(222 days)
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 100 pg cotinine/mg hair with presence of hydroxycotinine at or above 10 pg/mg hair.
The Psychemedics Microplate EIA For Cotinine in Hair consists of two parts; a pre-analytical hair treatment procedure (to convert the solid matrix of hair to a measurable liquid matrix) and the screening assay, the Psychemedics Microplate EIA for Cotinine. The screening portion of the test system consists of (1) microplate wells coated with cotinine conjugated to bovine serum albumin (BSA), polyclonal rabbit anti-cotinine, goat antirabbit secondary antibody conjugated to HRP (horseradish peroxidase), substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify (and stop the reaction), and wash buffer for washing the plates. Absorbance in the wells is read with a microplate reader.
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.
Here's a structured summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:
Acceptance Criteria and Device Performance for Psychemedics Microplate EIA for Cotinine in Hair (K192517)
1. Table of Acceptance Criteria and Reported Device Performance:
The document primarily focuses on demonstrating substantial equivalence to a predicate device and provides performance data rather than explicitly stating pre-defined "acceptance criteria" as pass/fail thresholds for specific metrics. However, based on the performance testing results, we can infer the implied acceptance criteria.
| Performance Metric | Implied Acceptance Criteria (Inferred) | Reported Device Performance |
|---|---|---|
| Sensitivity (EIA) | No false negatives above the LC/MS/MS confirmation cutoff relative to the 200 pg/mg hair EIA cutoff. | No samples that screened negative were false negatives (i.e., no negative EIA result confirmed above 200 pg cotinine/mg hair by LC/MS/MS). |
| Specificity (EIA) | Minimal cross-reactivity with related compounds; no interference from common substances. | Specific cross-reactivity percentages documented (e.g., 50% for trans-3-hydroxycotinine, 0.4% for nicotine); 86 compounds showed no cross-reactivity/interference. |
| Precision (EIA) - Qualitative | Able to consistently classify samples at and around the cutoff (200 pg/mg hair) as negative or positive. | Intra-Assay: 10/10 samples correctly classified for all levels (-100% to +100% of cutoff, except -25% level where 10/10 classified as negative).Inter-Assay: 50/50 samples correctly classified for all levels (-100% to +100% of cutoff, except -25% level where 50/50 classified as negative). |
| Precision (LC/MS/MS) - Intra-Assay (around cutoff) | %CV < 10% for concentrations around cutoff (50, 75, 100, 125, 150 pg/mg hair). Results averaged 98-100.1% of targets. | %CV < 10% for all concentrations. Results averaged 98-100.1% of targets. |
| Precision (LC/MS/MS) - Intra-Assay (over range) | %CV < 10% for concentrations across assay range (10-1000 pg/mg hair). Results averaged 90.1-111.4% of targets. | %CV < 10% for all concentrations. Results averaged 90.1-111.4% of targets. |
| Precision (LC/MS/MS) - Inter-Assay | %CV < 10% for concentrations for 5 days. Averages within 15% of targets. | All sets of replicates had < 10% CV. Averages of all replicates were within 15% of target concentrations. |
| Linearity (LC/MS/MS) | Regression coefficient > 0.995; actual data points deviate < 10% from regression line. | Regression coefficient > 0.995. Average value of 5 determinations within 15% of predicted Y value. Actual data points deviated < 10% from regression line, demonstrating linearity from 10 to 1000 pg/mg hair. |
| Recovery (LC/MS/MS) | Recovery > 90% from hair; Recovery > 65% for cotinine and > 55% for hydroxycotinine with SPE. | Recovery from hair was > 91.6%; Recoveries of analytes and I.S. with SPE were > 65% for cotinine and > 55% for hydroxycotinine. |
| Carryover (LC/MS/MS) | No signal detected in negative sample following a high positive sample (up to 1000 pg/mg hair). | No signal detected in negative sample after 1000 pg/mg sample. |
| LLOQ/LOD Precision (LC/MS/MS) | S:N ratio > 19:1 to 20:1. Results within 15% of target for LLOQ/LOD spiked samples. | S:N ratios 161-302 for cotinine, 137-217 for hydroxycotinine (at 10 pg/mg hair). Results showed 109.5% and 110.9% of target, respectively. |
| Cosmetic Treatments | Negative samples remain negative; positive samples remain positive. | All negative samples remained negative; all positive samples remained positive. |
| Environmental Contamination | Confirmed positive samples (from contamination) should drop below LC/MS/MS cutoff after washing procedure. | Heavily smoke-exposed hair, after extensive washing, resulted in no samples above the LC/MS/MS cutoff of 100 pg cotinine/mg hair. |
2. Sample Size Used for the Test Set and Data Provenance:
- Comparison Study (EIA vs. LC/MS/MS): A total of 82 samples were utilized.
- Data Provenance: The document states "Samples were identified for inclusion in the study by screening with a screening assay for cotinine in serum (K974234)." It doesn't explicitly state the country of origin or if they were retrospectively or prospectively collected human hair samples, but implies they are human hair samples.
- Precision Studies (EIA): Not explicitly stated as "test set," but 10 samples were used for intra-assay and 50 samples for inter-assay precision at various levels around the cutoff. These were "negative hair spiked with previously LC/MS/MS-validated calibrator and control spiking solutions."
- Cosmetic Treatments: Sets of 5 cotinine-negative and 6 cotinine-positive samples (total of 11) were used.
- Environmental Contamination: 7 hair samples were exposed to smoke for this specific test.
- LC/MS/MS Recovery from Hair: 5 authentic samples.
- LC/MS/MS Precision, Linearity, LLOQ/LOD: These studies involved spiked samples rather than a separate "test set" of authentic patient samples, with varying numbers of replicates (e.g., 5 analyses, 5 samples for 5 days).
- Sample Stability during Shipping and Storage: 6 samples for shipping stability and another 6 samples for storage stability.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Experts:
The primary "ground truth" for the comparison study and for confirming the EIA results is the Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) method. This is a laboratory-based analytical method, not directly dependent on expert human interpretation of images or clinical signs. The document does not mention human experts establishing ground truth for the test set.
4. Adjudication Method for the Test Set:
Not applicable, as the ground truth is established by the LC/MS/MS analytical method, which is a quantitative measurement, not subject to human adjudication in the traditional sense of multiple readers interpreting a case. The LC/MS/MS confirms positive cotinine levels above a certain cutoff (100 pg cotinine/mg hair with presence of hydroxycotinine at or above 10 pg/mg hair).
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done:
No, an MRMC comparative effectiveness study was not done. The device is an in vitro diagnostic assay, not an imaging device or AI algorithm requiring human interpretation. The comparison is between the new EIA screening method and the confirmatory LC/MS/MS method.
6. If a Standalone Study (algorithm only without human-in-the-loop performance) was done:
Yes, a standalone study of the device (Psychemedics Microplate EIA for Cotinine in Hair) was performed in comparison to the LC/MS/MS confirmatory method, which serves as the "gold standard" ground truth. The EIA itself operates independently without direct human intervention in the interpretation of its results (beyond reading the microplate reader output). The final confirmed analytical result is obtained by LC/MS/MS.
7. The Type of Ground Truth Used:
The ground truth used is an alternative chemical method, specifically Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS), which is considered a highly specific and sensitive analytical technique for cotinine detection. This is a form of laboratory/analytical ground truth. The LC/MS/MS analysis uses a cutoff of 100 pg cotinine/mg hair (after extensive washing) with the presence of hydroxycotinine at or above 10 pg/mg hair.
8. The Sample Size for the Training Set:
The document does not explicitly mention a "training set" in the context of machine learning. The device is a traditional enzyme immunoassay (EIA). The performance studies involve validation of the assay's characteristics (precision, linearity, cross-reactivity, etc.) using spiked samples, negative controls, and a limited number of authentic samples for comparison studies. There isn't a "training set" in the sense of data used to train an AI model.
9. How the Ground Truth for the Training Set Was Established:
Not applicable, as there is no specific "training set" for an AI model. The calibrators and control materials for the EIA and LC/MS/MS are prepared using drug stocks from commercial vendors with certificates of analysis, and their concentrations are confirmed by LC/MS/MS.
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April 22, 2020
Psychemedics Corporation Virginia Hill Vice-President, Research and Development 5832 Uplander Way Culver City, CA 90230
Re: K192517
Trade/Device Name: Psychemedics Microplate EIA for Cotinine in Hair Regulation Number: 21 CFR 862.3220 Regulation Name: Carbon Monoxide Test System Regulatory Class: Class I, meets the limitation to the exemption 21 CFR 862.9(a) and 862.9(b) Product Code: MKU Dated: March 4, 2020 Received: March 11, 2020
Dear Virginia Hill:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration. listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR
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- for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE(@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Marianela Perez-Torres, Ph.D. Acting Deputy Director Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K192517
Device Name
Psychemedics Microplate EIA for Cotinine in Hair
Indications for Use (Describe)
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 ChromatographyMass 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 100 pg cotinine/mg hair with presence of hydroxycotinine at or above 10 pg/mg hair.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| | Prescription Use (Part 21 CFR 801 Subpart D)
|X Over-The-Counter Use (21 CFR 801 Subpart C)
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510(k) Summary
This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 862.3650.
The assigned 510(k) number is: K192517
Submitted By: Psychemedics Corporation
5832 Uplander Way Culver City, CA 90230 TEL: 310 216 7776 FAX: 310 216 6662
Submission Contact: Virginia Hill
Date Prepared: April 20, 2020
Device Trade Name: Psychemedics Microplate EIA for Cotinine in Hair, K192517
Predicate Device: STC Micro-Plate Cotinine EIA, K974534
Product Code: MKU
Regulation: 862.3220 Carbon monoxide test system
Device Classification/Name: Class I, exempt; meets the limitation to the exemption 21 CFR 862.9(a) and 862.9(b)
Intended 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.
Limitations: The assay is designed only for human body and head hair. The assay is designed to detect the habitual smoking of more than one cigarette/day.
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The results of the Psychemedics Assay for Cotinine in Hair should not be used to make decisions to initiate, change, or stop medical or mental health therapy.
Positive screening results only indicate the presumptive presence of cotinine and require additional analysis by mass spectrometry to obtain a confirmed result. Certain medication or dietary supplements that contain cotinine and/or compounds that may be metabolized to produce cotinine may cause a positive result. Studies to determine the correlation of the typical doses for those drugs and dietary supplements to concentrations of cotinine in hair have not been performed. The clinical performance of this assay has not been characterized with regard to the amount and frequency of nicotine ingestion needed to test positive for cotinine. Negative results should be interpreted with caution.
A negative screening test result does not necessarily rule out the possibility of nicotine 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 dyeing. There is a possibility that other substances and/or factors not listed above may interfere with the test and cause false results that cannot be confirmed by mass spectrometry.
Assay Description: The Psychemedics Microplate EIA For Cotinine in Hair consists of two parts; a pre-analytical hair treatment procedure (to convert the solid matrix of hair to a measurable liquid matrix) and the screening assay, the Psychemedics Microplate EIA for Cotinine. The screening portion of the test system consists of (1) microplate wells coated with cotinine conjugated to bovine serum albumin (BSA), polyclonal rabbit anti-cotinine, goat antirabbit secondary antibody conjugated to HRP (horseradish peroxidase), substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify (and stop the reaction), and wash buffer for washing the plates. Absorbance in the wells is read with a microplate reader.
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.
Sample Collection & Stability: A sample of hair should be cut as close as possible to the skin. The hair is placed in a V-shaped aluminum foil sample holder with the root end of the hair protruding beyond the slanted edge of the foil. The aluminum foil is crimped around the sample, securing the hair specimen firmly into place within the foil. The hair sample, crimped within the foil, is placed in a sample acquisition card envelope and the envelope is sealed with a tamper-
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evident seal. Hair specimens are kept at ambient temperature in a secure location until they are shipped without refrigeration to the laboratory. Stability of cannabinoids in hair samples stored at room temperature has been shown for over two months. Cannabinoids in samples shipped coast-tocoast twice were stable.
| Item | Proposed Device K192517 | Predicate Device K974534 |
|---|---|---|
| Indications/Intended use | The Psychemedics Microplate EIA For Cotininein Hair is an in vitro diagnostic device for thequalitative detection of cotinine in human headand body hair as an aid in the detection ofcotinine after use or exposure to tobaccoproducts. The assay is intended for a single siteand uses a cutoff calibrator of 200 pgcotinine/mg hair. This device is intendedexclusively for Psychemedics use only and is notintended for sale to anyone.The Psychemedics Microplate EIA For Cotininein Hair provides only a preliminary analyticaltest result. A more specific alternate chemicalmethod must be used to obtain a confirmedanalytical result. Liquid Chromatography/Massspectrometry/Mass spectrometry (LC/MS/MS)is the confirmatory method used byPsychemedics Corporation. The LC/MS/MSanalysis uses a cutoff, after extensive washingof the hair, of 100 pg cotinine/mg hair withpresence of hydroxycotinine at or above 10pg/mg hair. | Orasure EIA for Cotinine in Serum is aqualitative assay for detection ofcotinine in serumThe assay provides only a preliminaryanalytical test result. A more specificalternative chemical method must beused in order to obtain a confirmedanalytical result |
| ProductCode | MKU | MKU |
| Measurand | Cotinine in Hair | Cotinine |
| Test System | Psychemedics Microplate EIA for Cotinine inHair | STC Micro-Plate Cotinine EIA |
| Matrix | Human Hair | Serum |
| Measurement | Microplate reader | Microplate reader |
| Cutoff | 200 pg cotinine/mg hair | 10 ng cotinine/mL |
| Type of Test | Enzyme Immunoassay | Enzyme Immunoassay |
| Confirmation | LC/MS/MS | Mass Spectrometry |
Comparison with Predicate:
Materials required: Hair sample collection kit, Microplate for Cotinine, Microplate washer and reader, LC/MS/MS for confirmation.
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Summary of Performance Testing of the EIA
Precision Studies
The precision studies were performed by spiking negative hair with previously LC/MS/MSvalidated calibrator and control spiking solutions to achieve concentrations of negative, the cutoff of 200 pg/mg hair, and +/-75%, +/-50%, and +/- 25% of the cutoff.
| Summary -Intra-Assay | Summary-Inter-Assay | ||||
|---|---|---|---|---|---|
| LEVEL | NEG | POS | LEVEL | NEG | POS |
| Bo (-100%) | 10 | 0 | Bo (-100%) | 50 | 0 |
| -75% | 10 | 0 | -75% | 50 | 0 |
| -50% | 10 | 0 | -50% | 50 | 0 |
| -25% | 10 | 0 | -25% | રે રેણ | 0 |
| plus 25% | ○ | 10 | plus 25% | 0 | 50 |
| plus 50% | 0 | 10 | plus 50% | 0 | 50 |
| plus 75% | 0 | 10 | plus 75% | 0 | ર૦ |
| plus 100% | 0 | 10 | plus 100% | 0 | રે0 |
Comparison Testing
A total of 82 samples were utilized for a comparison study of the Psychemedics Cotinine EIA with the LC/MS/MS results. Samples were identified for inclusion in the study by screening with a screening assay for cotinine in serum (K974234). Results are provided below.
| Comparison of Immunoassay Screening Results with Psychemedics Cotinine EIA and | ||||
|---|---|---|---|---|
| LC/MS/MS | ||||
| EIA Result | LCMSMS Result (pg cotinine/mg hair) | |||
| < 100 | 100 - 200 | 200 - 300 | > 300 | |
| Positive | 0 | 5 | 10 | 27 |
| Negative | 40 | 0 | 0 | 0 |
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| Sample | EIA Result | Cotinine by LCMSMS (pg/mg hair) | Explanation |
|---|---|---|---|
| 1 | Positive | 183 | OHCotinine Cross reacted to give result above Cutoff |
| 2 | Positive | 140 | OHCotinine Cross reacted to give result near Cutoff |
| 3 | Positive | 187 | OHCotinine Cross reacted to give result above Cutoff |
| 4 | Positive | 153 | OHCotinine Cross reacted to give result near Cutoff |
| 5 | Positive | 194 | OHCotinine Cross reacted to give result above Cutoff |
Discordant Results Between EIA and LC/MS/MS on Samples Unwashed before Confirmation
Conclusions:
-
- No samples that screened negative were false negatives-i.e., no sample that screened negative confirmed above 200 pg cotinine/mg hair.
-
- Cutoffs are appropriately set such that the EIA predicts positivity at 200 pg/mg hair with confirmation by LCMSMS at 100pg/mg.
Cosmetic Treatments
Sets of five cotinine-negative and 6 cotinine-positive samples were treated with cosmetic products and the results were compared to those for the same samples without the treatments. The treatments studied included dye, perm, relaxer, and shampoo. All of the negative samples remained negative after the treatments, and all of the positive samples remained positive.
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Summary of Cross-reactivity and Interference Studies
| Compound | Percent Cross-reactivity | Expected ConcentrationEquivalent to 200 pgcotinine/mg hair |
|---|---|---|
| Cotinine | 100 | 200 |
| Trans-3-hydroxycotinine | 50 | 400 |
| Nicotine | 0.4 | 50,000 |
| (S) - N- nitrosoanatabine | < 1% | >100,0000 |
| (+)-anabasine | < 1% | >100,000 |
| (+/-)-nornicotine | < 1% | >100,000 |
| (S)-nitrosoanabasine | < 1% | >100,000 |
| (+/-)-N-Nitrosonornicotine | < 1% | >100,000 |
Cross-reactivity of Related Compounds in Cotinine EIA
The following compounds were shown to have no cross-reactivity in the Cotinine EIA assay:
Glutethimide, Meprobamate, Methyprylon, Flurazepam, 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, Naproxen, Naproxen, Nicotine, Nortriptyline, Propoxyphene, R.R. Pseudoephedrine, Cis-Tramadol, Venlafaxine hydochloride, 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-hydrox yindole-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, 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, Nortiptyline, Trimipramine, glutethimide, meprobromate, methylprylon, Amphetamine, Caffeine, Imipramine, Methamphetamine, Phencyclidine, Phenmetrazine, Phenylpropanolamine,
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Butabarital, Amobarbital, Secobarbital, Hexobarbital, Phenobarbital, Amitriptyline, Dextromethorphan, Lidocaine, Methocarbamol, Nordoxepin, Pentazocine, Phenylephrine, Triamterene, Amoxicillin, Propanolol, Promethazine, Phendimetrazine, Benzocaine, Ecgononine, Metanephrin, Ethylmorphine, Nalorphine.
Interference
The following compounds were shown to have no interference in the Cotinine EIA assay:
Glutethimide, Meprobamate, Methyprylon, Flurazepam, 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, Naltrexone, Naproxen, Nicotine, Nortriptyline, Propoxyphene, R,R Pseudoephedrine, Cis-Tramadol, Venlafaxine hydochloride, 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, 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, Nortiptyline, Protriptyline, Trimipramine, glutethimide, meprobromate, 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.
Calibrator
Psychemedics prepares calibrators and control materials using drug stocks purchased from a commercial vendor. Each lot of drug is received with its specific certificate of analysis. The commercially obtained stock is made into the calibrators and controls to the desired concentrations. The Certified Reference Material used for the EIA calibrator is obtained from Restek.The concentrations are confirmed by LC/MS/MS. The Certified Reference Material for the LCMSMS analysis is obtained from Cerilliant.
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Sample Stability During Shipping and Storage
Six samples showed no significant differences between results before and after shipping twice cross-country. Another six samples were tested before and after 2 months in storage at room temperature in the original shipping wrappers; no differences were seen after 2 months of storage.
Recovery
The hair sample preparation for the screening assay is by a nonproteolytic digestion procedure (US Patent 8,084,215) carried out at 37°C. Recovery of cotinine in the method was shown to be essentially 100% complete at 2 hours.
Environmental Contamination
The Psychemedics screening assay does not distinguish between samples containing cotinine due to ingestion vs. from environmental contamination such as from sidestream or exhaled smoke. This determination is performed only on presumptive positive samples (identified by the screening assay) using a decontamination process (extensive sequential washing of the hair sample), described below.
Post-harvest treatment of tobacco, i.e., curing, fermentation and aging, results in an accumulation of various degradation products, including cotinine (1-3). This degradation may be ascribed to the action of enzymes present in the tobacco leaf, of bacteria contaminating the leaf and of nonenzymic reactions.
The Aqueous Buffer Wash Procedure
- Add 2 mL of dry isopropanol to the tube with hair in it and shake in waterbath for 15 i. minutes at 37°C with shaking @ 100-120 oscillations/minute. Remove isopropanol.
- ii. Add 2 mL of Wash Buffer (0.01 M phosphate buffer, pH 6.0, containing 0.1% BSA) to the tube and shake in waterbath for 30 minutes at 37°C with shaking @ 100 -120 oscillations/minute. Remove Buffer. Repeat Step ii. two more times.
- iii. Add 2 mL of Wash Buffer to the tube containing hair and shake in waterbath for 60 minutes at 37℃ with shaking @ 100 -120 oscillations/minute. Remove Buffer. Repeat Step iii, one more time.
A contamination experiment was carried out in which cotinine-EIA-negative hair locks were suspended in a large Erlenmeyer flask equipped to draw smoke through the flask from a burning cigarette. The exposure in this set-up is very extreme, as seen by the amounts of both nicotine and cotinine on the hair after exposure and before washing. The hair locks may not all be exposed to the same extent since they hang freely but may hang near and against one another and block the smoke in variable ways. Also, hair that is more porous may absorb more smoke. The data shown below demonstrate that extended washing of the extremely contaminated hair resulted in no samples above the LCMSMS cutoff of 100 pg cotinine/mg hair.
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| Hair Samples Exposed to Smoke and Analyzed by LC/MS/MS With and Without Washing | ||||||||
|---|---|---|---|---|---|---|---|---|
| NO WASH | WASHED | |||||||
| Sample | Nicotine | Cotinine | Nornicotine | Hydroxycotinine | Nicotine | Cotinine | Nornicotine | Hydroxycotinine |
| pg /mg hair | ||||||||
| 1 | 28258 | 1197 | 3177 | 99 | 6937 | 14 | 116 | 4.6 |
| 2 | 85944 | 1897 | 3656 | 171 | 404 | 20 | 111 | 5 |
| 3 | 43884 | 957 | 2063 | 82 | 4565 | 14 | 95 | 4.4 |
| 4 | 86302 | 1561 | 3884 | 142 | 28185 | 59 | 342 | 13.5 |
| 5 | 61477 | 1081 | 2525 | 105 | 15331 | 25 | 188 | 12.2 |
| 6 | 10011 | 1026 | 2433 | 99 | 459 | 64 | 167 | 13.3 |
| 7 | 11343 | 1543 | 3712 | 120 | 147 | 63 | 333 | 12.2 |
Summary of Performance Testing of the LCMSMS for Cotinine and Hydroxycotinine in Hair
Recovery
Recovery from Hair
Recovery of cotinine and hydroxycotinine from hair acidic methanol, tested with 5 authentic samples, was greater than 91.6 %.
Recovery of Analytes and Internal Standard with SPE
Comparison of (1) analysis of analytes and Internal Standards directly without extraction (extraction of matrix BEFORE addition of analytes and internal standards) with (2) analysis of analytes and Internal Standards extracted from matrix was carried out. Recoveries of analytes and I.S. were greater than 65% for cotinine and 55% for hydroxycotinine, and precisions showed less than 5% CV. Average quantitations of 5 samples were 99 - 101% of target concentrations.
Precision
Intra-Assay Precision Around the Cutoff
Precision of 5 analyses of concentrations around the cutoff was evaluated at 50, 75, 100,125, and 150 pg/mg hair. Percent CV was less than 10 for all concentrations, and results averaged 98 - 100.1 % of targets.
Intra-Assay Precision over the Range of the Assay
The concentrations evaluated were 5 samples each of 10, 20, 35, 50 100, 300, 500, 700, and 1000 pg/mg hair. Percent CV was less than 10 for all concentrations, and results averaged 90.1 - 111.4 % of targets.
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Intra-Assay Precision Around the Cutoff
The concentrations evaluated were 5 samples each, for 5 days, at 50, 100, and 150 pg/mg mg hair. All sets of replicates had < 10% CV. Averages of all replicates were within 15% of target concentrations.
Linearity
Linearity was tested by analyzing 5 samples each of cotinine and hydroxycotinine at the following concentrations: 10, 20, 35, 50 100, 300, 500, 700, and 1000 pg/mg hair.
The following results of the study are true for both analytes. All results were within 15 percent of target concentrations. Agreement of 5 determinations at each concentration was < 10% CV. The regression coefficient was > 0.995. The average value of 5 determinations was within 15% of the predicted Y value. The actual data points deviated less than 10% from the regression line. Linearity of the Cotinine/hydroxycotinine assay from 10 to 1000 pg/mg hair was demonstrated.
Validity of Dilution was demonstrated by analyzing a 1:2 dilution of a 200 pg/100 uL solution of cotinine and hydroxycotinine added to 10 mg hair, resulting in a target result of 100 pg/mg hair. Agreement of 5 determinations were 2.2% CV. The averages of 5 determinations at each dilution was within 15% of the target values.
Carryover
The ULOL was set at 1000 pg/mg hair in the linearity study. Carryover from this highest point of the assay range was tested by following the 1000 pg/mg sample with a negative sample. No signal was detected in the negative sample, indicating that up to 1000 pg/mg hair, carryover is not a factor. Samples following a sample greater than 1000pg/mg hair may require repeating.
Specificity
Matrix Effects. Suppression of ±25% was exceeded. As stipulated, the laboratory has demonstrated CVs of the area counts are less than 10% and the percent CVs of the quantitations with and without matrix are less than 10%. Further, precision, linearity, and reproducibility studies demonstrate there is no impact on other critical validation parameters.
Interference from Internal Standard or nondeuterated analytes. Samples containing no internal standard (D3) did not show presence of any peaks in the D3 window, showing that there is no D3 contaminant in the Do analytes. Samples containing no analytes showed no presence of any peaks in the Do window, showing that there is no Do contaminant in the D3 analytes.
Interference from other compounds. Eighty-six compounds were tested for interference in the LCMSMS assay. No interference was detected.
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Accuracy
Accuracy was supported by linearity, precision and specificity results. Carryover was shown to be non-existent under 1000 pg cotinine or hydrxycotinine/mg hair.
LLOQ Precision. LLOQ was determined to be at 10 pg/mg hair. LOD was administratively also set at that level. Signal-to-noise ratios for 5 samples spiked at 10 pg/mg hair were 161 - 302 for cotinine and 137 - 217 for hydroxycotinine, both much greater than the required 19:1 to 20:1 S:N recommended by CLSI document C62-A. Results showed 109.5% and 110.9% of target, respectively for cotinine and hydroycotinine.
Averages of agreement among triplicate analyses of 4 authentic positive hair samples averaged less than 15% CV.
Reproducibility among samples of multiple hair types spiked at LLOQ/LOD showed 96.8 and 104.8% of target concentrations for cotiine and hydroxycotinine, respectively, and <11% CV precision.
Conclusion:
The Psychemedics Microplate EIA and LCMSMS confirmation for Cotinine and Hydroxycotinine in Hair is substantially equivalent based on acceptable performance studies.
§ 862.3220 Carbon monoxide test system.
(a)
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.(b)
Classification. Class I (general controls). 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.