IMMUNALYSIS COCAINE ELISA FOR HAIR

K050465 · Immunalysis Corporation · DIO · Aug 4, 2005 · Clinical Toxicology

Device Facts

• Record ID: K050465
• Device Name: IMMUNALYSIS COCAINE ELISA FOR HAIR
• Applicant: Immunalysis Corporation
• Product Code: DIO · Clinical Toxicology
• Decision Date: Aug 4, 2005
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3250
• Device Class: Class 2

Indications for Use

The Immunalysis Cocaine ELISA Kit for Hair test system utilizes an Enzyme Linked Immunoassay (ELISA) for the qualitative detection of cocaine in head hair samples using a cutoff of 500 pg/mg (0.5 ng/mg) of hair for the purpose of identifying chronic cocaine use. This process has not been evaluated for use with hair specimens other than head. This in vitro diagnostic device is intended for clinical laboratory use only. The Immunalysis Cocaine ELISA Kit for Hair test system provides only a preliminary analytical test result. For confirmation of the results, presumptive positives are analyzed using a gas chromatograph - mass spectrometer operating in electron impact mode and using deuterated internal standards. Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are obtained. This device is for prescription use only.

Device Story

Immunalysis Cocaine ELISA Kit for Hair is an in vitro diagnostic test for identifying chronic cocaine use. Input: head hair samples. Principle: Enzyme Linked Immunoassay (ELISA) targeting cocaine metabolites. Output: qualitative preliminary analytical result based on 500 pg/mg cutoff. Context: clinical laboratory setting; performed by laboratory personnel. Presumptive positive results require confirmation via gas chromatography-mass spectrometry (GC-MS) using deuterated internal standards. Clinical judgment required for interpretation. Benefits: provides screening tool for chronic cocaine exposure.

Clinical Evidence

No clinical data provided. Device evaluation based on analytical performance characteristics typical of in vitro diagnostic ELISA test systems.

Technological Characteristics

Solid-phase microtiter plate ELISA; polyclonal sheep anti-cocaine antibodies. Extraction: aqueous (phosphate buffer) or methanolic (methanol/acetic acid). Detection: colorimetric (450/620 nm). Confirmation: GC-MS (electron impact mode). Form factor: 96-well plate. No specific ASTM standards cited.

Indications for Use

Indicated for qualitative detection of cocaine in head hair samples to identify chronic cocaine use. Intended for clinical laboratory use. Not evaluated for non-head hair specimens. Requires confirmatory testing via GC-MS for presumptive positives.

Regulatory Classification

Identification

A cocaine and cocaine metabolite test system is a device intended to measure cocaine and a cocaine metabolite (benzoylecgonine) in serum, plasma, and urine. Measurements obtained by this device are used in the diagnosis and treatment of cocaine use or overdose.

Special Controls

*Classification.* Class II (special controls). A cocaine and cocaine metabolite 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).

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY DEVICE ONLY TEMPLATE

A. 510(k) Number:
k050465

B. Purpose for Submission:
New Device

C. Measurand:
Cocaine

D. Type of Test:
Qualitative immunoassay

E. Applicant:
Immunalysis Corporation

F. Proprietary and Established Names:
Immunalysis Cocaine ELISA Kit for Hair

G. Regulatory Information:
1. Regulation section:
21 CFR § 862.3250
Enzyme Immunoassay, Cocaine and Cocaine Metabolites
2. Classification:
Class II
3. Product Code:
DIO
4. Panel:
Toxicology (91)

H. Intended Use:
1. Intended use(s):
Refer to Indications for use.
2. Indication(s) for use:
The Immunalysis Cocaine ELISA Kit for Hair test system utilizes an Enzyme Linked Immunoassay (ELISA) for the qualitative detection of cocaine in head hair samples using a cutoff of 500 pg/mg (0.5 ng/mg) of hair for the purpose of identifying chronic cocaine use. This process has not been evaluated for

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use with hair specimens other than head. This in vitro diagnostic device is intended for clinical laboratory use only.

The Immunalysis Cocaine ELISA Kit for Hair test system provides only a preliminary analytical test result. For confirmation of the results, presumptive positives are analyzed using a gas chromatograph - mass spectrometer operating in electron impact mode and using deuterated internal standards. Clinical and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are obtained.

This device is for prescription use only.

Characterization of the performance of the device was limited to two distinct study populations; individuals who were self-reported to be cocaine users and individuals who were self-reported to be non-users.

3. Special condition for use statement(s):

The Immunalysis Cocaine ELISA Kit for Hair combines a screening method (ELISA) with a confirmation method (GC-MS) in one test system. A negative screening result is reported as negative. A presumptive positive screening result is not reported until it has been confirmed by GC-MS.

The assay is not designated for use in point-of-care settings.

4. Special instrument Requirements:

See device description below.

I. Device Description:

The Immunalysis Cocaine ELISA Kit for Hair is a complete system for detecting cocaine in hair using a combination of an immunoassay and a GC-MS confirmation procedure. The screening assay uses a standard 96 well microtiter plate and reader. Confirmation testing is done by GC-MS operating in electron impact mode using deuterated internal standards.

J. Substantial Equivalence Information:

1. Predicate device name(s): DRI Cocaine Metabolite EIA
2. Predicate K number(s): k960187
3. Comparison with predicate

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| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Analyte | Same | Cocaine |
| Methodology | Same (for screening test) | Immunoassay |
| Differences | | |
| Item | Device | Predicate |
| Antibody | Polyclonal anti-cocaine | Monoclonal anti-benzoylecgonine |
| Instrumentation | Microplate Reader | Automated Clinical Chemistry Analyzers |
| Matrix | Hair (Cutoff 500 pg/mg) | Urine (Cutoff 300 ng/mL) |

K. Standard/Guidance Document Referenced (if applicable):
The sponsor did not reference any standards in their submission.

L. Test Principle:
The Immunalysis Cocaine ELISA Kit for Hair is a complete system for detecting cocaine in hair using the combination of an immunoassay and a GC-MS confirmation procedure. The screening assay is a solid-phase microtiter plate ELISA. The test is performed in microwells coated with polyclonal antibodies of sheep origin to cocaine. Hair samples are collected as close to the scalp as possible and preferably from the back of the head near the crown. The sponsor states that 100 hair strands with a length of 4 cm will give approximately 100 mg of hair. The 4 cm lengths are then cut into smaller segments of 3-5 mm, mixed to ensure homogeneity, and weighed into 10 mg aliquots. Prior to extraction, the hair is washed briefly with methanol, the solvent is decanted, and the hair is allowed to dry.

The sponsor has included two distinct extraction steps in their labeling. Users may choose one or the other depending on their needs. For the aqueous extraction, 0.5 mL of a 0.025 M monobasic phosphate buffer is added to the hair samples, the tubes are capped and incubated at 60 °C for one hour. 0.5 M dibasic phosphate buffer is added to neutralize the acid environment and the liquids are transferred to corresponding clean glass tubes. All specimens, calibrators and controls are then diluted 1:5 by adding 400 uL of phosphate buffer saline (100 mM PBS with 0.1% BSA, pH 7.0) to 100 uL of hair extract. For the methanolic extraction, 2 mL of Methanol with 1% v/v of Glacial Acetic Acid is added to the hair samples, the tubes are capped and incubated at 60 °C for one hour. After an hour the methanol is transferred to clean tubes and evaporated under nitrogen. The tubes are then reconstituted with 550 uL of 100 mM Phosphate buffered Saline pH 7.0 containing 0.1% BSA. All specimens, calibrators and controls are then diluted 1:5 by adding 400 uL of phosphate buffer saline (PBS with 0.1% BSA, pH 7.0) to 100 uL of the reconstituted hair extract.

To begin the ELISA procedure, 20 µL aliquots of controls, calibrators, and patient samples are added to the sample well, followed by the Enzyme Conjugate. During

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the 60 minute incubation that follows, the enzyme conjugate competes with the analyte in the samples for binding sites on the antibody-coated microwells. After the incubation the wells are washed six times with DI water to remove any unbound materials such as excess conjugate or residual sample. Enzyme substrate is then added to each well and incubated for 30 minutes at room temperature, preferably in the dark. At 30 minutes the stop solution is added to each well, which changes the color from blue to yellow. The absorbance of each well is read at 450 and 620 nm and users are instructed to read the plates within 30 minutes of yellow color development. If the average sample absorbance is greater than the average absorbance of the cutoff calibrator of 500 pg/mg, the sample is interpreted as NEGATIVE for Cocaine at 500 pg/mg of hair. A negative interpretation means that the sample does not contain cocaine or cocaine is present at a concentration below the cutoff level for this assay. If the average sample absorbance is equal to or less than the average absorbance of the laboratory cutoff calibrator of 500 pg/mg, the sample is interpreted as presumptive POSITIVE for Cocaine at 500 pg/mg of hair. A presumptive positive interpretation means that the sample contains cocaine at a concentration at or above the cutoff concentration.

Other structurally similar compounds can produce positive results. Compounds that are not structurally similar to cocaine have not been observed to produce positive results, however false positive screening results may occur because of non-specific binding or other technical problems.

Presumptive positives are carried forward to the confirmation procedure utilizing GC-MS. A 20 mg aliquot of hair from the original hair specimen is weighed out and incubated with methanol at room temperature for 1-5 minutes, after which the methanol is discarded. Deuterated internal standards are added to each calibrator, control, or unknown tube followed by a two hour incubation with hydrochloric acid at 75 °C. After the incubation the acid is decanted into clean glass tubes, 0.1 M phosphate buffer is added, and the pH is adjusted with 0.1 M sodium hydroxide. Solid phase extraction columns are conditioned with a mixture of methylene chloride, isopropanol, ammonium hydroxide, methanol, and phosphate buffer, after which the samples are allowed to flow through the columns with no vacuum. After washing with deionized water, hydrochloric acid, and methanol, the columns are dried at high pressure for five minutes. The drug is then eluted into collection tubes with a mixture of fresh methylene chloride, isopropanol, and ammonium hydroxide, and the extracts are evaporated to dryness. The extracts are then derivatized with pentafluoropropanol and pentafluoropropionic anhydride, evaporated to dryness, and reconstituted with pyridine/ethyl acetate prior to transfer into auto-sampling vials for GC-MS analysis.

Interpretation of Confirmatory Testing Results: Samples are considered positive for cocaine use if*:

- Cocaine is present at or above 500 pg/mg AND the benzoylecgonine / cocaine ratio is ≥ 0.05

OR

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- Norcocaine is present at or above 50 pg/mg

OR

- Cocaethylene is present at or above 50 pg/mg

* SAMHSA proposed guidelines April 13, 2004

Interpretation of results must take into account that drug concentrations detected in hair from a single individual can vary extensively depending on the site of collection. Positive screening results only indicate the presumptive presence of cocaine, and require additional analysis by mass spectrometry to obtain a confirmed result. A negative screening result does not necessarily rule out the possibility of cocaine use, i.e., time of collection, frequency of use, mode of ingestion, dosage used, hair types and other factors may influence results. It is not possible to document all possible effects due to treatments such as bleaching, straightening and dying. There is a possibility that other substances and/or factors that were not evaluated in the interference studies may interfere with the test and cause false results that cannot be confirmed by mass spectrometry, e.g. technical or procedural errors.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

**Within-Run Precision Using Spiked (Contrived) Samples**

The intra-assay analytical precision of the screening assay was evaluated by analyzing sixteen replicate samples at five different concentrations in one run. To prepare the samples, a pool of previously washed certified negative hair was spiked with cocaine in methanol at concentrations of 250, 500, 750, and 1000 pg/mg of cocaine, which correspond to 50%, 100%, 150%, and 200%, respectively of the sponsor's recommended cutoff concentration. A zero concentration sample was also included in the experiment. The study used one lot of reagent and was performed by two of the sponsor's employees at the sponsor's quality control laboratory. The results are summarized as follows:

| Spiked Concentration (pg/mg) | Mean Optical Density | Standard Deviation | % Coefficient of Variation | Lower 95% Confidence Interval | Upper 95% Confidence Interval of Mean Optical Density |
| --- | --- | --- | --- | --- | --- |
| 0 | 2.991 | 0.037 | 1.25 | 2.971 | 3.012 |
| 250 | 2.016 | 0.083 | 4.13 | 1.97 | 2.062 |
| 500 | 1.558 | 0.047 | 3.04 | 1.531 | 1.584 |
| 750 | 1.442 | 0.049 | 3.40 | 1.415 | 1.469 |
| 1000 | 1.223 | 0.041 | 3.39 | 1.201 | 1.246 |

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## Between-Run Precision Using Spiked (Contrived) Samples

The between-run analytical precision of the screening assay was similarly evaluated by analyzing four different concentrations in eight runs over three days. The preparation of the samples was the same as for Within-Run Precision above, and the concentrations used were 0, 250, 500, 750, and 1000 pg/mg. The study used two lots of reagents and was performed by two of the sponsor's employees at the sponsor's Quality Control laboratory. The results are summarized as follows:

| Spiked Concentration (pg/mg) | Mean Optical Density | Standard Deviation | % Coefficient of Variation | Lower 95% Confidence Interval | Upper 95% Confidence Interval of Mean Optical Density |
| --- | --- | --- | --- | --- | --- |
| 0 | 3.209 | 0.062 | 1.94 | 3.157 | 3.261 |
| 250 | 2.152 | 0.136 | 6.30 | 2.038 | 2.266 |
| 500 | 1.688 | 0.098 | 5.81 | 1.606 | 1.770 |
| 750 | 1.472 | 0.071 | 4.84 | 1.413 | 1.514 |
| 1000 | 1.318 | 0.065 | 4.92 | 1.264 | 1.372 |

## Between-Run Precision Using Previously Analyzed Positive Hair Samples

The between-run analytical precision of the screening assay was also evaluated using hair specimens which had previously been assayed and had produced readings between the low control (250 pg/mg) and high control (1000 pg/mg). These specimens span a range of colors including black, gray, brown and blond. Since the sponsor's procedure includes the option of either an aqueous or methanolic extraction, both were evaluated. Five replicates of four previously positive samples were assayed using the aqueous extraction, and five replicates of five previously positive samples were assayed using the methanolic extraction. The results are summarized as follows:

### Aqueous Extraction

| Sample # | DT0086 | DT0112 | DT0147 | DT0110 |
| --- | --- | --- | --- | --- |
| Replicate 1 OD | 1.152 | 1.344 | 1.352 | 0.883 |
| Replicate 2 OD | 1.288 | 1.301 | 1.287 | 0.995 |
| Replicate 3 OD | 1.199 | 1.180 | 1.309 | 0.926 |
| Replicate 4 OD | 1.356 | 1.306 | 1.351 | 0.819 |
| Replicate 5 OD | 1.409 | 1.388 | 1.333 | 0.975 |
| Mean | 1.281 | 1.304 | 1.326 | 0.910 |
| Standard Deviation | 0.107 | 0.078 | 0.028 | 0.080 |
| % Coefficient of Variation | 8.33 | 5.95 | 2.12 | 8.85 |

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| Controls and Calibrators for Aqueous Extraction | Zero | Low Control | Calibrator | High Control |
| --- | --- | --- | --- | --- |
| Optical Density | 2.553 | 1.672 | 1.294 | 0.948 |

## Methanolic Extraction

| Sample # | DT0002 | DT0011 | DT0079 | DT0108 | DT 0290 |
| --- | --- | --- | --- | --- | --- |
| Replicate 1 OD | 1.331 | 1.008 | 1.115 | 1.009 | 1.509 |
| Replicate 2 OD | 1.296 | 0.942 | 1.342 | 0.882 | 1.382 |
| Replicate 3 OD | 1.402 | 0.999 | 1.255 | 0.941 | 1.241 |
| Replicate 4 OD | 1.257 | 1.102 | 1.199 | 0.962 | 1.366 |
| Replicate 5 OD | 1.508 | 1.255 | 1.203 | 0.951 | 1.392 |
| Mean | 1.359 | 1.055 | 1.223 | 0.949 | 1.378 |
| Standard Deviation | 0.099 | 0.111 | 0.083 | 0.046 | 0.095 |
| % Coefficient of Variation | 7.29 | 10.51 | 6.82 | 4.81 | 6.91 |
| Controls and Calibrators for Methanolic Extraction | Zero | Low Control | Calibrator | High Control |
| --- | --- | --- | --- | --- |
| Optical Density | 2.422 | 1.622 | 1.278 | 0.933 |

b. Linearity/assay reportable range:
Not applicable. The assay is intended for qualitative use.

c. Traceability (controls, calibrators, or method):
Calibrators and controls are required but not supplied. The sponsor has included instructions in their labeling for preparing calibrators and controls from commercially available stock cocaine solutions.

d. Detection limit:

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The analytical sensitivity was estimated by measuring the absorbance of negative hair samples, and spiked samples at 15, 30, 60, 125, 250, and 500 pg/mg. The means and standard deviations were calculated. The concentration corresponding to the mean minus two standard deviations was calculated to be approximately 12 pg/mg. The sponsor has chosen to use the more conservative figure of 15 pg/mg in their labeling.

e. Analytical specificity:

Cross-Reactivity with structurally unrelated compounds: To assess the potential for positive and/or negative interference the sponsor prepared negative hair matrix, negative hair matrix spiked at the cutoff minus 50%, and negative hair matrix spiked at the cutoff plus 50%. The sponsor then spiked the equivalent of 500,000 pg/mg of the following compounds into the prepared matrices. There was no deviation from the expected results; i.e., the negative samples all had an immunoassay response below the detection limit of the assay, the samples at the cutoff minus 50% read negative, and the samples at the cutoff plus 50% read positive.

Acetaminophen, Acetylsalicylic acid, Amphetamine, Aminopyrine, Amitryptyline, Ampicillin, Amobarbital, Ascorbic acid, Atropine, Barbital, Butabarbital, Bromazepam, Caffeine, Carbamazepine, Codeine, Chloroquine, Chlorpheniramine, Chlorpromazine, Carbromal, Desipramine, Dextromethorphan, Dextropropoxyphene, Diacetylmorphine, 5,5Diphenylhydantoin, 10-1 11 - Dihydrocarbamazepine, Diazepam, Doxepin, Doxylamine, Ethosuximide, Ethotoin, Flurazepam, Glutethimide, Hexobarbital, Hydrocodone, Hydromorphone, Ibuprofen, Imipramine, Lidocaine, Lorazepam, LSD, Medazepam, Methadone, Methadone - primary metabolite, Methaqualone, Methamphetamine, Metharbital, Mephenytoin, Methylphenidate, Methyl-propylsuccinimide, Mephobarbital, Methyl PEMA, Methsuximide, 4-Methylprimidone, Morphine, Meperidine, Nalorphine, Niacinamide, Nordoxepin, Norethindrone, N-Normethsuximide, Nortriptyline, Oxazepam, Oxycodone, PEMA, Pentobarbital, Phencyclidine, Pheniramine, Phenobarbital, Phensuximide, Phenothiazine, Phenylpropanolamine, Primidone, Procaine, Promethazine, Protriptyline, Quinine, Secobarbital, Temazepam, Tetracycline, Tetrahydrozoline, THCCOOH, Trimipramine

Cross-Reactivity with structurally similar compounds: To assess cross-reactivity, serial dilutions were prepared in negative hair matrix for each of the compounds listed in the table below. The concentration of compound that produced absorbance readings

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similar to the cutoff calibrator was recorded and from this a percent cross-reactivity was calculated

| Structurally Similar Compound | Concentration equivalent to 500 pg/mg (cutoff concentration) | Percent Cross-Reactivity |
| --- | --- | --- |
| Cocaine | 500 pg/mg | 100 |
| Benzoylecgonine | 5500 pg/mg | 9 |
| Cocaethylene | 375 pg/mg | 133 |
| m-hydroxybenzoylecgonine | 9000 pg/mg | 5.5 |
| Benzoylecgonine Isopropyl Ester | 250 pg/mg | 200 |
| Norcocaine | 10,000 pg/mg | 5 |
| Norbenzoylecgonine | 20,000 pg/mg | 2.5 |
| Norcocaethylene | 9000 pg/mg | 5.5 |
| Ecgonine | >50,000 pg/mg | <1 |
| Ecgonine Methyl Ester | >50,000 pg/mg | <1 |

There is the possibility that other substances and/or factors not listed above may interfere with the test and cause false results, e.g., technical or procedural errors.

f. Assay cut-off:

Characterization of how the device performs analytically around the claimed cutoff concentration appears in the precision section, above.

g. Environmental Contamination Study

To assess the influence of environmental contamination on the assay, the sponsor performed the following experiment. Cocaine was added to an artificially synthesized sweat solution, and samples of drug-free hair were incubated in this solution. The sponsor studied the variables of hair color, cocaine concentration, length of incubation, and washing. Results were as follows:

| Hair Color | Concentration of Soaking Solution (ng/mL) | Incubation Time (hrs) | Wash with Methanol | ELISA result | GC-MS Confirmation Result |
| --- | --- | --- | --- | --- | --- |
| Light | 100 | 4 | Yes | + | + |
| Light | 100 | 4 | No | + | + |
| Black | 100 | 4 | Yes | - | - |
| Black | 100 | 4 | No | - | - |
| Light | 10 | 4 | Yes | - | - |
| Light | 10 | 4 | No | - | - |

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| Hair Color | Concentration of Soaking Solution (ng/mL) | Incubation Time (hrs) | Wash with Methanol | ELISA result | GC-MS Confirmation Result |
| --- | --- | --- | --- | --- | --- |
| Black | 10 | 4 | Yes | - | - |
| Black | 10 | 4 | No | - | - |
| Light | 100 | Overnight* | Yes | - | - |
| Light | 100 | Overnight | No | + | - |
| Black | 100 | Overnight | Yes | - | + |
| Black | 100 | Overnight | No | - | + |
| Light | 10 | Overnight | Yes | - | - |
| Light | 10 | Overnight | No | - | - |
| Black | 10 | Overnight | Yes | - | - |
| Black | 10 | Overnight | No | - | - |

*exact time not specified

Of the eight samples that were soaked in a 10 ng/mL solution, none tested positive by the ELISA assay or GC-MS. Of the eight samples that were soaked in a 100 ng/mL solution, five were positive by ELISA and/or GC-MS.

h. Stability Study

The sponsor obtained from a reference laboratory 41 hair samples which had previously been confirmed positive by GC-MS at a cutoff of 500 pg/mg cocaine. The samples were stored at room temperature for 12-14 months, re-extracted by the aqueous and/or methanolic methods, and analyzed by the sponsor’s ELISA method. If enough hair was available the samples were also re-analyzed by GC-MS.

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| Aqueous Extraction | Prior to storage | | | | After Storage | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Hair color | cocaine | BZE | norcocaine | cocaethylene | Immunolysis ELISA | cocaine | BZE | norcocaine | cocaethylene |
| BLACK | 8906 | 2578 | 0 | 0 | + | 6310 | 2440 | 0 | 0 |
| BROWN | 1276 | 3006 | 0 | 0 | + | 1040 | 2890 | 0 | 0 |
| BROWN | 731 | 70 | 0 | 0 | + | 690 | 65 | 0 | 0 |
| BLACK | 4843 | 581 | 0 | 0 | + | 4610 | 520 | 0 | 0 |
| BLACK | 1490 | 169 | 0 | 0 | + | 1130 | 140 | 0 | 0 |
| BLACK | 4281 | 199 | 0 | 0 | + | 3150 | 174 | 0 | 0 |
| BLACK | 1634 | 99 | 0 | 0 | - | 1510 | 110 | 0 | 0 |
| GREY | 10811 | 472 | 0 | 0 | + | 9955 | 635 | 0 | 0 |
| BROWN | 3149 | 172 | 75 | 0 | + | 2810 | 195 | 61 | 0 |
| BLACK | 3120 | 410 | 0 | 0 | + | 2530 | 390 | 0 | 0 |
| BLACK | 14530 | 1040 | 0 | 0 | + | 9980 | 995 | 0 | 0 |
| BLONDE | 4843 | 477 | 0 | 0 | + | 4340 | 559 | 0 | 0 |
| BROWN | 540 | 150 | 0 | 0 | + | 530 | 126 | 0 | 0 |
| BLACK | 22960 | 1710 | 0 | 3390 | + | 19880 | 1630 | 0 | 3170 |
| BLACK | 11310 | 960 | 0 | 220 | + | 10540 | 1050 | 0 | 185 |
| DK BRN | 13710 | 960 | 0 | 1700 | + | 12980 | 880 | 0 | 1495 |
| BLACK | 6860 | 1850 | 0 | 0 | + | 6340 | 2010 | 0 | 0 |
| DK BRN | 7480 | 810 | 0 | 0 | + | 7190 | 960 | 0 | 0 |
| BLACK | 580 | Not Performed | Not Performed | Not Performed | + | Insufficient Sample For GC-MS Analysis | | | |
| GREY | 4450 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 1990 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 9130 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 2000 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 4210 | Not Performed | Not Performed | Not Performed | + | 3350 | 730 | 0 | 115 |
| BLACK | 1400 | Not Performed | Not Performed | Not Performed | + | 1310 | 240 | 0 | 0 |
| BLACK | 4200 | Not Performed | Not Performed | Not Performed | + | 4090 | 310 | 95 | 0 |
| BLACK | 1790 | Not Performed | Not Performed | Not Performed | + | 1450 | 159 | 0 | 65 |
| BLACK | 4924 | 0 | 143 | 0 | - | 3370 | 195 | 120 | 0 |
| BROWN | 16185 | 0 | 512 | 0 | + | 15600 | 126 | 390 | 0 |
| BLACK | 19088 | 0 | 453 | 0 | + | 17200 | 225 | 425 | 0 |
| BLACK | 13074 | 0 | 0 | 0 | + | 10900 | 740 | 0 | 0 |
| BLACK | 271071 | 27281 | 0 | 0 | + | 223500 | 31500 | 0 | 0 |
| BLACK | 2767 | 363 | 41 | 152 | + | 2350 | 426 | 65 | 99 |
| BLACK | 24211 | 2082 | 0 | 9599 | + | 22100 | 1850 | 0 | 8875 |
| BLACK | 3875 | 299 | 2237 | 15.6 | + | 3670 | 245 | 2100 | 0 |
| BLACK | 23850 | 2563 | 1035 | 5031 | + | 19700 | 2375 | 990 | 4750 |
| BLONDE | 2539 | 289 | 36 | 0 | + | 2200 | 207 | 0 | 0 |
| BLONDE | 3478 | 226 | 0 | 0 | + | 3180 | 269 | 0 | 0 |
| BLACK | 299654 | 48105 | 17289 | 3398 | + | 260500 | 56700 | 16850 | 2840 |
| BLACK | 25943 | 2971 | 834 | 7814 | + | 23700 | 3590 | 935 | 7680 |
| GREY | 10330 | 746 | 285 | 2372 | + | 9950 | 880 | 250 | 2250 |

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Using the aqueous extraction method, two out of the 41 previously positive samples produced negative results after storage at room temperature. Both samples contained cocaine but at levels below the cutoff of the ELISA assay.

| Methanolic Extraction | Prior to storage | | | | After Storage | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| COLOR | COC | BE | NORCOC | COCAETH | ELISA | COC | BE | NORCOC | COCAETH |
| BLACK | 8906 | 2578 | 0 | 0 | + | 6310 | 2440 | 0 | 0 |
| BROWN | 1276 | 3006 | 0 | 0 | + | 1040 | 2890 | 0 | 0 |
| BROWN | 731 | 70 | 0 | 0 | + | 690 | 65 | 0 | 0 |
| BLACK | 4843 | 581 | 0 | 0 | + | 4610 | 520 | 0 | 0 |
| BLACK | 1490 | 169 | 0 | 0 | + | 1130 | 140 | 0 | 0 |
| BLACK | 4281 | 199 | 0 | 0 | + | 3150 | 174 | 0 | 0 |
| BLACK | 1634 | 99 | 0 | 0 | - | 1510 | 110 | 0 | 0 |
| GREY | 10811 | 472 | 0 | 0 | + | 9955 | 635 | 0 | 0 |
| BLACK | 3120 | 410 | 0 | 0 | + | 2530 | 390 | 0 | 0 |
| BLACK | 14530 | 1040 | 0 | 0 | + | 9980 | 995 | 0 | 0 |
| BLONDE | 4843 | 477 | 0 | 0 | + | 4340 | 559 | 0 | 0 |
| BLACK | 22960 | 1710 | 0 | 3390 | + | 19880 | 1630 | 0 | 3170 |
| DK BRN | 7480 | 810 | 0 | 0 | + | 7190 | 960 | 0 | 0 |
| BLACK | 580 | Not Performed | Not Performed | Not Performed | + | Insufficient Sample For GC-MS Analysis | | | |
| GREY | 4450 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 1990 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 9130 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 2000 | Not Performed | Not Performed | Not Performed | + | | | | |
| BLACK | 4210 | Not Performed | Not Performed | Not Performed | + | 3350 | 730 | 0 | 0 |
| BLACK | 1790 | Not Performed | Not Performed | Not Performed | + | 1450 | 159 | 0 | 0 |
| BLACK | 19088 | 0 | 453 | 0 | + | 17200 | 225 | 425 | 0 |
| BLACK | 13074 | 0 | 0 | 0 | + | 10900 | 740 | 0 | 0 |
| BLACK | 271071 | 27281 | 0 | 0 | + | 223500 | 31500 | 0 | 0 |
| BLACK | 2767 | 363 | 41 | 152 | + | 2350 | 426 | 65 | 99 |
| BLACK | 24211 | 2082 | 0 | 9599 | + | 22100 | 1850 | 0 | 8875 |
| BLACK | 3875 | 299 | 2237 | 15.6 | + | 3670 | 245 | 2100 | 0 |
| BLACK | 23850 | 2563 | 1035 | 5031 | + | 19700 | 2375 | 990 | 4750 |
| BLONDE | 2539 | 289 | 36 | 0 | + | 2200 | 207 | 0 | 0 |
| BLONDE | 3478 | 226 | 0 | 0 | + | 3180 | 269 | 0 | 0 |
| BLACK | 299654 | 48105 | 17289 | 3398 | + | 260500 | 56700 | 16850 | 2840 |
| BLACK | 25943 | 2971 | 834 | 7814 | + | 23700 | 3590 | 935 | 7680 |
| GREY | 10330 | 746 | 285 | 2372 | + | 9950 | 880 | 250 | 2250 |

Using the methanolic extraction method, one out of the 32 previously positive samples produced negative results after storage at room temperature. The sample contained cocaine but at levels below the cutoff of the ELISA assay.

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i. Hair Treatment Effect on Negative and Positive Hair Samples

The effects of hairspray, hair gel, and bleaching on the screening assay were examined. Six negative samples of varying colors were selected and treated with hairspray, hair gel, and bleach prior to analysis. An untreated sample was also analyzed. Results were as follows:

| Hair Sample | Brown | Black #1 | Black #2 | White | Blond #1 | Blond #2 |
| --- | --- | --- | --- | --- | --- | --- |
| Untreated Absorbance | 3.16 | 3.142 | 3.175 | 3.146 | 3.208 | 3.226 |
| Hairspray Absorbance | 3.162 | 3.145 | 3.144 | 3.144 | 3.209 | 3.205 |
| Hair Gel Absorbance | 3.15 | 3.147 | 3.176 | 3.16 | 3.242 | 3.218 |
| Bleach Absorbance | 3.169 | 3.156 | 3.149 | 3.152 | 3.237 | 3.209 |
| Mean | 3.16025 | 3.1475 | 3.161 | 3.1505 | 3.224 | 3.2145 |
| Standard Deviation | 0.007848 | 0.006028 | 0.016872 | 0.007188 | 0.018019 | 0.009399 |
| % Coefficient of Variation | 0.248319 | 0.191508 | 0.533757 | 0.228153 | 0.558887 | 0.292381 |

Ten previously confirmed positive samples were treated with hairspray, hair gel, and bleach. An untreated sample was also analyzed. Results were as follows:

| No treatment | Hair Spray | | | Hair Gel | | | Bleach | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| B/B0* | B/B0 | Diff (%) | ELISA | B/B0 | Diff (%) | ELISA | B/B0 | Diff (%) | ELISA |
| 39.79 | 41.7 | 4.70 | + | 38.45 | -3.37 | + | 45.62 | 14.65 | + |
| 49.49 | 48 | -2.93 | + | 50.1 | 1.23 | + | 54.12 | 9.36 | - |
| 23.51 | 22.1 | -6.17 | + | 24.88 | 5.83 | + | 27.65 | 17.61 | + |
| 19.54 | 18.8 | -4.04 | + | 20.66 | 5.73 | + | 22.45 | 14.89 | + |
| 33.62 | 34 | 1.10 | + | 35.21 | 4.73 | + | 37.68 | 12.08 | + |
| 38.55 | 39.2 | 1.79 | + | 37.81 | -1.92 | + | 43.55 | 12.97 | + |
| 16.83 | 15.6 | -7.55 | + | 15.92 | -5.41 | + | 19.27 | 14.50 | + |
| 44.33 | 45.8 | 3.41 | + | 44.91 | 1.31 | + | 47.33 | 6.77 | + |
| 29.71 | 28.4 | -4.31 | + | 29.29 | -1.41 | + | 32.93 | 10.84 | + |
| 24.05 | 25.7 | 6.69 | + | 24.98 | 3.87 | + | 26.73 | 11.14 | + |

*B/B0 = absorbance of cutoff calibrator / absorbance of zero calibrator

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The sponsor noted a negative bias in the samples treated with bleach. In one case, a previously positive specimen tested negative after treatment with bleach.

2. Comparison studies:

a. Method comparison with predicate device:

A total of 157 samples (74 self-reported negative and 83 self-reported positive) were evaluated by the candidate device and by GC/MS and the predicate device.

Number of study sites: one

Type of study site: Manufacturer's facility

Operator description: Manufacturer's staff

Positive Agreement Study

This study enrolled 83 drug users who admitted using cocaine. The participants indicated frequency of usage ranging from daily use to once per month. The subjects ranged in age from 21 to 65 years old, and of the 83 subjects 17 were female and 66 were male. Hair color was distributed as follows: black – 56%, brown – 22%, gray – 20%, light brown – 1%, and blonde – 1%. Each subject provided a urine and a head hair sample.

Positive Agreement Study Results

| Number of Subjects | Self-Reported | Urine Screen Result* | Hair Screen Result – Aqueous Extraction | Hair Screen Result – Methanolic Extraction | Hair GC/MS Result** |
| --- | --- | --- | --- | --- | --- |
| 59 | + | + | + | + | + |
| 17 | + | - | + | + | + |
| 1 | + | - | - | - | + |
| 1 | + | + | - | + | + |
| 3 | + | - | - | - | - |
| 2 | + | - | - | + | + |

*300 ng/mL cutoff

**SAMHSA proposed cutoffs

Negative Agreement Study

This study enrolled 74 volunteers who self-reported as non cocaine users. The subjects ranged in age from 19 to 97 years old, and of the 74 subjects 33 were female and 41 were male. Hair color was distributed as follows: black – 45%, brown – 24%, blonde – 18%,

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gray – 5%, white – 5%, and red – 3%. Each subject provided a urine and a head hair sample.

## Negative Agreement Study Results

| Number of Subjects | Self-Reported | Urine Screen Result* | Hair Screen Result – Aqueous Extraction | Hair Screen Result – Methanolic Extraction | Hair GC/MS Result** |
| --- | --- | --- | --- | --- | --- |
| 70 | - | - | - | - | - |
| 3 | - | - | - | - | + |
| 1 | - | - | + | + | + |

*300 ng/mL cutoff

**SAMHSA proposed cutoffs

b. Matrix comparison:
Not applicable. The assay is intended for only one sample matrix.

3. Clinical studies:
a. Clinical sensitivity:
See comparison study above.
b. Clinical specificity:
See comparison study above.
c. Other clinical supportive data (when a and b are not applicable):

4. Clinical cut-off:
Not applicable.

5. Expected values/Reference range:
Cocaine should not be detectable in the hair of persons who have not used cocaine.

## N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10

## O. Conclusion:

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