(269 days)
The Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair. The assay is intended for use in workplace settings for the qualitative analysis of human head and body hair. The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.
The immunoassay consists of two parts; a pre-analytical hair treatment procedure (to convert the solid matrix of hair to a measurable liquid matrix) and the screening assay, the Psychemedics Microplate EIA for Benzodiazepines. The screening portion of the test system consists of microplate wells coated with Oxazepam conjugated to bovine serum albumin (BSA), the prepared hair sample, a cutoff calibrator added to the sample at a concentration of 1 ng Oxazepam/10 mg hair, monoclonal mouse anti-Oxazepam antibody, goat anti-mouse secondary antibody conjugated to HRP (horseradish peroxidase), substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify (and stop the reaction), and wash buffer for washing the plates. Absorbance in the wells is read with a microplate reader.
The confirmation assay consists of a AB Sciex API 3200 LC/MS/MS (Serial numbers AA24661109 and AA28841310) linked to two Shimazu LC-20AD Micro pumps and a Leap Technologies PAL autosampler.
The provided text describes the performance characteristics of the "Psychemedics Microplate EIA for Benzodiazepines in Hair" (the "device") and its supporting studies. The information is presented in the context of a 510(k) premarket notification to the FDA, demonstrating substantial equivalence to a predicate device.
Here's a breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance
The document doesn't explicitly state "acceptance criteria" for each performance metric in a pass/fail format. Instead, it describes validated ranges and outcomes that were considered acceptable for demonstrating the device's performance. The reported performance is directly from the tables and text provided.
| Performance Characteristic | Acceptance Criteria (Implicit) | Reported Device Performance |
|---|---|---|
| Immunoassay Precision | Consistent positive/negative calls across concentrations and replicates, especially near the cutoff. | Intra-Assay:- Negative, 25%, 50%, 75% of cutoff: 15/0 (Negative/Positive) - Cutoff: 7/8 (Negative/Positive) - 125%, 150%, 175%, 200% of cutoff: 0/15 (Negative/Positive) Inter-Assay:- Negative, 25%, 50%, 75% of cutoff: 75/0 (Negative/Positive) - Cutoff: 43/32 (Negative/Positive) - 125%, 150%, 175%, 200% of cutoff: 0/75 (Negative/Positive) |
| LC-MS/MS Precision | %CV of 10% or less for each level tested; concentrations within ± 15% of target; correlation coefficient >0.995; mean of replicates within ± 15% of predicted. | Alprazolam: %CV range 2.96% - 11.45% (n=5 samples, triplicate reps) Lorazepam: %CV range 4.83% - 7.42% (n=4 samples, triplicate reps) Diazepam: %CV range 0.91% - 12.77% (n=5 samples, triplicate reps) Nordiazepam: %CV range 3.81% - 7.75% (n=4 samples, triplicate reps) Oxazepam: %CV range 2.25% - 10.82% (n=4 samples, triplicate reps) Temazepam: %CV range 1.99% - 13.12% (n=5 samples, triplicate reps) |
| LC-MS/MS Linearity | %CV ≤ 10%, concentrations within ± 15% of target, correlation coefficient >0.995, mean of replicates within ± 15% of predicted value based on linear regression. | All conditions met, establishing a linear range of 0.05 to 20.0 ng/10 mg hair for Alprazolam, Lorazepam, Diazepam, Nordiazepam, and Temazepam. |
| LC-MS/MS Detection Limit (LLOQ) | Analyte response at LLOQ ≥ 5 times blank response; identifiable, discrete, reproducible peak; precision (CV) ≤ 20%; accuracy within 20% of nominal. | LLOQ of 0.05 ng/10 mg hair for all six benzodiazepines, with all acceptance criteria satisfied. |
| Immunoassay Specificity (Cross-reactivity) | N/A (listed cross-reactivity percentages) | Varies significantly by compound (e.g., Clobazam 550%, Oxazepam 100%, Lorazepam 13%, 7-aminoclonazepam <0.3%). This data informs potential false positives/negatives based on specific benzodiazepines. |
| Immunoassay Specificity (Interference) | No positive or negative interference observed. | No positive or negative interference observed from 70+ structurally unrelated compounds tested at 100 ng/10 mg hair. |
| LC-MS/MS Specificity (Cross-reactivity) | No cross-reactivity observed with structurally related compounds. | No cross-reactivity observed for Clonazepam, Estazolam, Flurazepam, Meprobromate, Midazolam, Nitrazepam, Prazepam, Triazolam, and Zolpidem. |
| LC-MS/MS Specificity (Interference) | No interference observed with structurally unrelated compounds. | No interference observed from 30+ structurally unrelated compounds including common drugs and metabolites. |
| Immunoassay Accuracy (Method Comparison) | High concordance with confirmatory LC/MS/MS method with clear explanation of discordant results based on established cutoffs. | 382 hair samples (318 head, 64 body) compared to LC/MS/MS. - True Negative: 233 - True Positive (high): 107 - False Positive (-0.5x cutoff): 5 - False Positive (near cutoff 0.5x-1x): 19 - False Negative (near cutoff 1x-1.5x): 0 - False Negative (+1.5x cutoff): 0 Discordant results (24 samples) were analyzed, and concentrations were calculated based on cross-reactivities, showing that positive immunoassay results for samples with "LC/MS/MS Result" below the 1 ng cutoff were frequently due to the summed concentration of benzodiazepines present in the sample being above the effective cutoff when accounting for cross-reactivity. Examples show calculated effective concentrations ranging from 0.255 to 0.961 ng/10 mg hair for these discordant samples. |
2. Sample Size Used for the Test Set and Data Provenance
- Test Set Sample Size:
- Immunoassay Precision: 15 replicates per concentration for Intra-Assay (9 concentrations), 75 replicates per concentration for Inter-Assay (9 concentrations over 5 days).
- LC-MS/MS Precision: For each of the six benzodiazepines, between 4 and 5 authentic samples were tested in triplicate.
- LC-MS/MS Linearity: 8 concentrations plus a zero concentration sample were evaluated. The number of replicates per concentration is stated as five (implied for linearity validation criteria).
- LC-MS/MS LLOQ: Not explicitly stated how many samples were used, but acceptance criteria were met.
- Immunoassay Specificity (Cross-reactivity): Various concentrations of 27 benzodiazepines. Not specified how many unique samples/tests per compound.
- Immunoassay Specificity (Interference): At least 70 unique compounds, each tested at 100 ng/10 mg hair on samples spiked with oxazepam at cutoff and ±50% of cutoff. Not specified how many replicates per compound.
- LC-MS/MS Specificity (Cross-reactivity): 9 structurally related compounds. Not specified how many unique samples/tests per compound.
- LC-MS/MS Specificity (Interference): 30+ structurally unrelated compounds. Not specified how many unique samples/tests per compound.
- Accuracy (Method Comparison): 382 hair samples (318 head hair, 64 body hair).
- Data Provenance: The general context implies that the data was collected at the manufacturer's site ("Psychemedics plans to perform this test at one site"). The type of data appears to be prospective as it involves experimental testing of the device's performance characteristics. The document does not specify the country of origin of the data beyond the manufacturer's location in Culver City, CA, USA, and the FDA's US location.
- For the accuracy study, the samples represented diverse hair colors (159 black, 197 brown, 8 blond, 4 red, 14 gray) and ethnicities (175 Caucasian, 40 African-American, 125 Hispanic, 41 Asian subjects). This suggests real-world samples were used.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not mention the use of human experts or readers to establish ground truth for the analytical studies. The ground truth for the test set is established through confirmatory chemical methods, specifically Liquid Chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS), which is the gold standard for quantitative drug analysis in forensic and clinical toxicology. These methods are analytical and do not typically involve human expert interpretation for "ground truth" in the same way imaging studies might.
4. Adjudication Method for the Test Set
Not applicable. The ground truth is established by a definitive chemical analysis (LC/MS/MS), not through human review or adjudication of qualitative results.
5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study Was Done, If so, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance
Not applicable. This device is an in vitro diagnostic (IVD) test, specifically an immunoassay for drug detection. It is not an AI-powered image analysis tool that assists human readers/interpreters. Therefore, MRMC studies and human reader improvement are not relevant to this type of device.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the studies presented are essentially "standalone" performance evaluations of the device technology (immunoassay and LC/MS/MS). The immunoassay provides a preliminary screening result, and the LC/MS/MS provides a confirmed analytical result. Human involvement is in operating the instruments and interpreting the quantitative data, rather than forming a diagnostic opinion that is then augmented or compared to an AI algorithm.
7. The Type of Ground Truth Used
The primary ground truth used for evaluating the device's performance is chemical confirmation via Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS). This is considered the "confirmatory method" and is explicitly described as "a more specific alternate chemical method must be used to obtain a confirmed analytical result." This is a highly objective and quantitative method for identifying and measuring specific benzodiazepines. The document also points out that the immunoassay results (qualitative) were interpreted relative to the LC/MS/MS quantitative results, taking into account cross-reactivity for clarity in discordant cases.
8. The Sample Size for the Training Set
The document does not explicitly mention a "training set" in the context of machine learning or AI models. This device is an immunoassay, which is a biochemical assay, not an AI algorithm that requires training data in the traditional sense. The studies described demonstrate the analytical performance of the immunoassay and the confirmatory LC/MS/MS method.
9. How the Ground Truth for the Training Set Was Established
As there is no mention of a "training set" for an AI algorithm, this question is not applicable to the presented information. The ground truth for the validation/test samples (as discussed in point 7) was established through LC/MS/MS.
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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
September 15, 2017
PSYCHEMEDICS CORPORATION VIRGINIA HILL VICE PRESIDENT, RESEARCH AND DEVELOPMENT 5832 UPLANDER WAY CULVER CITY, CA 90230
Re: K163590
Trade/Device Name: Psychemedics Microplate EIA For Benzodiazepines in Hair Regulation Number: 21 CFR 862.3170 Regulation Name: Benzodiazepine test system Regulatory Class: II Product Code: JXM Dated: August 15, 2017 Received: August 15, 2017
Dear Ms. Hill:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA), You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the
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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Courtney H. Lias -S
Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K163590
Device Name
Psychemedics Microplate EIA for Benzodiazepines in Hair
Indications for Use (Describe)
The Psychemedics Microplate EIA For Benzodiazepines in Hair is an in vitro diagnostic device for the qualitative detection of benzodiazepines in hair. The assay is intended for use in workplace settings for the qualitative analysis of human head and body hair. The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.
Psychemedics plans to perform this test at one site. Psychemedics has not performed an evaluation of reproducibility at different laboratories.
The Psychemedics Microplate EIA For Benzodiazepines in Hair provides only a preliminary analytical test result. A more specific alternate chemical method must be used to obtain a confirmed analytical result. Liquid Chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS) using deuterated internal standards in multiple reaction monitoring (MRM) mode is the confirmatory method used by Psychemedics Corporation. This confirmatory method uses a cutoff of 0.2 ng of the identified benzodiazepine/10 mg hair.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) SUMMARY
This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR § 807.92.
The assigned 510(k) number is: K163590
| Submitted By: | Psychemedics Corporation5832 Uplander WayCulver City, CA 90230TEL: 310 216 7776FAX: 310 216 6662 |
|---|---|
| Submission Contact: | Virginia Hill |
| Date Prepared: | September 15, 2017 |
| Device Trade Name: | Psychemedics Microplate EIA for Benzodiazepines in Hair |
| Common/Usual Name: | Benzodiazepine Enzyme Immunoassay |
| Predicate Device: | Orasure Benzodiazepine Intercept MICRO-PLATE EIA |
| Product Code: | JXM |
| Device Classification and Name: | 21 CFR§862.3170, Benzodiazepine Test System, Class II |
| Intended Use: | The Psychemedics Microplate EIA For Benzodiazepines in Hair isan in vitro diagnostic device for the qualitative detection ofbenzodiazepines in hair. The assay is intended for use in workplacesettings for the qualitative analysis of human head and body hair.The assay uses a cutoff calibrator of 1 ng oxazepam/10 mg hair.Psychemedics plans to perform this test at one site. Psychemedicshas not performed an evaluation of reproducibility at differentlaboratories.The Psychemedics Microplate EIA For Benzodiazepines in Hairprovides only a preliminary analytical test result. A more specificalternate chemical method must be used to obtain a confirmedanalytical result. Liquid Chromatography/Mass spectrometry/Massspectrometry (LC/MS/MS) using deuterated internal standards inmultiple reaction monitoring (MRM) mode is the confirmatory |
| Psychemedics Corporation | |
| Assay Description: | The immunoassay consists of two parts; a pre-analytical hairtreatment procedure (to convert the solid matrix of hair to ameasurable liquid matrix) and the screening assay, thePsychemedics Microplate EIA for Benzodiazepines. Thescreening portion of the test system consists of microplate wellscoated with Oxazepam conjugated to bovine serum albumin(BSA), the prepared hair sample, a cutoff calibrator added to thesample at a concentration of 1 ng Oxazepam/10 mg hair,monoclonal mouse anti-Oxazepam antibody, goat anti-mousesecondary antibody conjugated to HRP (horseradish peroxidase),substrate [3, 3', 5, 5' tetramethylbenzidine (TMB)], HCl to acidify(and stop the reaction), and wash buffer for washing the plates.Absorbance in the wells is read with a microplate reader. |
| The confirmation assay consists of a AB Sciex API 3200LC/MS/MS (Serial numbers AA24661109 and AA28841310)linked to two Shimazu LC-20AD Micro pumps and a LeapTechnologies PAL autosampler. | |
| Sample Collectionand Stability: | Psychemedics provides sample collection kits, which arerecommended but not required for sample collection. A sample ofhair should be cut as close as possible to the skin. The hair isplaced in a V-shaped aluminum foil sample holder with the rootend of the hair protruding beyond the slanted edge of the foil. Thealuminum foil is crimped around the sample, securing the hairspecimen firmly into place within the foil. The hair sample,crimped within the foil, is placed in a sample acquisition cardenvelope and the envelope is sealed with a tamper-evident seal.Hair specimens are kept at ambient temperature in a securelocation until they are shipped without refrigeration to thelaboratory. Stability of benzodiazepines in hair samples stored atroom temperature has been shown to last for over one year.Benzodiazepines in samples shipped coast-to-coast twice werestable. |
| Materials required: | Hair sample collection kit Liquid Handling Equipment,Microplate washer and reader AB Sciex API 3200 LC/MS/MS (Serial numbers AA24661109 and AA28841310). |
| BSA-Oxazepam coated MicroplateCutoff Calibrator (.8 ng Oxazepam/37 uL) added to 8 mg hairControls (minus 50% of cutoff, and plus 100% of cutoff) |
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Primary antibody against Oxazepam and other benzodiazepines HRP-labeled secondary antibody directed against the primary antibody species Substrate TMB (tetramethylbenzidine) Acidic stop solution (2 N HCl)
Comparison with Predicate:
| Item | Proposed Device | Orasure OTI Benzodiazepine Assay |
|---|---|---|
| Indications/Intended use | The Psychemedics MicroplateEIA for Benzodiazepines is anenzyme immunoassay (EIA) forthe preliminary qualitativedetection of benzodiazepines inhuman head and body hair usingan Oxazepam calibrator at 1 ng/10 mg hair cutoff for identifyingbenzodiazepine use. ThePsychemedics Microplate EIABenzodiazepine assay providesonly a preliminary analytical testresult. A more specificalternative chemical methodmust be used in order to obtain aconfirmed analytical result.Liquid Chromatography / Massspectrometry /Massspectrometry (LC/MS/MS) usingdeuterated internal standards inmultiple reaction monitoring(MRM) mode is theconfirmatory method used byPsychemedics Corporation. | The OTI Benzodiazepine test isintended for use by clinicallaboratories in the qualitativedetermination of benzodiazepinesin oral fluid using a 1 ng/mL(Nordiazepam) cutoff. For in vitrodiagnostic use. This assayprovides only a preliminaryanalytical test result. A morespecific alternative chemicalmethod must be used in order toobtain a confirmed analyticalresult. Gas Chromatography/MassSpectrometry/Mass Spectrometry(GC/MS) is the preferredconfirmatory method. |
| Product Code | JXM | JXM |
| Measurand | Benzodiazepines in Hair | Benzodiazepines in Oral Fluid |
| Test System | Psychemedics Microplate EIAfor Benzodiazepines in Head andBody Hair | Orasure OTI Benzodiazepine test |
| Sample Matrix | Human Hair | Human oral fluid |
| Method of Measurement | Microplate reader, read at 450nm | Microplate reader, read at 450 nm |
| Cutoff | 1 ng Oxazepam/10 mg hair | 1 ng/mL Nordiazepam |
| Type of Test | Enzyme Immunoassay | Enzyme Immunoassay |
| Extraction Method | Patented Digestion method | Not applicable |
| Confirmation Method | LC/MS/MS | GC/MS/MS |
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Summary of Performance Characteristics
Precision of the immunoassay screening method
Intra-Assay Precision was demonstrated by measuring nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) in replicates of 15 within a single run. Results are summarized below.
| Concentration relativeto cutoff (1 ng/10 mghair) | No. Samples | Negative / Positive |
|---|---|---|
| Negative | 15 | 15/0 |
| 25% | 15 | 15/0 |
| 50% | 15 | 15/0 |
| 75% | 15 | 15/0 |
| Cutoff | 15 | 7/8 |
| 125% | 15 | 0/15 |
| 150% | 15 | 0/15 |
| 175% | 15 | 0/15 |
| 200% | 15 | 0/15 |
Inter-Assay Precision of the assay was evaluated by measuring nine concentrations of oxazepam (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff) over the course of five days, in replicates of 15 per day. Results are summarized below.
| Concentration relativeto cutoff (1 ng/10 mghair) | No. Samples | Positive / Negative |
|---|---|---|
| Negative | 75 | 75/0 |
| 25% | 75 | 75/0 |
| 50% | 75 | 75/0 |
| 75% | 75 | 75/0 |
| Cutoff | 75 | 43/32 |
| 125% | 75 | 0/75 |
| 150% | 75 | 0/75 |
| 175% | 75 | 0/75 |
| 200% | 75 | 0/75 |
Precision of the LC-MS/MS method
A study was performed to evaluate the precision of triplicate analyses of authentic (not spiked) benzodiazepine-positive samples. All six benzodiazepines measured with the confirmation method were included in the study. Each replicate measurement was made from a separate hair sample that was taken through the entire extraction process prior to analysis. Results for the six benzodiazepines measured by the LC-MS/MS assay are summarized below.
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| Alprazolam (ng/10 mg hair) | |||||
|---|---|---|---|---|---|
| Sample # | 1 | 2 | 3 | 4 | 5 |
| Rep 1 | 6.477 | 0.199 | 2.941 | 7.408 | 3.802 |
| Rep 2 | 6.713 | 0.202 | 2.886 | 7.702 | 4.746 |
| Rep 3 | 5.711 | 0.243 | 3.159 | 7.269 | 4.189 |
| Mean | 6.300 | 0.215 | 2.995 | 7.460 | 4.246 |
| SD | 0.524 | 0.025 | 0.144 | 0.221 | 0.475 |
| %CV | 8.31 | 11.45 | 4.82 | 2.96 | 11.18 |
| Lorazepam (ng/10 mg hair) | |||||
| Sample # | 1 | 2 | 3 | 4 | |
| Rep 1 | 1.174 | 0.555 | 0.536 | 0.341 | |
| Rep 2 | 1.222 | 0.643 | 0.57 | 0.375 | |
| Rep 3 | 1.102 | 0.591 | 0.59 | 0.333 | |
| Mean | 1.166 | 0.596 | 0.565 | 0.350 | |
| SD | 0.060 | 0.044 | 0.027 | 0.022 | |
| %CV | 5.18 | 7.42 | 4.83 | 6.38 | |
| Diazepam (ng/10 mg hair) | |||||
| Sample # | 1 | 2 | 3 | 4 | 5 |
| Rep 1 | 0.229 | 8.365 | 1.417 | 4.642 | 0.77 |
| Rep 2 | 0.235 | 8.443 | 1.572 | 3.75 | 0.903 |
| Rep 3 | 0.205 | 8.519 | 1.215 | 3.929 | 0.818 |
| Mean | 0.223 | 8.442 | 1.401 | 4.107 | 0.830 |
| SD | 0.016 | 0.077 | 0.179 | 0.472 | 0.067 |
| %CV | 7.12 | 0.91 | 12.77 | 11.49 | 8.11 |
| Nordiazepam (ng/10 mg hair) | |||||
| Sample # | 1 | 2 | 3 | 4 | |
| Rep 1 | 9.227 | 3.377 | 0.884 | 0.25 | |
| Rep 2 | 9.928 | 3.725 | 0.923 | 0.226 | |
| Rep 3 | 10.258 | 3.202 | 0.954 | 0.252 | |
| Mean | 9.804 | 3.435 | 0.920 | 0.243 | |
| SD | 0.527 | 0.266 | 0.035 | 0.014 | |
| %CV | 5.37 | 7.75 | 3.81 | 5.96 | |
| Oxazepam (ng/10 mg hair) | |||||
| Sample # | 1 | 2 | 3 | 4 | |
| Rep 1 | 0.176 | 0.262 | 0.161 | 0.073 | |
| Rep 2 | 0.179 | 0.267 | 0.149 | 0.071 | |
| Rep 3 | 0.146 | 0.274 | 0.143 | 0.066 | |
| Mean | 0.167 | 0.268 | 0.151 | 0.070 | |
| SD | 0.018 | 0.006 | 0.009 | 0.004 | |
| %CV | 10.82 | 2.25 | 6.07 | 5.15 |
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| Temazepam (ng/10 mg hair) | |||||
|---|---|---|---|---|---|
| Sample# | 1 | 2 | 3 | 4 | 5 |
| Rep 1 | 1.342 | 0.22 | 0.141 | 0.133 | 0.136 |
| Rep 2 | 1.327 | 0.22 | 0.147 | 0.129 | 0.139 |
| Rep 3 | 1.291 | 0.233 | 0.114 | 0.136 | 0.132 |
| Mean | 1.320 | 0.224 | 0.134 | 0.133 | 0.136 |
| SD | 0.026 | 0.008 | 0.018 | 0.004 | 0.004 |
| %CV | 1.99 | 3.35 | 13.12 | 2.65 | 2.59 |
Linearity of the LC-MS/MS method
The linearity of the confirmation method was evaluated by spiking each of the benzodiazepine analytes (Alprazolam, Lorazepam, Diazepam, Nordiazepam, and Temazepam) into a negative hair digest matrix over the desired linear range of 0.05 to 20.0 ng/10 mg hair. Eight concentrations within this range plus a zero concentration sample were evaluated. In order for the linear range to be considered validated, the following conditions needed to be met: the %CV for each level tested must be 10% or less, the concentrations tested must quantitate within ± 15% of the target value, the correlation coefficient must be >0.995, and the mean of the five replicates for each level must be within ± 15% of the predicted value based upon the linear regression line. All of these conditions were met, and the linear range of 0.05 to 20.0 ng/10 mg hair was claimed.
Detection Limit of the LC-MS/MS method
A study was performed to determine the Lower limit of Quantitation (LLOO) of the LC-MS/MS assay with acceptance criteria for analyte and blank response, and acceptable chromatography:
- . The analyte response at the LLOO should be at least five times the response compared to blank response.
- Analyte peak (response) should be identifiable, discrete, and reproducible, and the ● concentrations measured should have precision that does not exceed 20% of the CV and accuracy within 20% of the nominal concentration.
The acceptance criteria were satisfied, demonstrating an LLOO of 0.05 ng/10 mg hair for all six drugs.
Specificity of the immunoassay screening method
Cross-reactivity with structurally related compounds
The cross-reactivity characteristics of the screening immunoassay were evaluated by spiking various concentrations of benzodiazepines into drug-free hair samples and comparing the result to the cutoff calibrator. The table below lists the percent cross-reactivity and the approximate concentration of each compound required to produce a response approximately equivalent to the cutoff concentration of the assay.
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| Compound | PercentCross-reactivity | ExpectedConcentration inng/10 mg hairequivalent to cutoffcalibrator |
|---|---|---|
| Clobazam | 550 | 0.18 |
| Diazepam | 550 | 0.18 |
| Nimetazepam | 550 | 0.18 |
| Estazolam | 400 | 0.25 |
| Temazepam | 312 | 0.32 |
| Alprazolam | 277 | 0.36 |
| Flunitrazepam | 222 | 0.45 |
| Nordiazepam | 220 | 0.45 |
| Nitrazepam | 220 | 0.45 |
| N-Desmethylflunitrazepam | 172 | 0.58 |
| Lormetazepam | 133 | 0.75 |
| Oxazepam | 100 | 1 |
| Midazolam | 42 | 2.4 |
| Clonazepam | 33 | 3 |
| Triazolam | 31 | 3.2 |
| alpha-Hydroxyalprazolam | 22 | 4.5 |
| Lorazepam | 13 | 7.5 |
| Bromazepam | 8.3 | 12 |
| Chlordiazepoxide | 4 | 25 |
| 7-Aminoflunitrazepam | 3.1 | 32 |
| Hydroxymethylflurazepam | 2 | 50 |
| Alpha-Hydroxytriazolam | 1.7 | 60 |
| desalkylflurazepam | 1 | 100 |
| 7-Aminonitrazepam | 0.6 | 170 |
| 7-aminoclonazepam | <0.3 | >300 |
| Flurazepam | <0.3 | >300 |
| Prazepam | <0.3 | >300 |
| Zolpidem Tartrate | <0.3 | >300 |
Interference from structurally unrelated compounds
The following potentially interfering compounds were tested using the screening immunoassay on samples spiked with oxazepam at the cutoff and at ± 50% of the cutoff to assess possible positive or negative interference. All potential interferents listed below were tested at a concentration of 100 ng/10 mg hair. No positive or negative interference was observed in this study.
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| 11-nor-9-Carboxy-delta-9-THC | Dextromethorphan | Naproxen |
|---|---|---|
| 1S, 2R Ephedrine | Doxepin | Nicotine |
| 5-hydroxyindole-3-acetic acid | Doxylamine succinate | Normetanephrine (+/-) |
| 8(-)-11-nor-9-Carboxy-delta-9THC | Ecgononine | Nortriptyline |
| Acetaminophen | Erythromycin | O-Desmethyvenlafaxine |
| Amitriptyline | Ethosuximide | Oxycodone |
| Amobarbital | Ethylmorphine | Penicillin G |
| Amoxicillin | Fentanyl | Pentazocine |
| Anhydroecgonine methyl ester | Haloperidol | Phendimetrazine |
| Atropine | Hexobarbital | Phendimetrazine bitartrate |
| Benzocaine | Hydrocodone | Phenmetrazine |
| Benzocaine | Hydromorphone | Phenobarbital |
| Buprenorphine | Ibuprofen | Procaine |
| Bupropion | Imipramine | Promethazine |
| Butabarbital | Lidocaine | Propanolol |
| Caffeine | LSD | Propoxyphene |
| Cannabinol | Meperidine | Protriptyline |
| Chlorpheniramine maleate | Mepivacaine | R-(-)Phenylephrine |
| Cis-tramadol HCl | Metanephrine (+/-) | R,R(-)-Pseudoephedrine |
| Codeine | Methadone (+/-) | Secobarbital |
| Cotinine | Methanol | Streptomycin |
| Synthetic cannabinoids CP 47,497 (+/-) | Methaqualone | Synthetic cannabinoid AM2201 |
| Delta 8-THC | Methyl phenidate | Synthetic cannabinoid HU-211 |
| Desipramine | Morphine | Synthetic cannabinoids JWH-019,081, 122, 200, and 250 |
| Desmethyldoxepin | Nalorphine | Thioridazine |
| Desipramine | Naloxone | Trimipramine |
| Despropionyl fentanyl | Naltrexone | Vanilmandelic acid(+/-) |
Specificity of the LC-MS/MS method
Cross-reactivity with structurally related compounds
The cross-reactivity characteristics of the LC-MS/MS confirmation assay were evaluated by measuring contrived samples containing the following structurally related compounds: Clonazepam, Estazolam, Flurazepam, Meprobromate, Midazolam, Nitrazepam, Prazepam, Triazolam, and Zolpidem. No cross-reactivity was observed for these compounds.
Interference from structurally unrelated compounds
The specificity of the LC-MS/MS confirmation assay with structurally unrelated compounds was evaluated by measuring contrived samples containing the following compounds: Cotinine,
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Nicotine, Caffeine, Ibuprofen, Naproxen, Phentermine, Pseudoephedrine, Morphine, Hydrocodone, Oxycodone, Codeine, Cocaine, Phencyclidine, Methamphetamine, Methadone, Phenobarbital, Phenylephrine, Carbamazepine, Gabapentin, Salicylic Acid, Valproic Acid, Oxcarbazepine, Propoxyphene, Acetaminophen, Norfloxacin, Psilocybin, Nimesulide, Etifoxine, Chlorcyclizine, and Teriflunomide. No interference was observed for these compounds.
Accuracy of the immunoassay screening method
Method comparison studies were performed by comparing the results from 382 hair samples on the immunoassay screening method to the confirmatory LC/MS/MS method. The 382 samples included 318 head hair samples and 64 body hair samples.
The color of the hair samples included 159 black samples, 197 brown (from light brown to dark brown), 8 blond, 4 red, and 14 gray. The ethnicities represented included 175 Caucasian subjects, 40 African-American subjects, 125 Hispanic subjects, and 41 Asian subjects.
| ImmunoassayScreeningTest Result | Negative bythe predicatedevice or lessthan half thecutoffconcentrationbyLC/MS/MSanalysis | Near CutoffNegative(Between50% belowthe cutoff andthe cutoffconcentration) | Near CutoffPositive(Between thecutoff and50% abovethe cutoffconcentration) | High Positive(greater than50% abovethe cutoffconcentration) |
|---|---|---|---|---|
| (<0.500 ngoxazepamequivalents/10mg hair) | (0.500 - 1.000ng oxazepamequivalents/10mg hair) | (1.001 - 1.500ng oxazepamequivalents/10mg hair) | (>1.500 ngoxazepamequivalents/10mg hair) | |
| Positive | 5* | 19** | 17 | 107 |
| Negative | 233 | 1 | 0 | 0 |
Discordant Results
| ImmunoassayResult | LC/MS/MSResult | Sum ofconcentrationsbased on cross-reactivities |
|---|---|---|
| POS* | 0.092 ng Alprazolam | 0.255 |
| POS* | 0.093 ng Alprazolam | 0.258 |
| POS* | 0.196 ng Nordiazepam | 0.431 |
| POS* | 0.156 ng Alprazolam | 0.432 |
| POS* | 0.111 ng Nordiazepam, 0.217ng Oxazepam | 0.461 |
| POS** | 0.191 ng Alprazolam | 0.529 |
| POS** | 0.201 ng Alprazolam | 0.557 |
| POS** | 0.493 ng Lorazepam | 0.584 |
| POS** | 0.24 ng Alprazolam | 0.665 |
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| ImmunoassayResult | LC/MS/MSResult | Sum ofconcentrationsbased on cross-reactivities |
|---|---|---|
| POS** | 0.121 ng Diazepam | 0.666 |
| POS** | 0.073 ng Diazepam, 0.133 ngNordiazepam | 0.694 |
| POS** | 0.266 ng Alprazolam | 0.737 |
| POS** | 0.267 ng Alprazolam | 0.74 |
| POS** | 0.267 ng Alprazolam | 0.74 |
| POS** | 0.272 ng Alprazolam | 0.753 |
| POS** | 0.347 ng Nordiazepam | 0.763 |
| POS** | 0.283 ng Alprazolam | 0.784 |
| POS** | 0.293 ng Nordiazepam, 0.158ng Oxazepam | 0.803 |
| POS** | 0.099 ng Diazepam, 0.131 ngNordiazepam | 0.833 |
| POS** | 0.069 ng Alprazolam, 0.303 ngNordiazepam | 0.858 |
| POS** | 0.266 ng Alprazolam, 0.023 ngDiazepam | 0.863 |
| POS** | 0.322 ng Alprazolam | 0.892 |
| POS** | 0.427 ng Nordiazepam | 0.939 |
| POS** | 0.308 ng Temazepam | 0.961 |
Conclusion: The Psychemedics Microplate EIA for Benzodiazepines in Hair is substantially equivalent to the predicate, based on performance studies including precision, specificity, interference, and accuracy.
§ 862.3170 Benzodiazepine test system.
(a)
Identification. A benzodiazepine test system is a device intended to measure any of the benzodiazepine compounds, sedative and hypnotic drugs, in blood, plasma, and urine. The benzodiazepine compounds include chlordiazepoxide, diazepam, oxazepam, chlorzepate, flurazepam, and nitrazepam. Measurements obtained by this device are used in the diagnosis and treatment of benzodiazepine use or overdose and in monitoring levels of benzodiazepines to ensure appropriate therapy.(b)
Classification. Class II (special controls). A benzodiazepine test system is not exempt if it is intended for any use other than employment or insurance testing or is intended for Federal drug testing programs. The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9, provided the test system is intended for employment and insurance testing and includes a statement in the labeling that the device is intended solely for use in employment and insurance testing, and does not include devices intended for Federal drug testing programs (e.g., programs run by the Substance Abuse and Mental Health Services Administration (SAMHSA), the Department of Transportation (DOT), and the U.S. military).