Psychemedics Microplate EIA for Benzodiazepines in Hair
Device Facts
| Record ID | K163590 |
|---|---|
| Device Name | Psychemedics Microplate EIA for Benzodiazepines in Hair |
| Applicant | Psychemedics Corporation |
| Product Code | JXM · Clinical Toxicology |
| Decision Date | Sep 15, 2017 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.3170 |
| Device Class | Class 2 |
Indications for Use
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.
Device Story
Device performs qualitative screening for benzodiazepines in human hair samples; intended for workplace testing. Process involves two stages: pre-analytical hair digestion to convert solid matrix to liquid, followed by enzyme immunoassay (EIA). Screening uses microplate wells coated with Oxazepam-BSA conjugate; competitive binding between sample benzodiazepines and conjugate for monoclonal mouse anti-Oxazepam antibody; secondary goat anti-mouse HRP-conjugated antibody; TMB substrate; absorbance read at 450 nm. Preliminary positive results require confirmation via LC/MS/MS (AB Sciex API 3200) using deuterated internal standards in MRM mode. Samples collected in V-shaped aluminum foil, sealed in envelopes, and shipped at ambient temperature. Healthcare providers use preliminary results to identify potential drug use, necessitating confirmatory testing for clinical/workplace decision-making.
Clinical Evidence
Bench testing only. No clinical data. Analytical performance evaluated via precision studies (intra-assay and inter-assay), linearity (0.05–20.0 ng/10 mg hair), analytical specificity (cross-reactivity and interference testing), and cosmetic treatment effects. Method comparison performed on 382 hair samples (318 head, 64 body) against LC/MS/MS confirmation.
Technological Characteristics
Screening: BSA-oxazepam coated microplate, monoclonal mouse anti-oxazepam antibody, HRP-labeled secondary antibody, TMB substrate. Confirmation: Perkin Elmer Sciex API 3200 LC/MS/MS, Shimazu LC-20AD pumps, Leap Technologies PAL autosampler. Energy: Microplate reader (450/630 nm). Connectivity: Standalone. Software: AB Sciex MultiQuant v1.5.1/1.6.2 with SignalFinder Integration Algorithm.
Indications for Use
Indicated for qualitative detection of benzodiazepines in human head and body hair in workplace settings. Intended for use as a preliminary analytical test; requires confirmation by LC/MS/MS.
Regulatory Classification
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.
Special Controls
*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).
Predicate Devices
- Orasure Technologies Benzodiazepines Intercept Micro-plate EIA (k013882)
Related Devices
- K111927 — PSYCHEMEDICS METHAMPHETAMINE EIA · Psychemedics Corp. · May 1, 2012
- K123799 — PSYCHEMEDICS MICROPLATE EIA FOR OXYCODONE IN HAIR · Psychemedics Corp. · Feb 8, 2013
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k163590
B. Purpose for Submission:
New device
C. Measurand:
Benzodiazepines
D. Type of Test:
Qualitative screening test: enzyme immunoassay (EIA)
Quantitative confirmatory test: LC/MS/MS
E. Applicant:
Psychemedics Corporation
F. Proprietary and Established Names:
Psychemedics Microplate EIA for Benzodiazepines in Hair
G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| JXM | Class II | 21 CFR 862.3170 | Toxicology (91) |
H. Intended Use:
1. Intended use(s):
Refer to Indications for Use below.
2. Indication(s) for use:
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
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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.
3. Special conditions for use statement(s):
This assay is for over the counter use.
The Psychemedics Microplate EIA for Benzodiazepines in Hair combines a screening method (immunoassay) with a confirmation method (LC/MS/MS) in one test system.
4. Special instrument requirements:
The device is for use with a microplate reader capable of measuring at 450 and 630 nm. Plate washing also requires an instrument specifically designed to effectively and reproducibly wash all wells uniformly.
For confirmation testing, Psychemedics uses a AB Sciex API 3200 LC/MS/MS (Serial numbers AA24661109 and AA28841310), in multiple reaction monitoring (MRM) mode, linked to two Shimazu LC-20AD Micro pumps and a Leap Technologies PAL autosampler, along with deuterated internal standards.
I. Device Description:
The screening assay consists of:
- BSA-oxazepam coated Microplate
- Cutoff Calibrator (.8 ng oxazepam/37 uL) added to 8 mg hair
- Controls (minus 50% of cutoff, and plus 100% of cutoff)
- 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)
- Hair sample collection kit
The confirmation assay consists of a Perkin Elmer Sciex API 3200 LC/MS/MS (Serial
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numbers AA24661109 and AA28841310) linked to two Shimazu LC-20AD Micro pumps and a Leap Technologies PAL autosampler, an internal standard (0.2 ng oxazepam/10 mg hair), Signal peak identification and integration are performed with AB Sciex MultiQuant Software, version 1.5.1 and 1.6.2, using the SignalFinder Integration Algorithm that is a feature of the MultiQuant Software.
# J. Substantial Equivalence Information:
1. Predicate device name(s):
Orasure Technologies Benzodiazepines Intercept Micro-plate EIA
2. Predicate 510(k) number(s):
k013882
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device: Psychemedics Microplate EIA for Benzodiazepines in Hair | Predicate: k013882 Orasure Benzodiazepine Intercept Micro-plate EIA |
| Intended Use | Same | Qualitative detection of benzodiazepines |
| Methodology | Same | Enzyme Immunoassay read at 450 nm |
| Calibrator Drug | Same | Oxazepam |
| Differences | | |
| --- | --- | --- |
| Item | Device: Psychemedics Microplate EIA for Benzodiazepines in Hair | Predicate: k013882 Orasure Benzodiazepine Intercept Micro-plate EIA |
| Matrix | Hair | Oral Fluid |
| Confirmation Method | LC/MS/MS | GC/MS/MS |
| Cutoff | 1 ng / 10 mg hair | 1 ng / mL oral fluid |
| Intended User | Intended to be used in house at Psychemedics only | Intended to be marketed to individual laboratories |
| Extraction Step | Required; performed in-house | Not required |
# K. Standard/Guidance Document Referenced (if applicable):
None referenced
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L. Test Principle:
The Psychemedics Microplate EIA For Benzodiazepines in Hair consists of (1) a screening immunoassay and (2) a confirmation test by LC-MS/MS.
The screening immunoassay consists of two parts; a pre-analytical hair treatment procedure (to remove benzodiazepines from solid hair matrix to a measurable liquid matrix), and the qualitative assay. The qualitative assay is an Enzyme-Linked ImmunoSorbent Assay (ELISA) based upon the competitive binding of benzodiazepines in the sample and solid-phase oxazepam in the microplate wells to the assay detection antibody. Sample is added to the microplate well, followed by primary antibody (monoclonal mouse anti-oxazepam), and then (after washing the plate) goat anti-mouse-horseradish peroxidase conjugate is added. Enzyme substrate is then added and the plate is read on a microplate reader at 450 nm.
Confirmatory testing:
Confirmation testing of samples is performed on a 6-12 mg aliquot of the original hair specimen. The hair is washed with isopropanol, followed by phosphate buffer. After washing, samples are then spiked with deuterated benzodiazepines (Alprazolam-D5, Lorazepam-D4, Diazepam-D5, Nordiazepam-D5, Oxazepam-D5, and Temazepam-D5) to a concentration of 1.0 ng/10 mg hair, processed by solid phase extraction and analyzed using the Perkin Elmer Sciex API 3200 LC/MS/MS operating in the Positive Multiple Reaction Mode. To account for variability in drug recovery during solid phase extraction the recovered concentration of the deuterated internal standards in each sample is used to normalize the data for that sample.
M. Performance Characteristics:
1. Analytical performance:
a. Precision/Reproducibility:
Precision of the immunoassay screening method
Hair samples previously tested and shown to be negative for benzodiazepines were spiked with oxazepam at the following concentrations: 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2 ng/10 mg hair (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff). Intra-Assay Precision was demonstrated by measuring these nine concentrations of oxazepam in hair samples in replicates of 15 within a single run. Results are summarized below.
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| Concentration relative to cutoff (1 ng/10 mg hair) | 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 |
Hair samples previously tested and shown to be negative for benzodiazepines were spiked with oxazepam at the following concentrations: 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2 ng/10 mg hair (negative, ±75%, ±50%, ±25%, cutoff, and +100% of the cutoff). Inter-Assay Precision of the assay was evaluated by measuring these nine concentrations of oxazepam in hair samples over the course of five days, in replicates of 15 per day. Results are summarized below.
| Concentration relative to cutoff (1 ng/10 mg hair) | 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
Intermediate precision around the LC/MS/MS cutoff was conducted using drug negative hair samples spiked with 0.05, 0.1, 0.15, 0.2, 0.25, or 0.3 ng/10 mg hair. Samples were prepared in replicates of 5 per each concentration, and run over 5 days for 25 determinations per drug. Results for the six benzodiazepines measured by the LC-MS/MS assay are summarized below.
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| | ALPRAZOLAM (ng/10 mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Concentration | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 |
| Average | 0.0510 | 0.1031 | 0.1513 | 0.2088 | 0.2551 | 0.3128 |
| S.D. | 0.0049 | 0.0093 | 0.0100 | 0.0109 | 0.0111 | 0.0188 |
| % CV | 9.66 | 8.99 | 6.59 | 5.23 | 4.37 | 6.00 |
| Percent of Target | 102.1 | 103.1 | 100.9 | 104.4 | 102.0 | 104.3 |
| | LORAZEPAM (ng/10mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Concentration | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 |
| Average | 0.05088 | 0.10128 | 0.1502 | 0.19632 | 0.24548 | 0.29756 |
| S.D. | 0.0046 | 0.0060 | 0.0104 | 0.0144 | 0.0123 | 0.0164 |
| % CV | 9.03 | 5.93 | 6.92 | 7.34 | 5.02 | 5.52 |
| Percent of Target | 101.8 | 101.3 | 100.1 | 98.2 | 98.2 | 99.2 |
| | DIAZEPAM (ng/10 mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Concentration | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.3 |
| Average | 0.0498 | 0.1035 | 0.1587 | 0.2095 | 0.2661 | 0.3179 |
| S.D. | 0.0047 | 0.0062 | 0.0113 | 0.0143 | 0.0131 | 0.0190 |
| % CV | 9.51 | 6.01 | 7.12 | 6.84 | 4.92 | 5.99 |
| Percent of Target | 99.7 | 103.5 | 105.8 | 104.8 | 106.4 | 106.0 |
| | NORDIAZEPAM (ng/10mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Concentration | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 |
| Average | 0.0490 | 0.0990 | 0.1502 | 0.1960 | 0.2478 | 0.3044 |
| S.D. | 0.0043 | 0.0068 | 0.0103 | 0.0097 | 0.0149 | 0.0171 |
| % CV | 8.72 | 6.83 | 6.86 | 4.93 | 6.01 | 5.62 |
| Percent of Target | 98.0 | 99.0 | 100.1 | 98.0 | 99.1 | 101.5 |
| | OXAZEPAM (ng/10mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Concentration | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 |
| Average | 0.0497 | 0.1016 | 0.1554 | 0.1991 | 0.2576 | 0.3102 |
| S.D. | 0.0042 | 0.0082 | 0.0120 | 0.0105 | 0.0130 | 0.0115 |
| % CV | 8.50 | 8.10 | 7.71 | 5.29 | 5.06 | 3.72 |
| Percent of Target | 99.4 | 101.6 | 103.6 | 99.5 | 103.1 | 103.4 |
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Within run precision throughout the LC/MS/MS range was conducted using drug negative hair samples spiked with 0.05, 0.1, 0.15, 0.2, 1.0, 10.0, 15.0, or 20.0 ng/10 mg hair. Samples were prepared in replicates of 5, per each concentration, and concentrations were determined in one run. Results for the six benzodiazepines measured by the LC-MS/MS assay are summarized below.
| | Target Concentration (ng/10 mg hair) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | 0.05 | 0.10 | 0.15 | 0.20 | 1.0 | 10 | 15 |
| Alprazolam concentration | 0.056 | 0.100 | 0.158 | 0.234 | 0.947 | 10.395 | 15.898 |
| | 0.051 | 0.092 | 0.148 | 0.217 | 0.953 | 10.279 | 15.885 |
| | 0.054 | 0.093 | 0.162 | 0.222 | 0.903 | 10.430 | 15.934 |
| | 0.045 | 0.095 | 0.161 | 0.200 | 0.944 | 10.670 | 14.732 |
| | 0.053 | 0.090 | 0.164 | 0.198 | 0.947 | 10.196 | 15.577 |
| Average S.D. % CV | 0.0518 | 0.094 | 0.1586 | 0.2142 | 0.9388 | 10.394 | 15.605 |
| | 0.004 | 0.004 | 0.006 | 0.015 | 0.020 | 0.180 | 0.509 |
| | 8.12 | 4.05 | 3.98 | 7.10 | 2.16 | 1.73 | 3.26 |
| | 103.6 | 94.0 | 105.7 | 107.1 | 93.9 | 103.9 | 104.0 |
| | | | | | | | |
| Lorazepam concentration | 0.052 | 0.085 | 0.154 | 0.22 | 0.906 | 10.191 | 15.072 |
| | 0.044 | 0.092 | 0.147 | 0.21 | 0.923 | 10.165 | 14.604 |
| | 0.05 | 0.092 | 0.148 | 0.204 | 0.895 | 10.262 | 14.41 |
| | 0.05 | 0.102 | 0.137 | 0.2 | 0.92 | 10.429 | 15.425 |
| Average S.D. % CV | 7.48 | 7.29 | 6.39 | 4.11 | 1.27 | 3.03 | 3.06 |
| | 100.0 | 94.2 | 99.9 | 105.2 | 91.0 | 103.9 | 98.5 |
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| Diazepam concentration | 0.053 | 0.088 | 0.15 | 0.211 | 0.928 | 10.417 | 14.39 | 18.679 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 0.054 | 0.092 | 0.151 | 0.201 | 0.945 | 10.354 | 14.444 | 18.06 |
| | 0.044 | 0.084 | 0.163 | 0.203 | 0.96 | 10.561 | 14.943 | 19.458 |
| | 0.049 | 0.085 | 0.143 | 0.196 | 0.949 | 10.03 | 14.692 | 19.613 |
| | 0.054 | 0.094 | 0.167 | 0.209 | 0.936 | 10.439 | 14.661 | 19.133 |
| Average S.D. % CV | 0.0508 | 0.0886 | 0.1548 | 0.204 | 0.9436 | 10.360 | 14.626 | 18.9886 |
| | 0.004 | 0.004 | 0.010 | 0.006 | 0.012 | 0.199 | 0.221 | 0.630 |
| | 8.51 | 4.89 | 6.40 | 2.98 | 1.30 | 1.92 | 1.51 | 3.32 |
| | 101.6 | 88.6 | 103.2 | 102.0 | 94.4 | 103.6 | 97.5 | 94.9 |
| | Target Concentration (ng/10 mg hair) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 0.05 | 0.10 | 0.15 | 0.20 | 1.0 | 10 | 15 | 20 |
| Nordiazepam concentration | 0.049 | 0.086 | 0.151 | 0.201 | 0.904 | 10.659 | 14.942 | 19.881 |
| | 0.048 | 0.088 | 0.155 | 0.2 | 0.947 | 10.654 | 14.836 | 19.869 |
| | 0.056 | 0.092 | 0.14 | 0.201 | 0.937 | 10.278 | 14.917 | 20.328 |
| | 0.047 | 0.093 | 0.161 | 0.213 | 0.933 | 10.738 | 14.94 | 19.692 |
| | 0.044 | 0.095 | 0.163 | 0.208 | 0.902 | 10.132 | 14.717 | 19.635 |
| Average S.D. % CV | 0.0488 | 0.0908 | 0.154 | 0.2046 | 0.9246 | 10.492 | 14.870 | 19.881 |
| | 0.004 | 0.004 | 0.009 | 0.006 | 0.020 | 0.269 | 0.096 | 0.272 |
| | 9.10 | 4.08 | 5.95 | 2.78 | 2.20 | 2.57 | 0.65 | 1.37 |
| | 97.6 | 90.8 | 102.7 | 102.3 | 92.5 | 104.9 | 99.1 | 99.4 |
| | | | | | | | | |
| Oxazepam concentration | 0.058 | 0.101 | 0.168 | 0.21 | 0.926 | 9.841 | 15.053 | 19.451 |
| | 0.05 | 0.093 | 0.168 | 0.211 | 0.93 | 10.277 | 14.791 | 19.701 |
| | 0.049 | 0.099 | 0.161 | 0.198 | 0.903 | 10.146 | 14.561 | 19.889 |
| | 0.048 | 0.091 | 0.158 | 0.2 | 0.905 | 10.394 | 15.271 | 19.595 |
| | 0.055 | 0.093 | 0.162 | 0.198 | 0.878 | 10.417 | 15.164 | 19.593 |
| Average S.D. % CV | 0.052 | 0.0954 | 0.1634 | 0.2034 | 0.9084 | 10.215 | 14.968 | 19.6458 |
| | 0.004 | 0.004 | 0.004 | 0.007 | 0.021 | 0.235 | 0.289 | 0.162 |
| | 8.27 | 4.54 | 2.72 | 3.22 | 2.30 | 2.30 | 1.93 | 0.83 |
| | 104.0 | 95.4 | 108.9 | 101.7 | 90.8 | 102.2 | 99.8 | 98.2 |
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A second study was conducted for precision using triplicate analyses of authentic (non-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.
| Alprazolam (ng/10 mg hair) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample # | 1 | 2 | 3 | 4 | 5 | | |
| Replicate 1 | 6.477 | 0.199 | 2.941 | 7.408 | 3.802 | | |
| Replicate 2 | 6.713 | 0.202 | 2.886 | 7.702 | 4.746 | | |
| Replicate 3 | 5.711 | 0.243 | 3.159 | 7.269 | 4.189 | | |
| Mean | 6.3 | 0.2 | 3.0 | 7.5 | 4.2 | | |
| Standard Deviation | 0.5 | 0.0 | 0.1 | 0.2 | 0.5 | | |
| %CV | 8.3 | 11.5 | 4.8 | 3.0 | 11.2 | | |
| Lorazepam (ng/10 mg hair) | | | | | | | |
| Sample # | 1 | 2 | 3 | 4 | | | |
| Replicate 1 | 1.174 | 0.555 | 0.536 | 0.341 | | | |
| Replicate 2 | 1.222 | 0.643 | 0.57 | 0.375 | | | |
| Replicate 3 | 1.102 | 0.591 | 0.59 | 0.333 | | | |
| Mean | 1.2 | 0.6 | 0.6 | 0.3 | | | |
| Standard Deviation | 0.1 | 0.0 | 0.0 | 0.0 | | | |
| %CV | 5.2 | 7.4 | 4.8 | 6.4 | | | |
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| Diazepam (ng/10 mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample # | 1 | 2 | 3 | 4 | 5 |
| Replicate 1 | 0.229 | 8.365 | 1.417 | 4.642 | 0.77 |
| Replicate 2 | 0.235 | 8.443 | 1.572 | 3.75 | 0.903 |
| Replicate 3 | 0.205 | 8.519 | 1.215 | 3.929 | 0.818 |
| Mean | 0.2 | 8.4 | 1.4 | 4.1 | 0.8 |
| Standard Deviation | 0.0 | 0.1 | 0.2 | 0.5 | 0.1 |
| %CV | 7.1 | 0.9 | 12.8 | 11.5 | 8.1 |
| Nordiazepam (ng/10 mg hair) | | | | |
| --- | --- | --- | --- | --- |
| Sample # | 1 | 2 | 3 | 4 |
| Replicate 1 | 9.227 | 3.377 | 0.884 | 0.25 |
| Replicate 2 | 9.928 | 3.725 | 0.923 | 0.226 |
| Replicate 3 | 10.258 | 3.202 | 0.954 | 0.252 |
| Mean | 9.8 | 3.4 | 0.9 | 0.2 |
| Standard Deviation | 0.5 | 0.3 | 0.0 | 0.0 |
| %CV | 5.4 | 7.8 | 3.8 | 6.0 |
| Oxazepam (ng/10 mg hair) | | | | |
| --- | --- | --- | --- | --- |
| Sample # | 1 | 2 | 3 | 4 |
| Replicate 1 | 0.176 | 0.262 | 0.161 | 0.073 |
| Replicate 2 | 0.179 | 0.267 | 0.149 | 0.071 |
| Replicate 3 | 0.146 | 0.274 | 0.143 | 0.066 |
| Mean | 0.2 | 0.3 | 0.2 | 0.1 |
| Standard Deviation | 0.0 | 0.0 | 0.0 | 0.0 |
| %CV | 10.9 | 2.3 | 6.1 | 5.2 |
| Temazepam (ng/10 mg hair) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample # | 1 | 2 | 3 | 4 | 5 |
| Replicate 1 | 1.342 | 0.22 | 0.141 | 0.133 | 0.136 |
| Replicate 2 | 1.327 | 0.22 | 0.147 | 0.129 | 0.139 |
| Replicate 3 | 1.291 | 0.233 | 0.114 | 0.136 | 0.132 |
| Mean | 1.3 | 0.2 | 0.1 | 0.1 | 0.1 |
| Standard Deviation | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| %CV | 2.0 | 3.3 | 13.1 | 2.6 | 2.6 |
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b. Linearity/assay reportable range:
The screening immunoassay is a qualitative test only; therefore, a linearity evaluation is not applicable.
Linearity of the LC-MS/MS confirmation method
The linearity of the confirmation method was evaluated by spiking each of the benzodiazepine analytes (Alprazolam, Lorazepam, Diazepam, Nordiazepam, Oxazepam, and Temazepam) into a negative hair 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 (0, 0.05, 0.1, 0.15, 0.2, 1.0, 10.0, 15.0, or 20.0 ng/10 mg hair). All levels for all benzodiazepines demonstrated percent recoveries within +/- 10% of the expected value.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Controls and calibrators are prepared as a methanolic solution containing oxazepam, and are traceable to a commercial oxazepam source. The value of the cutoff for oxazepam is 1 ng/10 mg hair.
Stability studies for both controls and calibrators have been conducted. Protocols and acceptance criteria were described and found to be acceptable. The manufacturer claims the following expiration date for both controls and calibrators: When stored at less than or equal to -10° C the calibrators and controls are stable for one year.
Shipping stability: 19 hair samples containing benzodiazepines were shipped overnight to another location, held for four days, and then returned to Psychemedics by overnight shipping. Samples were tested by the routine LC/MS/MS procedure both before being shipped and after returning to Psychemedics. All six benzodiazepines were evaluated, and recoveries after shipping were found to be acceptable.
Sample storage stability: 21 hair samples were stored in the same collection foil and card-envelope provided with the hair collection kit, following the instructions provided in the package insert. Samples were tested by LC/MS/MS on day 0, and again at 12 months, following storage at 20° - 24° C (68° - 75° F). All six benzodiazepines were evaluated, and recoveries after storage were found to be acceptable.
d. Detection limit:
The screening immunoassay is a qualitative test only; therefore, a detection limit evaluation is not applicable. See section M.1.a. above for sensitivity around the immunoassay device cutoff.
11
{11}
12
# Detection Limit of the LC-MS/MS method
The sponsor performed a study to determine the Lower limit of Quantitation (LLOQ) of the LC-MS/MS assay for each of the six benzodiazepines in the assay. Four types of hair (Caucasian, Hispanic, African-American, and Asian) were obtained and were cut into three segments for a total of 12 hair segments to be analyzed per drug. Each of the 12 hair segments was independently spiked with each specific benzodiazepine and analyzed according to the LC-MS/MS procedure stated in the package insert.
The results from the study support an LLOQ of 0.05 ng/10 mg hair, and a reportable range of 0.05 to 20.0 ng/10 mg hair for all six drugs.
e. Analytical specificity:
## Specificity of the immunoassay screening method
### Immunoassay cross-reactivity with structurally related compounds
The cross-reactivity characteristics of the screening immunoassay was evaluated by spiking various concentrations of 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.
| Compound | Percent Cross-reactivity | Expected Concentration in ng/10 mg hair equivalent to cutoff calibrator |
| --- | --- | --- |
| 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 |
{12}
13
| Compound | Percent Cross-reactivity | Expected Concentration in ng/10 mg hair equivalent to cutoff calibrator |
| --- | --- | --- |
| Clonazepam | 33 | 3 |
| Triazolam | 31 | 3.2 |
| alpha-Hydroxyprazolam | 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 $\pm 50\%$ of the cutoff to assess possible positive or negative interference. All potential interferents listed below were tested at a concentration of $100\mathrm{ng} / 10\mathrm{mg}$ hair. No positive or negative interference was observed in this study.
| 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-9
THC | Ecgononine | Nortriptyline |
| Acetaminophen | Erythromycin | O-Desmethyvenlafaxine |
| Amitriptyline | Ethosuximide | Oxycodone |
| Amobarbital | Ethylmorphine | Penicillin G |
| Amoxicillin | Fentanyl | Pentazocine |
| Anhydroecgonine methyl ester | Haloperidol | Phendimetrazine |
{13}
| 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 |
| Despiramine | Naloxone | Trimipramine |
| Despropionyl fentanyl | Naltrexone | Vanilmandelic acid(+/-) |
| | | Venlafaxine hydrochloride |
## Effect of cosmetic treatments
To evaluate any effects of cosmetic treatments, 20 hair samples negative for benzodiazepines and 8 hair samples positive for benzodiazepines were treated with the following cosmetic treatments: permanent wave, relaxer, bleach, dye, and shampoo. After the treatments these samples and an aliquot of the same hair samples, untreated, were digested and assayed by the immunoassay screening method. All negative results remained negative and all positive results remained positive after the treatments.
The effect of cosmetic treatments was also evaluated using the LC-MS/MS method, for the same samples. The sponsor determined the concentrations of drug before and after treatment (% recovery in parentheses) and the results are provided below (ND = not detected in either untreated or treated samples).
{14}
| Effect of Dye Product #2 on Benzodiazepines in Hair – ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | 1.158 | 3.447 | 1.090 | ND | ND | 1 |
| 1.563 | 0.792 | ND | ND | ND | ND | 2 |
| 0.910 | ND | ND | ND | ND | ND | 3 |
| 0.992 | ND | ND | ND | ND | ND | 4 |
| Dyed | | | | | | |
| ND | 0.941 (81%) | 2.075 (60%) | 1.195 (110%) | ND | ND | 1 |
| .901(58%) | 0.740 (93%) | ND | ND | ND | ND | 2 |
| 1.258(133%) | ND | ND | ND | ND | ND | 3 |
| .623(63%) | ND | ND | ND | ND | ND | 4 |
| Effect of Bleach Product #1 on Benzodiazepines in Hair – ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | ND | ND | ND | ND | 0.847 | 1 |
| ND | ND | ND | 0.613 | ND | ND | 2 |
| 1.737 | ND | 0.403 (94%) | 1.128 | ND | ND | 3 |
| ND | ND | 3.826 | 5.057 | ND | ND | 4 |
| Bleached | | | | | | |
| ND | ND | ND | ND | ND | 0.996 (118%) | 1 |
| ND | ND | ND | 0.476 (78%) | ND | ND | 2 |
| 1.500 (86%) | ND | 0.378 (94%) | 1.326 (118%) | ND | ND | 3 |
| ND | ND | 2.930 (77%) | 4.526 (89%) | ND | ND | 4 |
{15}
| Effect of Bleach Product #2 on Benzodiazepines in Hair – ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | ND | 1.947 | 5.643 | 0.610 | 0.392 | 1 |
| 0.876 | ND | ND | ND | ND | ND | 2 |
| 1.410 | 0.567 | ND | ND | ND | ND | 3 |
| 0.992 | ND | ND | ND | ND | ND | 4 |
| Bleached | | | | | | |
| ND | ND | 2.25 (116%) | 5.941 (105%) | 0.92 (151%) | 0.46 (118%) | 1 |
| 0.819 (93%) | ND | ND | ND | ND | ND | 2 |
| 1.330 (94%) | 0.50 (88%) | ND | ND | ND | ND | 3 |
| .788 (79%) | ND | ND | ND | ND | ND | 4 |
| Effect of Relaxer Product #1 on Benzodiazepines in Hair – ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | ND | ND | ND | ND | 0.847 | 1 |
| ND | ND | ND | 0.613 | ND | ND | 2 |
| ND | ND | 1.101 | 1.702 | ND | ND | 3 |
| 0.226 | 1.158 | 3.447 | 1.070 | ND | ND | 4 |
| Treated | | | | | | |
| ND | ND | ND | ND | ND | 0.985 (116%) | 1 |
| ND | ND | ND | 0.641 (105%) | ND | ND | 2 |
| ND | ND | 1.11 (101%) | 2.051 (121%) | ND | ND | 3 |
| 0.314 (139%) | 1.37 (119%) | 2.113 (61%) | 0.970 (91%) | ND | ND | 4 |
{16}
| Effect of Relaxer Product #2 on Benzodiazepines in Hair - ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| 1.369 | ND | ND | ND | ND | ND | 1 |
| 5.957 | ND | .426 | .210 | ND | .496 | 2 |
| 1.027 | ND | ND | ND | ND | ND | 3 |
| ND | ND | 0.548 | 1.020 | ND | ND | 4 |
| Relaxed | | | | | | |
| 1.228 (90%) | ND | ND | ND | ND | ND | 1 |
| 3.821 (64%) | ND | .392 (92%) | .163 (78%) | ND | .319 (64%) | 2 |
| 1.05 (103%) | ND | ND | ND | ND | ND | 3 |
| ND | ND | 0.60 (109%) | 1.172 (115%) | ND | ND | 4 |
| Effect of Shampoo Product #1 on Benzodiazepines in Hair - ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | ND | ND | ND | ND | 0.601 | 1 |
| ND | ND | ND | 6.928 | ND | ND | 2 |
| ND | ND | ND | 0.281 | ND | ND | 3 |
| ND | ND | 1.947 | 5.643 | 0.610 | 0.392 | 4 |
| Shampoo | | | | | | |
| ND | ND | ND | ND | ND | 0.384 (64%) | 1 |
| ND | ND | ND | 8.245 (119%) | ND | ND | 2 |
| ND | ND | ND | 0.285 (101%) | ND | ND | 3 |
| ND | ND | 2.258 (116%) | 5.274 (93%) | 0.77 (127%) | 0.50 (128%) | 4 |
{17}
18
| Effect of Shampoo Product #2 on Benzodiazepines in Hair - ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| 1.563 | 0.792 | ND | ND | ND | ND | 1 |
| 1.410 | 0.567 | ND | ND | ND | ND | 2 |
| 0.992 | ND | ND | ND | ND | ND | 3 |
| ND | ND | 0.766 | 1.824 | ND | ND | 4 |
| Shampoo | | | | | | |
| 1.100 (70%) | 0.605 (76%) | ND | ND | ND | ND | 1 |
| 1.156 (82%) | 0.57 (101%) | ND | ND | ND | ND | 2 |
| 1.018 (103%) | ND | ND | ND | ND | ND | 3 |
| ND | ND | 0.613 (80%) | 1.493 (82%) | ND | ND | 4 |
| Effect of Perm Product #1 on Benzodiazepines in Hair - ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| ND | ND | ND | ND | ND | 0.601 | 1 |
| ND | ND | ND | ND | ND | 0.847 | 2 |
| ND | ND | 3.826 | 5.057 | ND | 0.120 | 3 |
| 1.800 | ND | 1.241 | 1.214 | ND | ND | 4 |
| Permed | | | | | | |
| ND | ND | ND | ND | ND | 0.274 (46%) | 1 |
| ND | ND | ND | ND | ND | 0.942 (111%) | 2 |
| ND | ND | 2.016 (53%) | 3.177 (63%) | ND | ND | 3 |
| 2.119 (118%) | ND | 1.000 (81%) | 1.182(97%) | ND | ND | 4 |
| Effect of Perm Product #2 on Benzodiazepines in Hair - ng/10 mg hair | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Alprazolam | Lorazepam | Diazepam | Nordiazepam | Oxazepam | Temazepam | Sample # |
| Untreated | | | | | | |
| 1.563 | 0.792 | ND | ND | ND | ND | 1 |
| 2.634 | ND | ND | ND | ND | ND | 2 |
| 5.957 | ND | 0.426 | ND | ND | 0.496 | 3 |
{18}
| 0.992 | ND | ND | ND | ND | ND | 4 |
| --- | --- | --- | --- | --- | --- | --- |
| Permed | | | | | | |
| 1.138
(73%) | 0.575
(73%) | ND | ND | ND | ND | 1 |
| 2.255
(86%) | ND | ND | ND | ND | ND | 2 |
| 3.014
(51%) | ND | 0.247
(58%) | ND | ND | 0.276 (56%) | 3 |
| 0.913
(92%) | ND | ND | ND | ND | ND | 4 |
## 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 hair samples spiked with benzodiazepines at the cutoff of 0.20 ng/10 mg/hair, and also containing the following structurally related compounds at a concentration of 0.20 ng/10 mg/hair: Clonazepam, Estazolam, Flurazepam, Meprobromate, Midazolam, Nitrazepam, Prazepam, Triazolam, and Zolpidem. All of the samples spiked with the potential cross-reactant and one of the target drugs at the cutoff produced a concentration within ± 15% of the spiked value of 0.20 ng/10 mg/hair. The sponsor concluded that these nine benzodiazepine compounds did not cross-react or cause interference with the LC-MS/MS assay.
### Interference from structurally unrelated compounds
The specificity of the LC-MS/MS confirmation assay with structurally unrelated compounds was evaluated by measuring samples spiked with benzodiazepines at the cutoff of 0.20 ng/10 mg/hair, containing the following compounds at the concentrations listed: Cotinine (500 ng/10 mg hair), Nicotine (500 ng/10 mg hair), Caffeine (500 ng/10 mg hair), Ibuprofen (50 ng/10 mg hair), Naproxen (50 ng/10 mg hair), Phentermine (20 ng/10 mg hair), Pseudoephedrine (20 ng/10 mg hair), Morphine (200 ng/10 mg hair), Hydrocodone (200 ng/10 mg hair), Oxycodone (200 ng/10 mg hair), Codeine (200 ng/10 mg hair), Cocaine (200 ng/10 mg hair), Phencyclidine (200 ng/10 mg hair), Methamphetamine (200 ng/10 mg hair), Amphetamine ((200 ng/10 mg hair), Methadone (200 ng/10 mg hair), Phenobarbital (200 ng/10 mg hair), Phenytoin (200 ng/10 mg hair), Phenylephrine(200 ng/10 mg hair), Carbamazepine (200 ng/10 mg hair), Gabapentin (500 ng/10 mg hair), Salicylic Acid (200 ng/10 mg hair), Valproic Acid (200 ng/10 mg hair), Oxcarbazepine (200 ng/10 mg hair), Propoxyphene (200 ng/10 mg hair), Acetaminophen (200 ng/10 mg hair), Norfloxacin (0.2 ng/10 mg hair), Psilocybin (200 ng/10 mg hair), Nimesulide (200 ng/10 mg hair), Etifoxine (200 ng/10 mg hair), Chlorcyclizine (200 ng/10 mg hair), and Teriflunomide (200 ng/10 mg hair).
19
{19}
All of the samples spiked with the potential interfering compounds and one of the target drugs at the cutoff produced a concentration within ± 15% of the spiked value of 0.20 ng/10 mg/hair. The sponsor concluded that the potentially interfering compounds listed above did not cross-react or cause interference with the LC-MS/MS assay.
## Studies to evaluate environmental contamination
Studies were performed to evaluate environmental contamination. Two different aliquots of 10 different hair specimens, previously shown to be negative for benzodiazepines, were soaked in either a water-based solution containing 5 ng benzodiazepines /mL or a saline-based solution containing 5 ng benzodiazepines /mL, resulting in a range of benzodiazepines on the hair from below the Limit of Quantitation (LOQ) to 0.452 ng of benzodiazepines /10 mg hair, prior to washing. After washing hair samples according to the instructions in the product insert, no samples contained an amount of drug above the LOQ of the confirmatory method.
### f. Assay cut-off:
Analytical performance of the device around the claimed cutoff is described in precision section M.1.a. above.
### 2. Comparison studies:
#### a. Method comparison with predicate device:
##### 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.
20
{20}
| Immunoassay Screening Test Result | Negative by the predicate device or less than half the cutoff concentration by LC/MS/MS analysis (<0.500 ng oxazepam equivalents/10 mg hair) | Near Cutoff Negative (Between 50% below the cutoff and the cutoff concentration) (0.500 – 1.000 ng oxazepam equivalents/10 mg hair) | Near Cutoff Positive (Between the cutoff and 50% above the cutoff concentration) (1.001 – 1.500 ng oxazepam equivalents/10 mg hair) | High Positive (greater than 50% above the cutoff concentration) (>1.500 ng oxazepam equivalents/10 mg hair) |
| --- | --- | --- | --- | --- |
| Positive | 5* | 19** | 17 | 107 |
| Negative | 233 | 1 | 0 | 0 |
Discordant Results
| Immunoassay Result | LC/MS/MS Result | Sum of concentrations based 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, 0.217 ng 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 |
| POS** | 0.121 ng Diazepam | 0.666 |
| POS** | 0.073 ng Diazepam, 0.133 ng Nordiazepam | 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, 0.158 ng Oxazepam | 0.803 |
{21}
The accuracy of the LC-MS/MS method is supported by the data collected to evaluate intra-assay precision around the cutoff (see section M.1.a.). The average recovery for all concentrations for all six benzodiazepines was within $\pm 7\%$ of the target value.
## Recovery study:
The sponsor conducted a study to evaluate recovery for all six benzodiazepines at concentrations of 0.2, 10, and $20\mathrm{ng} / 10\mathrm{mg}$ hair. For each combination of drug and concentration level, nine individual hair samples were prepared and analyzed. Results are summarized below.
| Sample # | Alprazolam | | Lorazepam | | Diazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 1 | 0.171 | 85.5 | 0.172 | 86 | 0.171 | 85.5 |
| 2 | 0.180 | 90.0 | 0.206 | 103 | 0.215 | 107.5 |
| 3 | 0.179 | 89.5 | 0.202 | 101 | 0.203 | 101.5 |
| 4 | 0.183 | 91.5 | 0.192 | 96 | 0.182 | 91.0 |
| 5 | 0.187 | 93.5 | 0.181 | 90.5 | 0.190 | 95.0 |
| 6 | 0.172 | 86.0 | 0.179 | 89.5 | 0.196 | 98.0 |
| 7 | 0.179 | 89.5 | 0.213 | 106.5 | 0.172 | 86.0 |
| 8 | 0.199 | 99.5 | 0.177 | 88.5 | 0.190 | 95.0 |
| 9 | 0.189 | 94.5 | 0.209 | 104.5 | 0.211 | 105.5 |
| | | | | | | |
| Average | 0.182 | 91.1 | 0.192 | 96.2 | 0.192 | 96.1 |
| S.D. | 0.009 | 4.35 | 0.016 | 7.79 | 0.016 | 7.86 |
| %CV | 4.78 | 4.78 | 8.10 | 8.10 | 8.18 | 8.18 |
{22}
| Sample # | Nordiazepam | | Oxazepam | | Temazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 1 | 0.198 | 99 | 0.179 | 89.5 | 0.172 | 86.0 |
| 2 | 0.207 | 103.5 | 0.227 | 113.5 | 0.185 | 92.5 |
| 3 | 0.229 | 114.5 | 0.22 | 110.0 | 0.183 | 91.5 |
| 4 | 0.193 | 96.5 | 0.216 | 108.0 | 0.186 | 93.0 |
| 5 | 0.192 | 96 | 0.186 | 93.0 | 0.183 | 91.5 |
| 6 | 0.177 | 88.5 | 0.2 | 100.0 | 0.172 | 86.0 |
| 7 | 0.196 | 98 | 0.228 | 114.0 | 0.172 | 86.0 |
| 8 | 0.214 | 107 | 0.225 | 112.5 | 0.173 | 86.0 |
| 9 | 0.172 | 86 | 0.221 | 110.5 | 0.184 | 92.0 |
| | | | | | | |
| Average | 0.198 | 98.8 | 0.211 | 105.7 | 0.179 | 89.4 |
| S.D. | 0.018 | 8.80 | 0.018 | 9.21 | 0.006 | 3.25 |
| %CV | 8.91 | 8.91 | 8.72 | 8.72 | 3.56 | 3.63 |
| Sample # | Alprazolam | | Lorazepam | | Diazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 10 | 9.19 | 91.9 | 9.67 | 96.7 | 10.35 | 103.4 |
| 11 | 8.91 | 89.1 | 8.68 | 86.8 | 9.11 | 91.1 |
| 12 | 9.02 | 90.2 | 8.82 | 88.2 | 10.17 | 101.7 |
| 13 | 8.92 | 89.2 | 9.50 | 95.0 | 9.14 | 91.4 |
| 14 | 8.93 | 89.3 | 9.83 | 98.3 | 9.72 | 97.2 |
| 15 | 10.51 | 105.1 | 8.68 | 86.8 | 9.23 | 92.3 |
| 16 | 9.30 | 93.0 | 8.87 | 88.7 | 10.00 | 100.0 |
| 17 | 9.84 | 98.4 | 8.75 | 87.5 | 9.79 | 97.9 |
| 18 | 9.25 | 92.5 | 9.40 | 94.0 | 9.41 | 94.1 |
| | | | | | | |
| Average | 9.320 | 93.2 | 9.133 | 91.3 | 9.657 | 96.6 |
| S.D. | 0.535 | 5.34 | 0.463 | 4.63 | 0.459 | 4.58 |
| %CV | 5.74 | 5.73 | 5.07 | 5.07 | 4.75 | 4.74 |
{23}
| Sample # | Nordiazepam | | Oxazepam | | Temazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 10 | 10.79 | 107.87 | 10.92 | 109.2 | 9.47 | 94.7 |
| 11 | 9.91 | 99.13 | 10.10 | 101.0 | 8.54 | 85.4 |
| 12 | 9.56 | 95.6 | 10.66 | 106.6 | 8.52 | 85.2 |
| 13 | 9.68 | 96.8 | 10.34 | 103.4 | 9.05 | 90.5 |
| 14 | 9.15 | 91.52 | 9.60 | 96.0 | 8.62 | 86.2 |
| 15 | 9.47 | 94.7 | 10.27 | 102.7 | 9.34 | 93.4 |
| 16 | 10.20 | 101.97 | 10.42 | 104.2 | 9.04 | 90.4 |
| 17 | 9.57 | 95.72 | 10.21 | 102.1 | 8.63 | 86.3 |
| 18 | 9.89 | 98.94 | 8.52 | 85.2 | 8.95 | 89.5 |
| | | | | | | |
| Average | 9.803 | 98.0 | 10.115 | 101.1 | 8.905 | 89.1 |
| S.D. | 0.475 | 4.75 | 0.702 | 7.02 | 0.351 | 3.51 |
| %CV | 4.85 | 4.85 | 6.94 | 6.94 | 3.94 | 3.94 |
| Sample # | Alprazolam | | Lorazepam | | Diazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 19 | 20.07 | 100.3 | 18.58 | 92.9 | 17.45 | 87.2 |
| 20 | 20.87 | 104.4 | 20.48 | 102.4 | 17.39 | 87.0 |
| 21 | 19.39 | 97.0 | 19.63 | 98.1 | 17.97 | 89.8 |
| 22 | 19.57 | 97.8 | 18.51 | 92.6 | 17.31 | 86.6 |
| 23 | 20.11 | 100.5 | 19.83 | 99.1 | 18.19 | 90.9 |
| 24 | 19.93 | 99.7 | 19.26 | 96.3 | 18.81 | 94.1 |
| 25 | 20.93 | 104.7 | 19.40 | 97.0 | 18.00 | 90.0 |
| 26 | 19.57 | 97.9 | 20.40 | 102.0 | 17.69 | 88.4 |
| 27 | 21.02 | 105.1 | 19.63 | 98.1 | 17.18 | 85.9 |
| | | | | | | |
| Average | 20.16 | 100.8 | 19.52 | 97.6 | 17.78 | 88.9 |
| S.D. | 0.631 | 3.16 | 0.689 | 3.44 | 0.520 | 2.60 |
| %CV | 3.13 | 3.13 | 3.53 | 3.53 | 2.92 | 2.92 |
{24}
| Sample # | Nordiazepam | | Oxazepam | | Temazepam | |
| --- | --- | --- | --- | --- | --- | --- |
| | ng/10 mg hair | % of target | ng/10 mg hair | % of target | ng/10 mg hair | % of target |
| 19 | 19.54 | 97.7 | 21.63 | 108.2 | 19.78 | 98.9 |
| 20 | 18.23 | 91.1 | 19.48 | 97.4 | 19.49 | 97.5 |
| 21 | 19.50 | 97.5 | 19.57 | 97.9 | 21.13 | 105.7 |
| 22 | 19.65 | 98.2 | 20.43 | 102.1 | 19.05 | 95.2 |
| 23 | 19.11 | 95.6 | 20.24 | 101.2 | 20.58 | 102.9 |
| 24 | 18.67 | 93.4 | 19.63 | 98.2 | 19.57 | 97.9 |
| 25 | 19.98 | 99.9 | 20.98 | 104.9 | 20.28 | 101.4 |
| 26 | 19.40 | 97.0 | 18.78 | 93.9 | 19.57 | 97.8 |
| 27 | 18.37 | 91.8 | 19.87 | 99.4 | 18.77 | 93.9 |
| | | | | | | |
| Average | 19.16 | 95.8 | 20.07 | 100.3 | 19.80 | 99.0 |
| S.D. | 0.610 | 3.05 | 0.860 | 4.30 | 0.745 | 3.72 |
| %CV | 3.18 | 3.18 | 4.29 | 4.29 | 3.76 | 3.76 |
b. Matrix comparison:
Not applicable. The assay is intended for only one sample matrix.
3. Clinical studies:
a. Clinical Sensitivity:
Not applicable.
b. Clinical specificity:
Not applicable.
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable.
4. Clinical cut-off:
Not applicable.
5. Expected values/Reference range:
Not applicable.
{25}
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.
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