Psychemedics Microplate EIA for Cotinine in Hair

K192517 · Psychemedics Corporation · MKU · Apr 22, 2020 · Clinical Toxicology

Device Facts

• Record ID: K192517
• Device Name: Psychemedics Microplate EIA for Cotinine in Hair
• Applicant: Psychemedics Corporation
• Product Code: MKU · Clinical Toxicology
• Decision Date: Apr 22, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3220
• Device Class: Class 1

Indications for Use

The Psychemedics Microplate EIA For Cotinine in Hair is an in vitro diagnostic device for the qualitative detection of cotinine in human head and body hair as an aid in the detection of cotinine after use or exposure to tobacco products. The assay is intended for a single site and uses a cutoff calibrator of 200 pg cotinine/mg hair. This device is intended exclusively for Psychemedics use only and is not intended for sale to anyone. The Psychemedics Microplate EIA For Cotinine 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) is the confirmatory method used by Psychemedics Corporation. The LC/MS/MS analysis uses a cutoff, after extensive washing of the hair, of 100 pg cotinine/mg hair with presence of hydroxycotinine at or above 10 pg/mg hair.

Device Story

Device performs qualitative screening for cotinine in human hair to identify nicotine use/exposure. Input: hair samples cut near skin; digested in dithiothreitol solution at pH 9.5. Operation: competitive enzyme immunoassay (EIA) using BSA-cotinine coated microplate; primary antibody against cotinine; HRP-labeled secondary antibody; TMB substrate. Output: absorbance read at 450nm/630nm; results normalized as B/B0 x 100. Presumptive positives require confirmation via LC/MS/MS (AB Sciex API-3200) using 100 pg/mg cutoff for cotinine and 10 pg/mg for hydroxycotinine. Used in laboratory setting by trained personnel. Benefits: provides objective evidence of tobacco use/exposure; distinguishes from incidental second-hand smoke exposure.

Clinical Evidence

No clinical trials performed. Analytical performance established via bench testing. Precision evaluated at cutoff (200 pg/mg) and across concentrations. Specificity tested against numerous structurally related and unrelated compounds; cosmetic treatments (dye, perm, relaxer, shampoo) showed no interference. Environmental contamination study confirmed smoke exposure does not exceed confirmatory cutoff. Literature provided to support clinical validity of cutoff relative to smoking frequency.

Technological Characteristics

In vitro diagnostic EIA. Materials: microplate wells coated with cotinine-BSA, polyclonal rabbit anti-cotinine, goat anti-rabbit HRP-conjugate, TMB substrate. Energy source: microplate reader. Connectivity: standalone laboratory equipment. Software: rule-based analysis of absorbance data. Sterilization: N/A. Matrix: human head/body hair.

Indications for Use

Indicated for qualitative detection of cotinine in human head and body hair to aid in identifying tobacco use or exposure. Intended for use by Psychemedics Corporation only.

Regulatory Classification

Identification

A carbon monoxide test system is a device intended to measure carbon monoxide or carboxyhemoglobin (carbon monoxide bound to the hemoglobin in the blood) in blood. Measurements obtained by this device are used in the diagnosis and treatment of or confirmation of carbon monoxide poisoning.

Predicate Devices

• STC Micro-Plate Cotinine EIA (k974534)

Submission Summary (Full Text)

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Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

k192517

B Applicant

Psychemedics Corporation

C Proprietary and Established Names

Psychemedics Microplate EIA for Cotinine in Hair

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| MKU | Class I, meets the limitation to the exemption 21 CFR 862.9(a) and 862.9(b) | 21 CFR 862.3220 - Carbon Monoxide Test System | TX - Clinical Toxicology |

## II Submission/Device Overview:

A Purpose for Submission:

New device

B Measurand:

Cotinine

C Type of Test:

Qualitative screening test: enzyme immunoassay (EIA)

Quantitative confirmatory test: LC/MS/MS

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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 Microplate EIA For Cotinine in Hair is an in vitro diagnostic device for the qualitative detection of cotinine in human head and body hair as an aid in the detection of cotinine after use or exposure to tobacco products. The assay is intended for a single site and uses a cutoff calibrator of 200 pg cotinine/mg hair. This device is intended exclusively for Psychemedics use only and is not intended for sale to anyone.

The Psychemedics Microplate EIA For Cotinine 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) is the confirmatory method used by Psychemedics Corporation. The LC/MS/MS analysis uses a cutoff, after extensive washing of the hair, of 100 pg cotinine/mg hair with presence of hydroxycotinine at or above 10 pg/mg hair.

C Special Conditions for Use Statement(s):

OTC - Over The Counter

D Special Instrument Requirements:

The Psychemedics Microplate EIA For Cotinine in Hair assay requires a Microplate Reader with 450nm and 630 nm wavelength absorbance capability.

The confirmation assay uses an AB Sciex API-3200 LC/MS/MS linked to a Shimadzu LC20-AD binary pump system with a CBM 20A communications bus module and a DGU-20A5R degassing unit. The autosampler is a PAL-HTC-xt.

IV Device/System Characteristics:

A Device Description:

The Psychemedics Microplate EIA for Cotinine in Hair is an enzyme immunoassay (EIA) for the preliminary qualitative detection of cotinine in human hair samples using a 2.0 ng cotinine/10 mg hair (200 pg/mg hair) cutoff for the purpose of identifying nicotine use. To confirm a preliminary positive result for the presence of cotinine, LC/MS/MS must be used in addition to this preliminary test.

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The Psychemedics Microplate EIA for Cotinine in Hair assay consists of:

- BSA-cotinine coated Microplate
- Cutoff calibrator and the controls around the cutoff
- Primary antibody against cotinine
- HRP-labeled secondary antibody directed against the primary antibody species
- Substrate TMB (tetramethylbenzidine)
- Acidic stop solution (2 N HCl)

The confirmation assay consists of calibrators and an AB Sciex API-3200 LC/MS/MS linked to a Shimadzu LC20-AD binary pump system with a CBM 20A communications bus module and a DGU-20A5R degassing unit. The autosampler is a PAL-HTC-xt.

## B Principle of Operation:

The Psychemedics Microplate EIA for Cotinine in Hair is based upon the competitive binding with the antibody to cotinine in the sample and solid-phase cotinine in the microplate wells, in proportion to their relative concentrations.

Hair samples are cut as close as possible to the skin, packaged and shipped at ambient temperature to the laboratory, where they are digested in a solution containing 0.3% dithiothreitol at pH 9.5 for two hours, then neutralized. Hair sample extracts and primary antibody are combined in the wells (coated with cotinine-BSA conjugate) and incubated. After washing, secondary antibody-HRP is added and the plate incubated 1 hour. After washing, substrate is added, and, after a final incubation, the wells are acidified and read with the microplate absorbance reader at 450 nm with background set at 630 nm. Results are normalized by expression as B/B0 x 100. If cotinine is present in the sample, less primary antibody will be bound to the solid-phase antigen, thereby resulting in less binding of HRP-labeled secondary antibody; the absorbance produced is inversely proportional to the amount of cotinine in the sample (specimen, calibrator or control).

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 cotinine, extracted by a different procedure, and confirmed by LC/MS/MS for the presence of cotinine and hydroxycotinine.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

Micro-plate Cotinine EIA

### B Predicate 510(k) Number(s):

K974534

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# C Comparison with Predicate(s):

| Device & Predicate Device(s): | k192517 | k974534 |
| --- | --- | --- |
| Device Trade Name | Psychemedics Microplate EIA for Cotinine in Hair | Orasure Cotinine Serum Microplate EIA |
| General Device Characteristic Similarities | | |
| Intended Use | Same | For the detection of Cotinine. The 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. |
| Product Code | Same | MKU |
| Measurand | Same | Cotinine |
| Method of Measurement | Same | Microplate reader, read at 450 nm |
| Type of Test | Same | Enzyme Immunoassay |
| Confirmation Method | Same | LC/MS/MS |
| General Device Characteristic Differences | | |
| Sample Matrix | Human Hair | Human Serum |
| Cutoff | 2 ng cotinine/10 mg hair (200 pg cotinine /mg hair) | 10 ng / mL |
| Extraction Method | Patented Digestion method | No extraction method |

# VI Standards/Guidance Documents Referenced:

None referenced.

# VII Performance Characteristics (if/when applicable):

## A Analytical Performance:

1. Precision/Reproducibility:

Precision of the Psychemedics Microplate EIA for Cotinine in Hair assay

The analytical precision around the cut-off (200 pg/mg hair) for cotinine was determined. Ten replicate hair samples each were spiked before digestion at the cutoff and at $\pm 25\%$,

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± 50%, ± 75% and ± 100% of the cutoff concentration of 200 pg cotinine/mg hair. The results of this intra-assay precision analysis are shown on the left side of the table below. The same procedure was carried out for 5 days to determine inter-assay precision. These results are shown in the table on the right.

| Summary -Intra-Assay Precision | | | | Summary-Inter-Assay Precision | | |
| --- | --- | --- | --- | --- | --- | --- |
| LEVEL | NEG | POS | | LEVEL | NEG | POS |
| B_{0}(-100%) | 10 | 0 | | B_{0}(-100%) | 50 | 0 |
| -75% | 10 | 0 | | -75% | 50 | 0 |
| -50% | 10 | 0 | | -50% | 50 | 0 |
| -25% | 10 | 0 | | -25% | 50 | 0 |
| plus 25% | 0 | 10 | | plus 25% | 0 | 50 |
| plus 50% | 0 | 10 | | plus 50% | 0 | 50 |
| plus 75% | 0 | 10 | | plus 75% | 0 | 50 |
| plus 100% | 0 | 10 | | plus 100% | 0 | 50 |

## Precision of the LC-MS/MS method

Within-run precision around the LC/MS/MS cutoff was conducted using drug negative hair samples spiked with 0, 10, 20, 35, 50, 80, 100, 300, 500, 700, 1000 pg/mg hair of cotinine. Samples were prepared in replicates of 5, per each concentration for cotinine and hydroxycotinine, and run over 5 days. Results for the cotinine and hydroxycotinine measured by the LC-MS/MS assay, are summarized below.

| Analyte | 0 | 10 | 20 | 35 | 50 | 80 |
| --- | --- | --- | --- | --- | --- | --- |
| Cotinine (pg/mg hair) | 0 | 11.51 | 19.75 | 37.67 | 52.29 | 82.11 |
| | 0 | 10.04 | 20.74 | 36.27 | 51.14 | 84.74 |
| | 0 | 10.16 | 21.61 | 38.72 | 56.48 | 85.71 |
| | 0 | 11.47 | 22.65 | 38.45 | 51.52 | 82.56 |
| | 0 | 11.72 | 20.98 | 37.5 | 51.1 | 81.08 |
| Average | 0 | 11.0 | 21.1 | 37.7 | 52.5 | 83.2 |
| S.D. | 0.000 | 0.41 | 1.77 | 2.58 | 1.60 | 2.78 |
| % CV | NA | 3.76 | 8.35 | 6.85 | 3.05 | 3.34 |
| 95% CI | NA | 10.62 - 11.34 | 19.6 - 22.69 | 35.46 - 39.99 | 51.1 - 53.91 | 80.81 - 85.68 |
| % of Target | NA | 109.8 | 105.7 | 107.8 | 105.0 | 104.1 |

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| Analyte | 80 | 100 | 300 | 500 | 700 | 1000 |
| --- | --- | --- | --- | --- | --- | --- |
| Cotinine (pg/mg hair) | 82.11 | 108.35 | 296.07 | 497.93 | 712.54 | 982.12 |
| | 84.74 | 102.85 | 295.45 | 491.4 | 706.34 | 1001.78 |
| | 85.71 | 100.42 | 310.71 | 503.78 | 694.01 | 973.54 |
| | 82.56 | 100.71 | 301.65 | 519.56 | 683.05 | 969.02 |
| | 81.08 | 105.58 | 316.6 | 491.73 | 691.6 | 1016.93 |
| Average | 83.2 | 103.6 | 304.1 | 500.9 | 697.5 | 988.7 |
| S.D. | 2.78 | 2.40 | 13.59 | 9.59 | 17.22 | 32.87 |
| % CV | 3.34 | 2.32 | 4.47 | 1.91 | 2.47 | 3.33 |
| 95% CI | 80.81-85.68 | 101.48 - 105.69 | 292.18 - 316.01 | 492.47 - 316.01 | 682.42 - 712.6 | 959.86 - 1017-49 |
| % of Target | 104.1 | 103.6 | 101.4 | 100.2 | 99.6 | 98.9 |
| Hydroxycotinine (pg/mg hair) | 0 | 11.1 | 21.0 | 34.6 | 48.2 | 76.7 |
| --- | --- | --- | --- | --- | --- | --- |
| | 0 | 10.7 | 19.7 | 32.2 | 49.4 | 77.8 |
| | 0 | 11.0 | 21.1 | 32.3 | 50.4 | 72.5 |
| | 0 | 11.0 | 23.9 | 31.7 | 49.7 | 71.2 |
| | 0 | 11.8 | 19.4 | 37.9 | 46.3 | 75.4 |
| Average | 0 | 11.1 | 21.0 | 33.7 | 48.8 | 74.7 |
| S.D. | 0.000 | 0.41 | 1.77 | 2.58 | 1.60 | 2.78 |
| % CV | NA | 3.71 | 8.41 | 7.65 | 3.28 | 3.72 |
| 95% CI | NA | 10.77 - 11.50 | 19.45 - 22.54 | 31.48 - 36.01 | 47.39 - 50.2 | 72.27 - 77.14 |
| % of Target | NA | 111.4 | 105.0 | 96.4 | 97.6 | 93.4 |
| Hydroxycotinine (pg/mg hair) | 76.7 | 97.3 | 280.2 | 482.4 | 610.0 | 848.2 |
| --- | --- | --- | --- | --- | --- | --- |
| | 77.8 | 94.2 | 249.6 | 462.2 | 646.6 | 911.9 |
| | 72.5 | 91.5 | 280.6 | 466.5 | 638.4 | 909.7 |
| | 71.2 | 93.2 | 281.8 | 468.1 | 637.7 | 897.3 |
| | 75.4 | 96.6 | 274.9 | 482.9 | 610.3 | 937.4 |
| Average | 74.7 | 94.6 | 273.4 | 472.4 | 628.6 | 900.9 |
| S.D. | 2.78 | 2.40 | 13.59 | 9.59 | 17.22 | 32.87 |
| % CV | 3.72 | 2.54 | 4.97 | 2.03 | 2.74 | 3.65 |
| 95% CI | 72.27-77.14 | 92.46 - 96.66 | 261.49 - 285.32 | 464.02 - 480.83 | 613.49 - 643.67 | 872.09 - 929.72 |
| % of Target | 93.4 | 94.6 | 91.1 | 94.5 | 89.8 | 90.1 |

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2. Linearity:

The screening immunoassay is a qualitative test only; therefore, a linearity evaluation is not applicable.

Linearity of the LC-MS/MS confirmation method

The linearity of the confirmation method was evaluated by spiking cotinine and hydroxycotinine into negative hair over the range of 0 to 1000 pg/mg hair. Eleven concentrations within this range were evaluated (0, 10, 20, 35, 50, 80, 100, 300, 500, 700, 1000 pg/mg hair). All levels for cotinine and hydroxycotinine demonstrated percent recoveries within +/- 15% of the expected value.

3. Analytical Specificity/Interference:

Specificity of the immunoassay screening method

Immunoassay: Cross-reactivity with structurally related compounds

The cross-reactivity characteristics of the screening immunoassay was evaluated by spiking various concentrations of cotinine 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 200 pg cotinine/mg hair |
| --- | --- | --- |
| Cotinine | 100 | 200 |
| Trans-3-hydroxycotinine | 50 | 400 |
| Nicotine | 0.4 | 50,000 |
| (S) - N- nitrosoanatabine | < 1% | >100,000 |
| (+)-anabasine | < 1% | >100,000 |
| (+/-)-nornicotine | < 1% | >100,000 |
| (S)-nitrosoanabasine | < 1% | >100,000 |
| (+/-)-N-Nitrosonornicotine | < 1% | >100,000 |

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The following compounds were found to have no cross-reactivity in the assay.

Glutethimide, Meprobamate, Methyprylon, Flurazepam, Lorazepam, Medazepam, Temazepam, Carbamazepine, Diazepam, Nordiazepam, Oxazepam, Acetaminophen, Caffeine, Dyphylline, Methaqualone, Theophylline, ethosuximide, a-methyl-a-propylsuccimide, metharbital, barbital, methsuximide, phensuximide, N-Normethsuximide, Mephenytoin, Ethotoin, Mephobarbital, PEMA, Phenobarbital, Methyl PEMA, 10,11-Dihydrocarbamazepine, Primidone, Carbamazepine, 5,5-Diphenylhydantoin, 4-Methylprimidone, Haloperidol, Ibuprofen, LSD, Meperidine, Methadone, Methaqualone, Methylphenidate, Naloxone, Naltrexone, Naproxen, Nicotine, Nortriptyline, Propoxyphene, R,R Pseudoephedrine, Thioridazine, Cis-Tramadol, Venlafaxine hydrochloride, 8(-)-11-nor-9-Carboxy-delta9 THC, 11-nor-9-Carboxy-delta9-THC, Delta 8-THC, Streptomycin solution, Benzocaine, Erythromycin, Penicillin G, Mepivacaine, Phendimetrazine bitartrate, Despropionyl, fentanyl, (+/-)epinephrine, (+/-)norepinephrine, (+/-)metanephrine, (+/-)normetanephrine, (+/-)vanilmandelic acid, 5-hydroxyindole-3-acetic acid, homovanillic acid, Benzoylecgonine, Cocaethylene, Ecgonine Methyl Ester, (+/-)Methamphetamine, Cocaine, Heroin, (+/-)methadone, codeine, hydrocodone, meperidine, oxycodone, AM2201 Spice, JWH-019 Spice, JWH-081 Spice, JWH-122 Spice, CP 47, 497 (+/-) Spice, C8 homologue Spice, HU-211 Spice, JWH-200 Spice, JWH-250 Spice, acetaminophen, caffeine, chlorphenirame maleate, ibuprofen, naproxen, R,R(-)-pseudoephedrine, Medazepam, Oxazepam, Lorazepam, Diazepam, Temazepam, Bromazepam, Cocaethylene, Cocaine, (+/-)methadone, buprenorphine, cis-tramadol HCl, codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, naloxone, naltrexone, oxymorphone, Amitriptyline, Desipramine, Doxepin,Imipramine, Nordoxepin, Nortriptyline, Protriptyline, Trimipramine, glutethimide, meprobamate, methylprylon, Amphetamine, Caffeine, Imipramine, Methamphetamine, Phencyclidine, Phenmetrazine, Phenylpropanolamine, Butabarital, Amobarbital, Secobarbital, Hexobarbital, Phenobarbital, Amitriptyline, Dextromethorphan, Lidocaine, Methocarbamol, Nordoxepin, Pentazocine, Phenylephrine, Triamterene, Amoxicillin, Propanolol, Promethazine, Phenmetrazine, Phendimetrazine, Benzocaine, Ecgononine, Metanephrin, Ethylmorphine, Nalorphine.

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

Cross-Reactivity of Cotinine-Related Drugs

| Compound | Percent Cross-reactivity | Expected Concentration Equivalent to 200 pg cotinine/mg hair |
| --- | --- | --- |
| Cotinine | 100 | 200 |
| Trans-3-hydroxycotinine | 50 | 400 |
| Nicotine | 0.4 | 50,000 |
| (S) - N- nitrosoanatabine | < 1% | >100,000 |
| (+)-anabasine | < 1% | >100,000 |
| (+/-)-nornicotine | < 1% | >100,000 |
| (S)-nitrosoanabasine | < 1% | >100,000 |
| (+/-)-N-Nitrosonornicotine | < 1% | >100,000 |

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# Immunoassay: Interference from structurally unrelated compounds

The following potentially interfering compounds were tested using the screening immunoassay on samples spiked with cotinine above and below 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-Dihydrocarbamazepine | Cocaethylene | HU-211 | metharbital | Phenmetrazine |
| --- | --- | --- | --- | --- |
| 11-nor-9-Carboxydelta9-THC | Cocaine | hydrocodone | Methocarbamol | Phenmetrazine |
| 4-Methylprimidone | Codeine | Hydromorphone | methsuximide | Phenobarbital |
| 5,5-Diphenylhydantoin | CP 47, 497 (+/-), C8 homologue | Ibuprofen | Methyl PEMA | phensuiximide |
| 5-hydroxyindole-3-acetic acid | Delta 8-THC | Imipramine | Methyl phenidate | Phenylephrine |
| 11-nor-9-Carboxydelta9 THC | Desipramine | JWH-019 | Methyprylon | Phenylpropanolamine |
| Acetaminophen | Despropionyl fentanyl | JWH-081 | Morphine | Primidone |
| AM2201 | Dextromethorphan | JWH-122 | Nalorphine | Procaine |
| a-methyl-a-propylsuccimide | Diazepam | JWH-200 | Naloxone | Promethazine |
| Amitriptyline | Doxepin | JWH-250 | Naltrexone | Propanolol |
| Amobarbital | Dyphylline | Lidocaine | Naproxen | Propoxyphene |
| Amoxicillin | Ecgonine Methyl Ester | Lorazepam | N-Normethsuximide | Protriptyline |
| Amphetamine | Ecgononine | LSD | Nordiazepam | R,R(-)-pseudoephedrine |
| barbital | epinephrine (+/-) | Medazepam | Nordoxepin | Secobarbital |
| Barbiturates | Erythromycin | Meperidine | Nordoxepin | Sedatives |
| Batubarbital | Ethosuximide | Mephenytoin | norepinephrine (+/-) | streptomycin |
| Benzocaine | Ethotoin | Mephobarbital | normetanephrine (+/-) | Temazepam |
| Benzoylecgonine | | Mepivacaine | Nortriptyline | Theophylline |

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# Immunoassay: Effect of cosmetic treatments

Hair samples negative for cotinine and samples positive for cotinine 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 digested for cotinine analysis and then assayed by the Psychemedics Microplate EIA for Cotinine in Hair. The EIA results with and without treatment are compared to detect interference in the assay or loss of drug due to the treatments. The study included 5 negative samples and 6 previously confirmed cotinine-positive samples with each type of treatment. Negative hair samples remained negative and positive samples remained positive after treatment with the cosmetic products for dye, shampoo, perm, or relaxing.

## Effects of Cosmetic Treatments on Cotinine EIA Analysis of Cotinine-Negative Hair Samples

| Effect of Perm on Cotinine-Negative Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with Permed Hair |
| 1 | Negative | Negative |
| 2 | Negative | Negative |
| 3 | Negative | Negative |
| 4 | Negative | Negative |
| 5 | Negative | Negative |
| Effect of Relaxer on Cotinine-Negative Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with Relaxed Hair |
| 1 | Negative | Negative |
| 2 | Negative | Negative |
| 3 | Negative | Negative |
| 4 | Negative | Negative |
| 5 | Negative | Negative |
| Effect of Dye on Cotinine-Negative Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with Dyed Hair |
| 1 | Negative | Negative |
| 2 | Negative | Negative |
| 3 | Negative | Negative |
| 4 | Negative | Negative |
| 5 | Negative | Negative |

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| Effect of Shampoo on Cotinine-Negative Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result after Shampoo |
| 1 | Negative | Negative |
| 2 | Negative | Negative |
| 3 | Negative | Negative |
| 4 | Negative | Negative |
| 5 | Negative | Negative |

## Effects of Cosmetic Treatments on Cotinine EIA Analysis of Cotinine-Positive Hair Samples

| Effect of Perm on Cotinine-Positive Hair Samples | | |
| --- | --- | --- |
| Sample # | Cotinine EIA Results | |
| | EIA Result with Untreated Hair | EIA Result with Permed Hair |
| 1 | Positive | Positive |
| 2 | Positive | Positive |
| 3 | Positive | Positive |
| 4 | Positive | Positive |
| 5 | Positive | Positive |
| 6 | Positive | Positive |
| Effect of Shampoo on Cotinine Positive Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with After Shampoo |
| 7 | Positive | Positive |
| 8 | Positive | Positive |
| 9 | Positive | Positive |
| 10 | Positive | Positive |
| 11 | Positive | Positive |
| 12 | Positive | Positive |
| Effect of Dye on Cotinine-Positive Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with Dyed Hair |
| 13 | Positive | Positive |
| 14 | Positive | Positive |
| 15 | Positive | Positive |
| 16 | Positive | Positive |
| 17 | Positive | Positive |
| 18 | Positive | Positive |

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| Effect of Relaxer on Cotinine-Positive Hair Samples | | |
| --- | --- | --- |
| Sample # | EIA Result with Untreated Hair | EIA Result with Relaxed Hair |
| 19 | Positive | Positive |
| 20 | Positive | Positive |
| 21 | Positive | Positive |
| 22 | Positive | Positive |
| 23 | Positive | Positive |
| 24 | Positive | Positive |

## Specificity of the LC-MS/MS method

An interference study was conducted for the LC-MS/MS method by spiking potential interferents into hair specimens containing cotinine and hydroxycotinine, and the results were compared to hair specimens containing cotinine and hydroxycotinine without potential interferent. There was no significant interference from potential interfering compounds (cotinine and hydroxycotinine were present at 40 pg/mg hair), as shown in the below table.

| Interferent Name | Interferent Amount (pg/mg hair) |
| --- | --- |
| Pseudoephedrine | 2000 |
| Phenobarbital | 2000 |
| Phenytoin | 2000 |
| Phentermine | 2000 |
| d-Amphetamine | 2000 |
| Methamphetamine | 2000 |
| Phencyclidine | 2000 |
| Triazolam | 2000 |
| Midazolam | 2000 |
| Meprobromate | 2000 |
| Flunitrazepam | 2000 |
| Flurazepam | 2000 |
| Estazolam | 2000 |
| Clobazepam | 2000 |
| Clonazepam | 2000 |
| Methyltestosterone | 2000 |
| Cocaine | 2000 |
| Salicylic acid | 2000 |
| Acetaminophen | 20,000 |
| Naproxen | 5000 |
| Ibuprofen | 5000 |
| Nicotine | 5000 |
| Propoxyphene | 5000 |
| Caffeine | 50,000 |
| (+/-) Methadone | 20,000 |

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| Interferent Name | Interferent Amount (pg/mg hair) |
| --- | --- |
| Buprenorphine | 20,000 |
| cis-Tramadol | 20,000 |
| Codeine | 20,000 |
| Fentanyl | 2000 |
| Hydrocodone | 20,000 |
| Hydromorphone | 20,000 |
| Meperidine | 20,000 |
| Morphine | 20,000 |
| Naloxone | 20,000 |
| Naltrexone | 20,000 |
| Oxycodone | 20,000 |
| Oxymorphone | 20,000 |
| Gabapentin | 5000 |
| Pregabalin | 5000 |
| Salicylic acid | 5000 |
| Valproic acid | 5000 |
| Vigabatrin | 5000 |
| (+)-Propoxyphene | 1000 |
| Aripiprazole | 2000 |
| Lacosamide | 2000 |
| Oxcarbazepine | 2000 |
| Rufinamide | 2000 |
| Warfarin | 200,000 |
| Alprazolam | 5000 |
| Cimetidine | 5000 |
| Citalopram HBr | 5000 |
| Clonazepam | 5000 |
| Clopidogrel bisulfate | 5000 |
| Dextromethorphan | 2500 |
| Fluconazole | 5000 |
| Hydrochlorothiazide | 5000 |
| Lamotrogine | 5000 |
| L-thyroxine | 250 |
| Methylphenidate | 5000 |
| Omeprazole | 5000 |
| Carbamazepine | 5000 |
| Levetiracetam | 5000 |
| Metformin HCl | 5000 |
| Phenobarbital | 5000 |
| Phenytoin | 5000 |
| R(-)-Phenylephrine HCl | 5000 |
| Sertraline HCL | 5000 |
| Topiramate | 5000 |
| Zolpidem tartrate | 5000 |

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| Interferent Name | Interferent Amount (pg/mg hair) |
| --- | --- |
| Zonisamide | 5000 |
| Carbamazepine | 5000 |
| Levetiracetam | 5000 |
| Amlodipine besylate | 250 |
| Atorvastatin calcium salt | 5000 |
| Azithromycin dihydrate | 5000 |
| Bupivacaine HCl.1H20 | 5000 |
| Cetirizine dihydrochloride | 2500 |
| Dimenhydrinate | 5000 |
| Lisinopril dihydrate | 5000 |
| Loratadine | 5000 |
| Montelukast sodium salt | 5000 |
| Pioglitazone hydrochloride | 5000 |
| Prednisolone | 5000 |
| Prednisone | 2500 |
| Procainamide HCl | 5000 |
| Simvastatin | 5000 |

## LC-MS/MS: Studies to evaluate environmental contamination

Studies were performed to evaluate environmental contamination. Seven samples of cotinine-EIA-negative hair locks were suspended in a large Erlenmeyer flask equipped to draw smoke through the flask from a burning cigarette. All of the washed samples had cotinine less than 100 pg/mg hair.

4. Assay Reportable Range:

Not applicable. The screening immunoassay is a qualitative test only.

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

Controls and calibrators are prepared as a methanolic solution containing cotinine and are traceable to certified cotinine and hydroxycotinine reference materials. Cotinine in methanol was shown to be stable up to 11.5 months.

Sample shipping and storage stability: Six samples containing cotinine (Samples were stored at ambient temperatures in the same collection foil and card-envelope in which they were placed at time of collection) were shipped overnight to another location and returned to the original location by overnight shipping. Six additional samples were tested before and after storage at ambient temperatures for 65 days. Samples were tested by the routine LC-MS/MS procedure both before being shipped and after returning to Psychemedics.

Neither shipping, nor storage for over 2 months, caused significant change in the cotinine EIA results.

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6. Detection Limit:

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

Detection Limit of the LC-MS/MS method

The sponsor performed a study to determine the Lower limit of Quantitation (LLOQ) of the LC-MS/MS assay for cotinine and hydroxycotinine.

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 is 10 pg/mg hair for cotinine and hydroxycotinine.

7. Assay Cut-Off:

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

B Comparison Studies:

1. Method Comparison:

Accuracy of the immunoassay screening method

A total of 153 samples were utilized for this study. Subjects were identified for inclusion in the study by screening using an independent screening assay (cotinine in oral fluid). The hair samples were then tested using the Psychemedics Microplate EIA for Cotinine in Hair assay and the Psychemedics LC-MS/MS confirmatory assay, results from both methods were compared. The comparison of Psychemedics Microplate EIA for Cotinine in Hair assay with LC-MS/MS confirmatory assay is shown in the following table.

Summary of Results with Positive and Negative Samples Reweighed and Confirmed without Washing

| EIA Result | LCMSMS Result (pg cotinine/mg hair) | | | |
| --- | --- | --- | --- | --- |
| | < 100 | 100-199 | 200 - 300 | > 300 |
| Positive | 0 | 6 | 13 | 23 |
| Negative | 40 | 0 | 0 | 0 |

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Discrepant results analysis:

| Positive Sample # | EIA result | MS Cotinine (pg/mg) | Comments |
| --- | --- | --- | --- |
| 3 | Positive | 183 | OHCotinine Cross reactivity is 50%; Sum of Cotinine + OHCotinine > 200 pg/mg |
| 9 | Positive | 187 | |
| 20 | Positive | 194 | |
| 4 | Positive | 140 | Sum of Cotinine + OHCotinine = 197 pg/mg; Very close to cutoff |
| 10 | Positive | 153 | 2nd weighing of sample below cutoff |
| 41 | Positive | 135 | 2nd weighing of sample below cutoff |

Recovery study for the LC-MS/MS:

The sponsor conducted a study to evaluate recovery for cotinine. For each cotinine concentration tested, 4 individual hair samples were prepared and analyzed. The recovery for cotinine ranged from 90.1 - 100% of Cotinine and Hydroxycotinine at the 2 hour extraction time of the method.

Recovery of Cotinine/Hydroxycotinine with 1:10 Trifluoroacetic acid:Methanol in Microwave

| Sample # | Time (Hours) | Cotinine (pg/mg hair)* | Hydroxycotinine (pg/mg hair)* |
| --- | --- | --- | --- |
| 1 | 1 | 410 | 65 |
| | 2 | 437 | 70 |
| | 3 | 449 | 74 |
| | % @ 2 hrs | 97.3 | 175 |
| 2 | 1 | 819 | 175 |
| | 2 | 985 | 205 |
| | 3 | 1075 | 222 |
| | % @ 2 hrs | 91.6 | 92.3 |
| 3 | 1 | 352 | 22 |
| | 2 | 378 | 24 |
| | 3 | 404 | 24 |
| | % @ 2 hrs | 93.6 | 100 |
| 4 | 1 | 391 | 28 |
| | 2 | 425 | 34 |
| | 3 | 436 | 37 |
| | % @ 2 hrs | 97.5 | 90.1 |
| *Cumulative | | | |

2. Matrix Comparison:

Not applicable. The assay is intended for only one sample matrix.

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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):
A balanced selection of scientific literature was provided to support the clinical validity of the cutoff, supporting that the device, as designed, can detect the approximate equivalent of one (1) cigarette smoked per day, every day. In addition, a clinical study was conducted in which 25 non-smokers were recruited, who were exposed to second hand smoke daily in the home setting. The amount of cotinine in the hair of all study subjects was below the confirmatory cutoff for the device, supporting that incidental exposure in these individuals did not result in high enough cotinine hair concentrations to be considered a positive result.

D Clinical Cut-Off:
Not applicable.

E Expected Values/Reference Range:
Not applicable.

VIII Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

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

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