BENZODIAZEPINE ENZYME IMMUNOASSAY

{0} 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY DEVICE ONLY TEMPLATE A. 510(k) Number: K032365 B. Analyte: Benzodiazepines C. Type of Test: Ready-to-use Enzyme Immunoassay for the qualitative and semi-quantitative analyses of benzodiazepines in human urine. Oxazepam calibrators. D. Applicant: Lin-Zhi International, Inc. E. Proprietary and Established Names: Benzodiazepine Enzyme Immunoassay Benzodiazepine Drugs of Abuse Calibrators and Controls F. Regulatory Information: 1. Regulation section: 21 CFR § 862.3170, Benzodiazepine test system § 862.3200, Clinical toxicology calibrator § 862.3280, Clinical toxicology control material 2. Classification: Class II 3. Product Code: JXM, DLJ, LAS 4. Panel: Toxicology (91) G. Intended Use: 1. Intended Use(s): Refer to the Indications for Use. 2. Indication(s) for use: The Benzodiazepine Enzyme Immunoassay is a homogenous immunoassay with a 200 ng/mL and/or 300 ng/mL cutoff. The assay is intended for use in the qualitative and semi-quantitative analyses of benzodiazepines in human urine. The assay is designed for professional use with a number of automated clinical chemistry analyzers. This immunoassay is calibrated using oxazepam at and around the 200 ng/mL and 300 ng/mL cutoff levels. The device is for in vitro diagnostic use, and intended for prescription use. {1} Page 2 of 10 The Benzodiazepine Drugs of Abuse Calibrators and Controls are intended for in vitro diagnostic use for the validation of Benzodiazepine enzyme immunoassays to detect benzodiazepines in human urine. 3. Special condition for use statement(s): The Benzodiazepine Enzyme Immunoassay provides only a preliminary analytical result. A more specific alternative chemical method must be used in order to obtain a confirmed analytical result. Gas Chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method. Other chemical confirmation methods are available. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when the preliminary test result is positive. Semi-quantitative analysis is helpful in estimating the concentrations of drugs in the samples. This can aid users in preparing dilutions of the samples for further analysis. 4. Special instrument Requirements: For professional use with a number of automated clinical chemistry analyzers that are capable of maintaining a constant temperature, pipetting sample, mixing reagents, measuring changes in absorbance at 340 nm and timing the reaction accurately. Performance for the assay as well as the calibrators and controls was demonstrated in this submission on the Hitachi 717 automated chemistry analyzer. H. Device Description: LZI's Benzodiazepine Enzyme Immunoassay contains two liquid reagents which contain the key components of the immunoassay. The assay uses monoclonal antibodies against benzodiazepine compounds and metabolites, substrate, and enzyme labeled-benzodiazepine. The Calibrators and Controls consist of a drug-free human urine matrix spiked with the following concentrations of oxazepam: | Reference Material | Benzodiazepine EIA | | --- | --- | | | Oxazepam | | Calibrator #2/Control I | 100 ng/mL | | Calibrator #3/Cutoff A/Control II | 200 ng/mL | | Calibrator #4/Cutoff B/Control III | 300 ng/mL | | Calibrator #5 | 1000 ng/mL | | Control IV | 400 ng/mL | {2} Page 3 of 10 # I. Substantial Equivalence Information: 1. Predicate device name(s): Syva EMIT® II Plus Benzodiazepine Assay Multi-Drug Urine Calibrators and Controls 2. Predicate K number(s): K993985 K935101 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Intended Use | Intended for qualitative and semi-quantitative determination of benzodiazepines in human urine | Intended for qualitative and semi-quantitative determination of benzodiazepines in human urine | | Assay cutoff | 200ng/ml or 300 ng/ml | 200ng/ml or 300 ng/ml | | Sensitivity | Functional sensitivity – 15 ng/ml | 15 ng/ml – distinguished from zero with 95% confidence | | Method principle | Enzymatic immunoassay | Enzymatic immunoassay | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Control levels | 100, 200, 300, and 400 ng/ml | 150, 225, 250, and 375 ng/ml | | Calibrator and Control | Oxazepam | Lormetazepam | Calibrators and controls use similar matrix compositions and concentrations of oxazepam. # J. Standard/Guidance Document Referenced (if applicable): Guidance for Prescription Use Drugs of Abuse Assays Premarket Notifications, published November 2000. # K. Test Principle: The benzodiazepine assay is a homogeneous enzyme immunoassay for use on clinical chemistry analyzers. Calibrators or cutoff controls, ranging in concentration from 0 – 1000 ng/mL, are run with the assay. The assay is based on competition between drug in the sample and drug labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for a fixed amount of antibody in the reagent. Enzyme activity decreases upon antibody binding, so that when free drug is present in the sample, the drug concentration is proportional to the increase in enzyme activity. This enzyme activity results in the {3} conversion of nicotinamide adenine dinucleotide (NAD) to NADH, resulting in an absorbance change that can be measured spectrophotometrically at $340~\mathrm{nm}$ . # L. Performance Characteristics (if/when applicable): # 1. Analytical performance: # a. Precision/Reproducibility: For both the qualitative and semi-quantitative assays, within run and run-to-run precision tests were performed on one single Hitachi 717 automated chemistry analyzer by one individual chemist. All calibrators and controls are diluted from a $1\mathrm{mg/mL}$ commercially purchased oxazepam standard solution, then spiked into a urine-based calibrator buffer matrix. All calibrators and controls values are confirmed by GC/MS $(+/-10\%)$ . Qualitative analysis: The five calibrators were evaluated. Within run precision was measured by running 21 replicates of six different analyte concentrations on the Hitachi 717 analyzer, while run-to-run precision was tested with 12 runs over a 3 week period. Typical results ( $\Delta \mathrm{mA} / \mathrm{min}$ ) are as follows: | | Within Run (n=21) | | | Run-to-Run (n=12) | | | | --- | --- | --- | --- | --- | --- | --- | | | Mean | SD | %CV | Mean | SD | %CV | | Negative | 361.0 | 3.1 | 0.86 | 360.0 | 2.5 | 0.70 | | 100 ng/mL | 417.8 | 3.5 | 0.84 | 417.9 | 2.5 | 4.42 | | 200 ng/mL | 455.5 | 3.5 | 0.76 | 457.0 | 1.9 | 0.51 | | 300 ng/mL | 483.3 | 3.5 | 0.73 | 483.6 | 2.4 | 0.55 | | 1000 ng/mL | 559.7 | 4.9 | 0.74 | 561.8 | 3.3 | 0.58 | Semi-quantitative analysis: The analyzers were first calibrated using five calibrators as reference (0, 100, 200, 300, and $1000\mathrm{ng / ml}$ ). Within run precision was determined by measuring the controls (100, 200, 300, and $400\mathrm{ng / ml}$ ) 21 times on the Hitachi 717 analyzer. Run-to-run precision was established using 12 runs over a 3 week period. The results $(\mathrm{ng / mL})$ are summarized below: | | Within Run (n=21) | | | Run-to-Run (n=12) | | | | --- | --- | --- | --- | --- | --- | --- | | | Mean | SD | %CV | Mean | SD | %CV | | 100 ng/mL | 105.7 | 7.6 | 7.2 | 95.8 | 6.4 | 6.7 | | 200 ng/mL | 203.6 | 5.9 | 2.9 | 192.0 | 6.5 | 3.4 | | 300 ng/mL | 281.8 | 11.1 | 3.9 | 294.8 | 15.5 | 5.3 | | 400 ng/mL | 373.9 | 15.3 | 4.1 | 398.6 | 15.4 | 3.9 | # b. Linearity/assay reportable range: The assay was challenged by spiking negative urine samples with known amounts of oxazepam at the following levels: 20, 50, 100, 180, 225, 375, 450, 600, and $800\mathrm{ng / ml}$ $(n = 5$ for each concentration). The assay correctly identified spiked levels $+ / - 25\%$ of the cutoffs using both the $200\mathrm{ng / mL}$ and $300\mathrm{ng / ml}$ levels. {4} For semi-quantitative analysis, 45 negative urine samples were spiked with various amounts of oxazepam (20-800 ng/ml, n=5 at each concentration). The average recovery is summarized in the following table: | Target Conc. (ng/mL) | Found (ng/mL) | % Recovery | | --- | --- | --- | | 20 | 18.9 | 94.4 | | 50 | 52.1 | 104.3 | | 100 | 108.2 | 108.2 | | 180 | 173.4 | 96.3 | | 225 | 218.8 | 97.3 | | 375 | 379.8 | 101.3 | | 450 | 468.1 | 104.0 | | 600 | 575.1 | 95.9 | | 800 | 791.2 | 98.9 | Linearity values for the semi-quantitative analysis (targeted value vs. measured value) are as follows: Slope $= 0.9838$ y intercept $= 3.4623$ $\mathrm{R}^2 = 0.9981$ # c. Traceability (controls, calibrators, or method): Calibrators and commercial controls are specified in the labeling but are sold separately from the kit. Calibrators and controls are obtaining clearance in this submission. A description of the concentrations is found in the table below. All calibrators and controls are diluted from a $1\mathrm{mg / mL}$ oxazepam standard solution, and are composed of drug-free human urine spiked with known concentrations of oxazepam. | Reference Material | Benzodiazepine EIA | | --- | --- | | | Oxazepam | | Calibrator #2/Control I | 100 ng/mL | | Calibrator #3/Cutoff A/Control II | 200 ng/mL | | Calibrator #4/Cutoff B/Control III | 300 ng/mL | | Calibrator #5 | 1000 ng/mL | | Control IV | 400 ng/mL | The calibrators contain five levels of calibrator material (calibrators #2-#5 plus a negative calibrator). These are used in the semi-quantitative determination of benzodiazepines in urine specimens. Qualitative evaluation is performed using the negative control and one of the cutoff controls, Cutoff A or Cutoff B respectively, for the 200 and $300\mathrm{ng / mL}$ assay cutoffs. There are four levels of control material (Controls I - IV) which are used in the validation of the assay. {5} Page 6 of 10 Traceability of calibrators and controls is established through GS/MS analysis with an acceptance criterion of +/- 10%. Real time stability studies of these calibrators and controls have been performed for 10 months and are ongoing. The assay reagents are aliquoted into two parts, one stored at 2-8°C and the other stored at room temperature. The rate performance of these populations are then assayed at various time points and compared. d. Detection limit: Functional sensitivity was demonstrated to be 15 ng/mL. Ten replicates each of 4 concentrations of oxazepam-spiked negative urine (from 0 - 25 ng/mL) were assayed (semi-quantitative), and the means and standard deviations were calculated. The %CV was below 20% at 15 ng/mL. e. Analytical specificity: To test cross-reactivity and potential interference, various potentially interfering substances were tested in the assay. Test compounds were spiked into the drug-free urine calibrator matrix to various concentrations and evaluated against the cutoff calibrator. The top table lists the concentration of each test compound that gave a (positive) response approximately equivalent to that of the cutoff calibrator. Potentially interfering non-benzodiazepine compounds are listed in the bottom table as the concentration that gave a (negative) response below the response of the cutoff calibrator. Response equivalent to cutoff: | Benzodiazepine Compounds (Cross-reactivity) | ng/mL at 200 ng/mL cutoff | ng/mL at 300 ng/mL cutoff | | --- | --- | --- | | Oxazepam | 200 | 300 | | Alprazolam | 75 | 100 | | Bromazepam | 2100 | 5000 | | Chlordiazepoxide | 65 | 125 | | Clobazam | 750 | 1300 | | Clonazepam | 65 | 125 | | Diazepam | 80 | 200 | | Flunitrazepam | 50 | 70 | | Flurazepam | 90 | 135 | | Lormetazepam | 50 | 75 | | Lorazepam | 90 | 165 | | Medazepam | 23 | 70 | | Nitrazepam | 150 | 220 | | Norfludiazepam | 15 | 25 | {6} | Benzodiazepine Compounds (Cross-reactivity) | ng/mL at 200 ng/mL cutoff | ng/mL at 300 ng/mL cutoff | | --- | --- | --- | | Prazepam | 75 | 105 | | Temazepam | 80 | 115 | | Triazolam | 45 | 105 | | Oxazepam-glucuronide | >10000 | >10000 | | Lorazepam-glucuroinde | >10000 | >10000 | | Temazepam-glucuronide | >10000 | >10000 | No interference seen at: | Non-benzodiazepines (Interference test) | (μg/mL) 200 ng/mL cutoff | (μg/mL) 300 ng/mL cutoff | | --- | --- | --- | | Acetaminophen | 1000 | 1000 | | Acetylsalicylic acid | 1000 | 1000 | | Amitriptyline | 180 | 400 | | Amobarbital | 1000 | 1000 | | Amphetamine | 1000 | 1000 | | Benzoylecgonine | 1000 | 1000 | | Caffeine | 1000 | 1000 | | Chlorpromazine | 200 | 500 | | Cocaine | 400 | 700 | | Codeine | 1000 | 1000 | | Dextromethorphan | 1000 | 1000 | | Ephedrine | 1000 | 1000 | | Imipramine | 300 | 500 | | Meperidine | 600 | 1000 | | Methadone | 1000 | 1000 | | Methamphetamine | 1000 | 1000 | | Methaqualone | 1000 | 1000 | | Morphine | 1000 | 1000 | | Nortriptyline | 600 | 1000 | | Phenobarbital | 1000 | 1000 | | Promethazine | 1000 | 1000 | | Propoxyphene | 1000 | 1000 | | Secobarbital | 1000 | 1000 | | Valproic Acid | 1000 | 1000 | | Lidocaine | 1000 | 1000 | | Chlorpheniramine | 100 | 150 | | Ecgonine | 1000 | 1000 | | Bupropion | 1000 | 1000 | | Ranitidine | 1000 | 1000 | It is possible that other substances and/or factors not listed above may interfere with the test and cause false positive results. {7} Page 8 of 10 The sponsor did not evaluate the effects of pH, specific gravity, or albumin on this assay. Glucuronide metabolites of oxazepam, lorazepam, and temazepam do not cross-react with the antibodies in this immunoassay. The cross-reactivity of other glucuronide metabolites with this assay is not known. f. Assay cut-off: The identified cutoff concentrations of the assay are 200 ng/ml and 300 ng/ml. Characterization of how the device performs around the claimed cutoff concentrations is included in the precision section above. 2. Comparison studies: a. Method comparison with predicate device: One hundred and sixteen (116) unaltered clinical urine specimens were tested with LZI’s benzodiazepine immunoassay and with the predicate device. No pre-determined criteria were applied in the selection of samples to be tested. All positive samples were confirmed with GC/MS or HPLC. A portion of samples having drug concentrations below the cutoff concentrations were also evaluated by GC/MS or HPLC. **200 ng/mL cutoff:** Using LZI’s assay, 66 samples were found to be positive, and 50 samples were found to be negative. | | Predicate Assay | | % Agreement | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | LZI Benzodiazepine EIA | Positive | 59 | 7* | 89.4% | | | Negative | 0 | 50 | 100% | **300 ng/mL cutoff:** Using LZI’s assay, 63 samples were found to be positive, and 53 samples were found to be negative. | | Predicate Assay | | % Agreement | | | --- | --- | --- | --- | --- | | | | Positive | | Negative | | LZI Benzodiazepine EIA | Positive | 57 | 6* | 90.5% | | | Negative | 0 | 53 | 100% | * All discrepant samples were confirmed as containing benzodiazepines by GC/MS or HPLC. The study included an acceptable number of samples that contained (apparent) benzodiazepine levels close to the cutoff values. Because of the number of {8} Page 9 of 10 benzodiazepine family members and metabolites, each with varying cross-reactivity to the antibodies in the immunoassay, it is sometimes difficult to find clinical samples meeting the criteria. To further challenge cutoff performance, analysis using spiked urine was performed at values +/- 25% of the cutoff values (see Linearity section above). The device as compared to GC/MS or HPLC analysis performed as follows: | 200 ng/mL cutoff | GC/MS or HPLC | | | | --- | --- | --- | --- | | | | positive | negative | | LZI Benzodiazepine EIA | positive | 64 | 2 | | | negative | 8 | 42 | 10 Discrepancies: LZI negative / HPLC 518 ng/mL Lorazepam LZI negative / HPLC 101 ng/mL Demoxepam 68 ng/mL Oxazepam 1889 ng/mL Temazepam LZI negative / HPLC 280 ng/mL Lorazepam LZI negative / HPLC 526 ng/mL Lorazepam LZI negative / HPLC 1811 ng/mL Lorazepam LZI negative / GC/MS 286 ng/mL Lorazepam LZI negative / GC/MS 261 ng/mL Alprazolam LZI negative / GC/MS 252 ng/mL Lorazepam LZI positive / HPLC 114 ng/mL Oxazepam LZI positive / GC/MS 185 ng/mL Oxazepam | 300 ng/mL cutoff | GC/MS or HPLC | | | | --- | --- | --- | --- | | | | positive | negative | | LZI Benzodiazepine EIA | positive | 61 | 2 | | | negative | 11 | 42 | 13 Discrepancies: LZI negative / 461 ng/mL Clonazepam LZI negative / HPLC 518 ng/mL Lorazepam LZI negative / HPLC 101 ng/mL Demoxepam 68 ng/mL oxazepam 1889 ng/mL Temazepam LZI negative / HPLC 280 ng/mL Lorazepam LZI negative / HPLC 526 ng/mL Lorazepam LZI negative / HPLC 1811 ng/mL Lorazepam LZI negative / GC/MS 307 ng/mL Oxazepam LZI negative / GC/MS 286 ng/mL Lorazepam LZI negative / GC/MS 261 ng/mL Alprazolam {9} Page 10 of 10 LZI negative / GC/MS 311 ng/mL Lorazepam LZI negative / GC/MS 252 ng/mL Lorazepam LZI positive / HPLC 114 ng/mL Oxazepam LZI positive / GC/MS 185 ng/mL Oxazepam The GC/MS and HPLC analysis included measurements of the following benzodiazepine compounds: clonazepam, demoxepam, alprazolam, oxazepam, lorazepam, temazepam, and norfludiazepam. Note that the sponsor claims that some of the false negative samples contained glucuronide-conjugated drug which their EIA antibody cannot detect. This was not analyzed by GC/MS or HPLC. EIA assays were performed at the manufacturer's facility. GC/MS and HPLC analysis was performed at VA Medical Center, TX. b. Matrix comparison: Not applicable. 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. M. Conclusion: I recommend that the Lin-Zhi International, Inc. Benzodiazepine Enzyme Immunoassay is substantially equivalent to the legally marketed predicate device.