, 125.0, and 150.0 ng/per 10 mg hair and oxycodone concentrations of 0.0, 0.25, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 10.0, 25.0, 50.0, 100.0, 125.0, and 150.0 ng per 10 mg hair were evaluated.

All concentrations for the morphine and oxycodone samples demonstrated percent recoveries within $\pm 20\%$ of the expected value.

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The LC-MS/MS assay is used only to determine if the concentration of the target analyte is above or below the cutoff. There are no clinical claims for concentrations other than the cutoff.

3. Analytical Specificity/Interference:

## Opiates Immunoassay: Cross-reactivity with structurally related compounds

The cross-reactivity characteristics of the opiates screening immunoassay were evaluated by spiking various concentrations of potential cross-reactants 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.

| Compound | Percent Cross-reactivity | Expected Concentration Equivalent to 2 ng morphine / 10 mg hair |
| --- | --- | --- |
| Codeine | 100 | 2.0 |
| 6-Acetylmorphine | 80 | 2.5 |
| Hydrocodone | 20 | 10 |
| Morphine 3-Glucuronide | 20 | 10 |
| Hydromorphone | 13 | 15 |
| Buprenorphine | 10 | 20 |
| Atropine | 2.7 | 75 |
| Oxycodone | <2 | >100 |
| Oxymorphone | <2 | >100 |
| Methadone | <2 | >100 |
| Naloxone | <2 | >100 |
| Naltrexone | <2 | >100 |
| Propoxyphene | <2 | >100 |
| Meperidine | <2 | >100 |

## Opiates Immunoassay: potential interference from structurally unrelated compounds

Potentially interfering compounds at a concentration of 100 ng/10 mg hair were added individually to spiked hair samples at +/- 50% of the morphine cutoff to evaluate which of them might interfere in the assay. The following compounds did not cause any positive or negative interference with the opiates screening assay.

Anhydroecgonine Methyl Ester, Homovanillic Acid, Bupropion, Cotinine, Cannabinol, O- Desmethylvenlafaxine, Desipramine, Doxylamine succinate, 1S, 2R Ephedrine, Naproxen, Nicotine, Nortriptyline, H-Propoxyphene, R,R-Pseudoephedrine, Thioridazine, Cis-Tramadol, Venlafaxine, (±)-11-nor-9-Carboxy-Δ9-THC, Pentazocine, Amoxicillin, Propranolol, Promethazine, Phenmetrazine, Phendimetrazine, Benzocaine, Ecgonine, Dextromethorphan, Amitriptyline, R-(-)-Phenylephrine, Glutethimide, Meprobamate, Lidocaine, Carbamazepine, Diazepam,

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Nordiazepam, AM-2201 (designer drug), JWH-019 (naphthoylindole), JWH-081 (naphthoylindole), JWH-122 (Synthetic Cannabimimetic), Imipramine, Acetaminophen, Caffeine, Dyphylline, Methaqualone, Theophylline. CP47.497 (Cannabinoid Receptor Agonist Drug), CP47.497 C8 Homologue, HU-211 (Dexanabinol), JWH-200, JWH-250, Ibuprofen, Ethosuximide, $(\pm)$-Epinephrine, Norepinephrine, Barbital, Metanephrine, Normetanephrine, Methocarbamol, Alprazolam, Citicoline, Citalopram, Clopidogrel, Bisulfate, Fluconazole, Hydrochlorothiazide, Lamotrigine, L-Thyroxine, MethylPhenidate, Omeprazole, Amlodipine Besylate, Atorvastatin, Azithromycin, Bupivacaine, Cetirizine, Dimenhydrinate, Lisinopril, Methsuximide, Phensuximide, N-Normethyl Suximide, Butabarbital, Amobarbital, Secobarbital, Hexobarbital, Phenobarbital, Mephenytoin, Ethotoin, Mephobarbital, PEMA (phenylethylmalonamide), 10, 11-Dihydrocarbamazepine, Medazepam, Chlorpromazine, Flurazepam, Lorazepam, Temazepam, Bromazepam, Primidone, 5,5-Diphenyl Hydantoin, Triamterene, Nordoxepin, Oxazepam, Levetiracetam, Metformin, Phenytoin, R-Phenylephrine, Sertraline, Topiramate, Zolpidem Tartrate, Vanillylmandelic acid, 5-Hydroxy Indole-3-Acetic Acid

## Opiates Immunoassay: Effect of cosmetic treatments

Hair samples negative for opiates and hair samples positive for opiates were treated with the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. After the treatments these samples and an aliquot of the same hair samples untreated were extracted for opioids analysis and assayed by the opiates screening assay. The screening results with and without treatment were compared to evaluate any interference in the assay or loss of drug due to the treatments. Fifteen (15) hair samples negative for opiates were evaluated before and after the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. Nine of the samples were described as brown, four as dark brown, and two as light brown.

All fifteen of the opiates negative samples produced a negative result both before and after the cosmetic treatments.

Forty (40) samples (ten for each of the four cosmetic treatments) positive for opiates were evaluated before and after the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. Thirty-one of the samples were described as black, seven as brown, and two as salt and pepper.

All forty of the opiates positive samples produced a positive result both before and after the cosmetic treatments.

## Oxycodone Immunoassay: Cross-reactivity with structurally related compounds

The cross-reactivity characteristics of the oxycodone screening immunoassay were evaluated by spiking various concentrations of potential cross-reactants 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

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compound required to produce a response approximately equivalent to the cutoff concentration of the assay.

Compounds related to oxycodone and tested for cross-reactivity with the antibody are listed in the table below.

Cross-Reactivity of Compounds structurally related to oxycodone

| Compound | Percent Cross-reactivity | Expected Concentration Equivalent to 2 ng oxycodone / 10 mg hair |
| --- | --- | --- |
| 6-Acetylcodeine | 100 | 2 |
| Hydrocodone | 80 | 2.5 |
| Ethylmorphine | 80 | 2.5 |
| Codeine | 67 | 3.0 |
| Dihydrocodeine | 67 | 3.0 |
| 6-Acetylmorphine | 27 | 7.5 |
| Morphine | 27 | 7.5 |
| Dihydromorphine | 20 | 10 |
| Morphine 3-Glucuronide | 20 | 10 |
| Atropine | 13 | 15 |
| Buprenorphine | 13 | 15 |
| Hydromorphone | 10 | 20 |
| Oxymorphone | 5 | 40 |
| Nalorphine | 2 | 100 |
| Imipramine | 2 | 100 |
| Meperidine | <2 | >100 |
| Methadone | <2 | >100 |
| Naloxone | <2 | >100 |
| Naltrexone | <2 | >100 |
| Propoxyphene | <2 | >100 |

## Oxycodone Immunoassay: Interference Testing

Potentially interfering compounds at a concentration of 100 ng/10 mg hair were added individually to spiked hair samples at +/- 50% of the oxycodone cutoff to evaluate which of them might interfere in the assay. The following compounds did not cause any positive or negative interference with the oxycodone screening assay.

Anhydroecgonine Methyl Ester, Homovanillic Acid, Bupropion, Cotinine, Cannabinol, O- Desmethylvenlafaxine, Desipramine, Doxylamine succinate, 1S, 2R Ephedrine, Naproxen, Nicotine, Nortriptyline, H-Propoxyphene, R,R-Pseudoephedrine, Thioridazine, Cis-Tramadol, Venlafaxine, (±)-11-nor-9-Carboxy-Δ9-THC, Pentazocine, Amoxicillin, Propranolol, Promethazine, Phenmetrazine, Phendimetrazine, Benzocaine, Ecgonine, Dextromethorphan. Amitriptyline, R-(-)-Phenylephrine, Glutethimide, Meprobamate, Lidocaine, Carbamazepine, Diazepam, Nordiazepam, AM-2201 (designer drug), JWH-019 (naphthoylindole), JWH-081 (naphthoylindole), JWH-122 (Synthetic Cannabimimetic), Acetaminophen, Caffeine, Dyphylline, Methaqualone,

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Theophylline, CP47.497 (Cannabinoid Receptor Agonist Drug), CP47.497 C8 Homologue. HU-211 (Dexanabinol), JWH-200, JWH-250, Ibuprofen, Ethosuximide, $(\pm)$-Epinephrine, Norepinephrine, Barbital, Metanephrine, Normetanephrine, Methocarbamol, Alprazolam, Citicoline, Citalopram, Clopidogrel, Bisulfate, Fluconazole, Hydrochlorothiazide, Lamotrigine, L-Thyroxine, MethylPhenidate, Omeprazole, Amlodipine Besylate, Atorvastatin, Azithromycin, Bupivacaine, Cetirizine, Dimenhydrinate, Lisinopril, Methsuximide, Phensuximide, N-Normethyl Suximide, Butabarbital, Amobarbital, Secobarbital, Hexobarbital, Phenobarbital, Mephenytoin, Ethotoin, Mephobarbital, PEMA (phenylethylmalonamide), 10, 11-Dihydrocarbamazepine, Medazepam, Chlorpromazine, Flurazepam, Lorazepam, Temazepam, Bromazepam, Primidone, 5,5-Diphenyl Hydantoin, Triamterene, Nordoxepin, Oxazepam, Levetiracetam, Metformin, Phenytoin, R-Phenylephrine, Sertraline, Topiramate, Zolpidem Tartrate, Vanillylmandelic acid, 5-Hydroxy Indole-3-Acetic Acid

Atropine and chlorpheniramine were found to cause positive interference in the Opiates and

Oxycodone screening assays.

## Oxycodone Immunoassay: Effect of cosmetic treatments

Hair samples negative for oxycodone and samples positive for oxycodone were treated with the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. After the treatments these samples and an aliquot of the same hair samples untreated were extracted for analysis and assayed by the oxycodone screening assay. The screening results with and without treatment are compared to evaluate any interference in the assay or loss of drug due to the treatments. The study included 15 negative samples and 10 oxycodone-positive samples with each of the treatments.

Fifteen (15) hair samples negative for oxycodone were evaluated before and after the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. Nine of the samples were described as brown, four as dark brown, and two as light brown.

All fifteen of the oxycodone negative samples produced a negative result both before and after the cosmetic treatments.

Forty (40) samples (ten for each of the four cosmetic treatments) positive for oxycodone were evaluated before and after the following cosmetic treatments: permanent wave, relaxer, dye (which includes bleach), and shampoo. Thirty-one of the samples were described as black, seven as brown, and two as salt and pepper. All forty of the oxycodone positive samples produced a positive result both before and after the cosmetic treatments.

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# Specificity of the LC-MS/MS method

An interference study was conducted for the LC-MS/MS method by spiking potential interferents into negative hair specimens that did not contain any drug, and individually into hair specimens containing 0.8 mg/10 mg hair of morphine, codeine 6-AM, hydrocodone, hydromorphone, oxycodone, and oxymorphone. All of the samples spiked with interferent but no drug produced a negative result and all of the samples spiked at 0.8 ng / 10 mg hair quantitated at ± 20% of the target value in the presence of interfering substances. The sponsor concluded that the potentially interfering compounds listed below did not cross-react or cause interference with the LC-MS/MS assay.

The following potential interferents were tested at a concentration of 2.5 ng/10 mg hair: Amlodipine besylate, Bupivacaine HCl•H20.

The following potential interferents were tested at a concentration of 20 ng/10 mg hair: Clonazepam, Estazolam, Flurazepam, Flunitrazepam, Midazolam, Nitrazepam, Prazepam, Triazolam, Zolpidem, Phentermine, R,R-(-)-Pseudoephedrine.

The following potential interferents were tested at a concentration of 25 ng/10 mg hair: Cetirizine dihydrochloride, Prednisolone, Prednisone.

The following potential interferents were tested at a concentration of 50 ng/10 mg hair: Ibuprofen, Meprobamate, Naproxen, (+)-Propoxyphene, Atorvastatin calcium salt, Azithromycin dihydrate, Dimenhydrinate, Lisinopril dihydrate, Loratadine, Montelukast sodium salt, Pioglitazone hydrochloride, Procainamide HCl, Simvastatin.

The following potential interferents were tested at a concentration of 100 ng/10 mg hair: Carbamazepine, Levetiracetam, Metformin HCl, Phenobarbital, Phenytoin, R (-)-Phenylephrine HCl, Sertraline hydrochloride, Topiramate, Zolpidem tartrate, Zonisamide.

The following potential interferents were tested at a concentration of 200 ng/10 mg hair: Morphine, Oxycodone, Codeine, Phencyclidine, S-(+)-Methamphetamine, S-(+)-Amphetamine, (±)-Methadone, Phenobarbital, Phenytoin, R-(-)-Phenylephrine HCl, Carbamazepine, Salicylic Acid, Valproic Acid, Buprenorphine (SYN), cis-Tramadol HCl, Fentanyl, Hydrocodone, Hydromorphone, Meperidine, Naloxone, Naltrexone, Oxymorphone, Aripiprazole, Lacosamide, Oxcarbazepine, Rufinamide.

The following potential interferents were tested at a concentration of 500 ng/10 mg hair: Oxcarbazepine, Gabapentin, Acetaminophen, (-)-Cotinine, S-(-)-Nicotine, Caffeine, Pregabalin, Salicylic acid, Valproic acid, Vigabatrin.

The following potential interferents were tested at a concentration of 2000 ng/10 mg hair: Warfarin.

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4. Assay Reportable Range:

An assay reportable range is not applicable for the opiates and oxycodone screening assays because they are qualitative only.

The claimed reportable range for the analytes measured by the LC-MS/MS method are as follows:

Morphine: 0.25 ng/10 mg hair to 100 ng/10 mg hair

Codeine, 6-Acetylmorphine, Hydrocodone, Hydromorphone, Oxycodone, and Oxymorphone: 0.25 ng/10 mg hair to 150 ng/10 mg hair

These reportable ranges are supported by the linearity, precision, and lower limit of quantitation (LLOQ) studies.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

Traceability:

Calibrators and control materials are prepared using drug stocks purchased from a commercial vendor with a certificate of analysis. The commercially obtained stock is made into calibrators and controls to the desired concentrations, which are confirmed by LC/MS/MS.

Sample shipping and storage stability:

Seven hair samples containing morphine, 6-AM, and codeine and six samples containing oxycodone and hydrocodone were stored in the same collection foil and card-envelope provided with the hair collection kit.

After a period of two months, the samples containing morphine, 6-AM, codeine, and hydrocodone were shipped overnight to another location and returned to the original location by overnight shipping. Samples were tested by the routine LC-MS/MS procedure both before being shipped and after their return. The sponsor stated that all samples that tested positive via the HEIA device and confirmed positive via LC-MS/MS before shipping and storage remained positive after shipping and storage.

After a period of four months, the samples containing oxycodone were shipped overnight to another location and returned to the original location by overnight shipping. Samples were tested by the routine LC-MS/MS procedure both before being shipped and after their return. The sponsor stated that all samples that tested positive via the HEIA device and confirmed positive via LC-MS/MS before shipping and storage remained positive after shipping and storage.

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6. Detection Limit:

The screening immunoassays are qualitative tests only; therefore, a detection limit evaluation is not applicable. See section VII.A.1. above for performance around the immunoassay device cutoff.

The sponsor performed a study to determine the lower limit of quantitation (LLOQ) of the LC-MS/MS assay for morphine, codeine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone.

The LLOQ is the lowest concentration that meets chromatographic and retention time criteria, and that can be quantitated within 20% of the expected value. The method LLOQ was determined to be 0.25 ng/10 mg for morphine, codeine, 6-MAM, oxycodone, oxymorphone, hydrocodone and hydromorphone. The LLOQ for this method has been administratively raised to 0.5 ng/10 mg for codeine, oxycodone, oxymorphone, hydrocodone, and hydromorphone.

7. Assay Cut-Off:

Analytical performance of the devices around the claimed cutoff is described in precision section VII.A.1 above.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Accuracy of the immunoassay screening methods

A total of 230 de-identified hair samples were analyzed for the opiates immunoassay and 219 samples were analyzed for the oxycodone assay. These samples were compared with results from the LC-MS/MS confirmatory assay. Results are summarized in the tables below:

| | LC/MS/MS Result | | | |
| --- | --- | --- | --- | --- |
| Opiates Immunoassay Result | < 1.0 (< 50% below cutoff) | 1.0 – 2.0 (≥ 50% below cutoff to cutoff) | 2.0 – 3.0 (cutoff to ≤ 50% above cutoff) | > 3.0 (> 50% above cutoff) |
| Positive | 43 | 3 | 14 | 42 |
| Negative | 118 | 10 | 0 | 0 |

The source, gender, and hair color of the opiates samples were as follows:

| Source | | Gender | | Color | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Head | Body | Male | Female | Brown | Black | Salt/Pepper | White/Gray | Blonde |
| 158 | 72 | 158 | 72 | 88 | 127 | 13 | 1 | 1 |

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The presence of codeine, 6-AM, and morphine was detected by LC-MS/MS in the 46 discordant results observed, and the presence of these analytes contributed to the positive results observed by the screening assay.

| | LC/MS/MS Result | | | |
| --- | --- | --- | --- | --- |
| Oxycodone Immunoassay Result | < 1.0 (< 50% below cutoff) | 1.0 – 2.0 (≥ 50% below cutoff to cutoff) | 2.0 – 3.0 (cutoff to ≤ 50% above cutoff) | > 3.0 (> 50% above cutoff) |
| Positive | 48 | 8 | 15 | 31 |
| Negative | 103 | 14 | 0 | 0 |

The source, gender, and hair color of the oxycodone samples were as follows:

| Source | | Gender | | Color | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Head | Body | Male | Female | Brown | Black | Salt/Pepper | White/Gray |
| 152 | 67 | 152 | 67 | 108 | 97 | 13 | 1 |

The presence of hydrocodone was detected by LC-MS/MS in the 56 discordant results observed, and the presence of this analyte contributed to the positive results observed by the screening assay.

## Recovery from LC-MS/MS Analysis

The sponsor conducted a study to evaluate recovery for morphine, codeine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone at a concentration of 2 ng/10 mg hair. Fifteen tubes were prepared and analyzed by LC/MS/MS. The recovery of the analytes ranged from 94.9% to 104.1%.

2. **Matrix Comparison:**

Not applicable. The assay is intended for only hair samples.

## C Clinical Studies:

1. **Clinical Sensitivity:**

Not applicable.

2. **Clinical Specificity:**

Not applicable.

3. **Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):**

Not applicable.

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D Clinical Cut-Off:
Not applicable.

E Expected Values/Reference Range:
Not applicable.

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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