RANDOX BENZODIAZEPINE ASSAY

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k092274 B. Purpose for Submission: New device C. Measurand: Benzodiazepines D. Type of Test: Qualitative and semi-quantitative immunoassay E. Applicant: Randox Laboratories Ltd. F. Proprietary and Established Names: Randox Benzodiazepine assay Randox Multidrug Calibrator/Control G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | JXM | Class II | 21 CFR § 862.3170, Benzodiazepine test system | 91-Toxicology | | DLJ | Class II | 21 CFR § 862.3200, Calibrators, Drug specific | 91-Toxicology | | LAS | Class I, reserved | 21 CFR 862.3280 Clinical Toxicology control material | 91-Toxicology | H. Intended Use: 1. Intended use(s): See Indications for use, below. 2. Indication(s) for use: The Randox Laboratory Ltd. Benzodiazepine Assay is an in vitro diagnostic test for the qualitative and semi-quantitative detection of Benzodiazepines in human urine. The cut off for both the qualitative and semi quantitative modes of the assay is 200ng/ml for oxazepam. The {1} Randox Benzodiazepine Assay has been developed for use on the Rx series analyzers, which includes the Rx Daytona and the Rx Imola. This in vitro diagnostic device is intended for prescription use only. The semi-quantitative mode is for purpose of (1) enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as GCMS Or (2) permitting laboratories to establish quality control procedures. The assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Gas chromatography /mass spectrometry (GC/MS) is the preferred confirmatory method. Clinical consideration and professional judgment should be exercised to any drug of abuse test result, particularly when the preliminary result is positive. The Randox Multidrug Calibrator Set consists of liquid calibrators containing Oxazepam. There are 5 levels of calibrator. They have been developed for use in the calibration of Benzodiazepines (Oxazepam) assays on the RX series analyzers, which includes the Rx Daytona and Rx Imola. This in vitro diagnostic device is intended for prescription use only. The Randox Multidrug Controls, level 1 and 2 are liquid controls containing Oxazepam. There are 2 levels of controls. They have been developed for use in the quality control of Benzodiazepine assays, on the RX series analyzers, which includes the Rx Daytona and the Rx Imola. This in vitro diagnostic device is intended for prescription use only. 3. Special conditions for use statement(s): The assay is for in vitro prescription use only. 4. Special instrument requirements: The Rx Daytona and Rx Imola analyzers were used to conduct performance studies below. I. Device Description: The assay consists of ready-to-use liquid reagents. Reagent 1 contains mouse monoclonal anti-oxazepam antibody, glucose-6-phosphate (G6P), nicotinamide adenine dinucleotide (NAD), stabilizers and Sodium Azide &lt;0.1% w/v). Reagent 2 contains oxazepam-labeled glucose-6-phosphate dehydrogenase (G6PDH) in buffer with Sodium Azide &lt;0.1% w/v. The calibrators and controls are ready to use human urine-based liquid. J. Substantial Equivalence Information: {2} 1. Predicate device names DRI Benzodiazepine Assay DRI Multi-Drug Calibrators and controls 2. Predicate 510(k) number(s): k930529 k983159 3. Comparison with predicate: | ITEM | Randox Benzodiazepines Assay | DRI Benzodiazepine Assay k930529 DRI Multi-Drug Calibrators and controls k983159 | | --- | --- | --- | | Cutoff | 200ng/ml | Same | | Intended Use | Qualitative and semi-quantitative analysis of benzodiazepines in human urine | Same | | Sample type | Human urine | Same | | Type of reagent | Liquid ready to use Two reagent assay | Same | | Calibrators | Liquid ready to use (0, 100, 200, 300, 1000 ng/mL) | Liquid ready to use (100, 200, 500, 1000 ng/mL) | | Controls | Liquid ready to use (150 and 250 ng/mL) | Liquid ready to use (150 and 300 ng/mL) | K. Standard/Guidance Document Referenced (if applicable): - CLSI Protocol EP7-A2: Interference Testing in Clinical Chemistry L. Test Principle: The Randox Laboratory Ltd. Benzodiazepine Assay is an immunoassay with ready-to-use liquid reagent. 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. In the absence of drug in the sample, the antibody binds the conjugated oxazepam-labeled G6PDH thus the enzyme activity is inhibited. When free drug is present on the sample, the antibody will bind to the free drug and the unbound oxazepam-labeled G6PDH exhibits its maximal enzyme activity. The G6PDH activity is measured spectrophotometrically at 340 nm because of conversion of NAD to NADH. M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: {3} Precision was determined by spiking oxazepam into drug free urine at various concentrations (-100%, -75%, -50%, -25%, +25%, at cutoff, +50%, +75% and +100% of the cut-off). Concentrations were confirmed by GC/MS. Testing for the intra-assay was performed once a day for 20 days. The between run testing was performed in replicate twice a day for 20 days. The qualitative and semi-quantitative results are presented below: Daytona: Total Precision for qualitative and semi-quantitative modes | Sample concentration (ng/mL) | No. observation | Results in Qualitative mode | Results in Semi-Quantitative mode | | --- | --- | --- | --- | | -100% cut off | 80 | 80 negative | 80 negative | | -75% cut off | 80 | 80 negative | 80 negative | | -50% cut off | 80 | 80 negative | 80 negative | | -25% cut off | 80 | 80 negative | 80 negative | | Cut off | 80 | 46 positive/34 negative | 53 positive/27 negative | | +25% cut off | 80 | 80 positive | 80 positive | | +50% cut off | 80 | 80 positive | 80 positive | | +75% cut off | 80 | 80 positive | 80 positive | | +100% cut off | 80 | 80 positive | 80 positive | Imola: Total Precision for qualitative and semi-quantitative mode | Sample concentration (ng/mL) | N | Results in Qualitative mode | Results in Semi-Quantitative mode | | --- | --- | --- | --- | | -100% cut off | 80 | 80 negative | 80 negative | | -75% cut off | 80 | 80 negative | 80 negative | | -50% cut off | 80 | 80 negative | 80 negative | | -25% cut off | 80 | 80 negative | 80 negative | | Cut off | 80 | 46 positive/34 negative | 26 positive/54 negative | | +25% cut off | 80 | 80 positive | 80 positive | | +50% cut off | 80 | 80 positive | 80 positive | | +75% cut off | 80 | 80 positive | 80 positive | | +100% cut off | 80 | 80 positive | 80 positive | b. Linearity/assay reportable range: The assay linearity was determined by testing the recoveries of a series of samples diluted from a high concentration oxazepam sample. A high urine sample containing around $1000\mathrm{ng / mL}$ oxazepam was serially diluted with analyte-free urine and tested by 3 replicates in semi-quantitative mode. The results were averaged and compared to the expected result and the percent recovery was calculated. Results are presented in the tables below: {4} Rx Daytona: | Expected Concentration (ng/mL) | Mean Observed Concentration (ng/mL) | Recovery (%) | | --- | --- | --- | | 0 | 12.12 | Not applicable | | 10 | 1.49 | 14.9 | | 20 | 0 | 0 | | 30 | 8.96 | 29.87 | | 40 | 18.61 | 46.53 | | 50 | 29.92 | 59.84 | | 60 | 28.80 | 48.00 | | 70 | 37.65 | 53.79 | | 80 | 65.12 | 81.40 | | 90 | 67.53 | 75.00 | | 100 | 90.95 | 90.95 | | 200 | 197.54 | 98.77 | | 300 | 284.40 | 94.80 | | 400 | 391.53 | 97.88 | | 500 | 496.88 | 99.38 | | 600 | 559.698 | 93.28 | | 700 | 701.35 | 100.19 | | 800 | 817.37 | 102.17 | | 900 | 827.44 | 91.93 | | 1000 | 971.32 | 97.13 | Rx Imola: | Expected Concentration (ng/mL) | Mean Observed Concentration (ng/mL) | Recovery (%) | | --- | --- | --- | | 0 | 2.16 | Not applicable | | 10 | 0 | 0 | | 20 | 2.38 | 11.90 | | 30 | 19.49 | 64.97 | | 40 | 29.32 | 73.30 | | 50 | 27.37 | 54.74 | | 60 | 53.81 | 89.68 | | 70 | 76.80 | 109.71 | | 80 | 85.35 | 106.69 | | 90 | 101.68 | 112.98 | | 100 | 111.94 | 111.94 | | 200 | 218.01 | 109.01 | | 300 | 331.49 | 110.50 | | 400 | 427.92 | 106.98 | | 500 | 521.54 | 104.31 | | 600 | 579.88 | 96.65 | {5} 6 | 700 | 638.93 | 91.28 | | --- | --- | --- | | 800 | 716.58 | 89.57 | | 900 | 885.61 | 98.40 | | 1000 | 959.52 | 95.95 | c. Traceability, Stability, Expected values (controls, calibrators, or methods): Traceability and value assignment The 5 levels calibrator (~0 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 1000 ng/mL) and 2 levels control (150 ng/mL and 250 ng/mL) materials are both traceable to master lots that have been GC/MS quantified. The master lots were made by spiking oxazepam into a human urine matrix. Oxazepam is supplied by Cerilliant Corporation, the accuracy of which is ensured by purity determination (GC/FID, HPLC and NMR) and gravimetric preparation using balances calibrated with NIST traceable standards. Each of the Randox calibrator/control level is value assigned using Rx Daytona and Rx Imola. The target value for each level is the median of the observed values. Stability Real time stability testing including shelf-life and on-board stability studies were performed for the assay, controls and calibrators. The acceptance criteria were found to be adequate. The Randox Benzodiazepine assay reagents, controls and calibrators are stable for 12 months when stored unopened at 2 – 8° C and 28 days on-board at approximately 10°C. d. Detection limit: Performance at low drug concentrations in the semi-quantitative assay was characterized by determination of recovery (see section b above). e. Analytical specificity: The Randox Laboratory Ltd. Benzodiazepine Assay was evaluated for interference according to the CLSI Guideline EP7-A2 recommendations. These studies were performed by spiking structurally related and unrelated compounds into drug-free and drug-containing urine samples. Drug-containing urine samples were tested at two different concentrations, +25% and -25% of the cut-off concentration of 200 ng/mL. Drug-free urine samples were used as controls. % cross-reactivity was calculated using the cross-reactant concentration that gives a reaction absorbance which matches the reaction absorbance obtained by the cut-off calibrator. The cut-off calibrator concentration divided by the cross-reactant concentration that achieved the matching reactant absorbance x 100% gives the % cross reactivity. These studies were performed on both, the Rx Daytona and Rx Imola analyzers. Similar results were obtained with both analyzers and in both qualitative and semi-quantitative modes. The percent cross-reactivity of the tested compounds are presented below: Structurally related compounds: Daytona | Compound | Tested | Cross-reactivity | | --- | --- | --- | {6} 7 | | concentration | (%) | | --- | --- | --- | | Oxazepam | 100 | 100 | | Diazepam | 54 | 371.92 | | Nordiazepam | 179 | 111.56 | | Temazepam | 75 | 266.25 | | Alprazolam | 77 | 260.42 | | Bromazepam | 4799 | 4.17 | | Chlordiazepoxide | 3377 | 5.92 | | Clobazam | 74 | 271.83 | | Clonazepam | 3420 | 5.85 | | Flunitrazepam | 15 | 1312.31 | | Flurazepam | 51 | 392.71 | | Lormetazepam | 490 | 39.99 | | Lorazepam | 2182 | 14.42 | | Medazepam | 328 | 61.01 | | Midazolam | 220 | 91.10 | | Nitrazepam | 143 | 139.80 | | Prazepam | 139 | 144.56 | | Triazolam | 964 | 20.74 | Imola: | Compound | Tested concentration | Cross-reactivity (%) | | --- | --- | --- | | Oxazepam | 100 | 100 | | Diazepam | 43 | 465.39 | | Nordiazepam | 170 | 117.84 | | Temazepam | 99 | 201.89 | | Alprazolam | 88 | 228.02 | | Bromazepam | 3568 | 5.61 | | Chlordiazepoxide | 5969 | 3.35 | | Clobazam | 82 | 243.83 | | Clonazepam | 7837 | 2.55 | | Flunitrazepam | 20 | 990.48 | | Flurazepam | 47 | 421.71 | | Lormetazepam | 1089 | 18.36 | | Lorazepam | 2263 | 8.84 | | Medazepam | 541 | 36.97 | | Midazolam | 255 | 78.46 | | Nitrazepam | 160 | 125.01 | | Prazepam | 180 | 111.05 | | Triazolam | 1296 | 15.43 | Structurally unrelated compounds: | Compound | Tested Concentration | Cross-reactivity (%) | | --- | --- | --- | {7} | | (ng/mL) | | | --- | --- | --- | | 11-hydroxy-delta9-THC | 100,000 | 0 | | 11-nor9-carboxy-delta9-THC | 100,000 | 0 | | 6 Acetyl morphine | 25,000 | 0 | | Amitriptyline | 125,000 | 0 | | Amobarbital | 100,000 | 0 | | Amphetamine D5 | 100,000 | 0 | | Ascorbic acid | 100,000 | 0 | | Aspartame | 100,000 | 0 | | Aspirin | 100,000 | 0 | | Atropine | 20,000 | 0 | | Benzilic acid | 100,000 | 0 | | Benzolyecgonine | 100,000 | 0 | | B-phenylethylamine | 100,000 | 0 | | Caffeine | 100,000 | 0 | | Cannabidiol | 100,000 | 0 | | Chlorpheniramine | 4800 | 0 | | Chlorquine | 100,000 | 0 | | Chloramphenicol | 100,000 | 0 | | Cocaethylene | 18,000 | 0 | | Cocaine | 100,000 | 0 | | Codeine | 100,000 | 0 | | Cotinine | 100,000 | 0 | | Delta9-THC | 100,000 | 0 | | Dihydrocodeine | 100,000 | 0 | | Ecgonine methyl ester | 20,000 | 0 | | EDDP | 13,500 | 0 | | EMDP | 7,500 | 0 | | d,l-Ephedrine | 100,000 | 0 | | d-Ephedrine | 100,000 | 0 | | R,R (-) Pseudoephedrine | 100,000 | 0 | | S,S (+) Pseudoephedrine | 100,000 | 0 | | Estrone | 100,000 | 0 | | Furosemide | 100,000 | 0 | | Heroin | 17,500 | 0 | | Ibuprofen | 100,000 | 0 | | Isoproterenol | 15,000 | 0 | | Ketamine | 100,000 | 0 | | LAAM | 100,000 | 0 | | Labetalol | 100,000 | 0 | | Loperamide | 30,000 | 0 | | MBDB | 100,000 | 0 | | MDA | 100,000 | 0 | | MDEA | 100,000 | 0 | | MDMA | 100,000 | 0 | 8 {8} 9 | Methadone | 25,000 | 0 | | --- | --- | --- | | d,l-Methamphetamine | 100,000 | 0 | | Morphine | 11,500 | 0 | | Oxycodone | 100,000 | 0 | | Paracetamol | 100,000 | 0 | | Pentobarbital | 75,000 | 0 | | Phencyclidine | 100,000 | 0 | | Pherphenazine | 100,000 | 0 | | Procaine | 100,000 | 0 | | Quinidine | 100,000 | 0 | | Ranitidine | 100,000 | 0 | | Secobarbital | 100,000 | 0 | Endogenous compounds: The following endogenous compounds were added into drug-free urine and urine containing oxazepam at the concentrations of +/- 25% surrounding the assay cutoff. These samples were tested using both, the Rx Daytona and Rx Imola analyzers. The substances listed in the table below were determined not to interfere at the concentration shown: | Compound | Tested Concentration | | --- | --- | | Total bilirubin | 15 mg/dL | | Direct bilirubin | 5 mg/dL | | Hemoglobin | 115 mg/dL | | Creatinine | 30 mg/dL | | Urea | 258 mg/dL | | Glucose | 2000 mg/dL | | HSA | 500 mg/dL | | Ethanol | 250 mg/dL | | Acetone | 1000 mg/dL | | Gamma globulin | 500 mg/dL | | Oxalic acid | 100 mg/dL | | Riboflavin | 7.5 mg/dL | | Sodium chloride | 3000 mg/dL | | Boric acid | 62.5 mg/dL | | Sodium azide | 250 mg/dL | | Sodium fluoride | 1000 mg/dL | In addition, the performance of the assay was evaluated under varying pH levels of: 3, 5, 7, 9 and 11, which also had no effect on results. Further, variations in specific gravity between 1.00 and 1.03 had no effect on results. The package insert includes the complete list of all structurally related and unrelated compounds and metabolites tested. f. Assay cut-off: {9} Analytical performance of the device around the claimed cutoff is described in precision section (1 a.) above. ## 2. Comparison studies: a. Method comparison with predicate device: Eighty unaltered clinical urine samples were evaluated by the Randox Benzodiazepine assay and compared to a GC/MS. Results from the study are presented below: Daytona – Semi-quantitative | GCMS for oxazepam (based on cross reactivity profile) → | Negative | Low Negative by GC/MS (concentration < 50% below the cutoff concentration for oxazepam) | Near Cutoff Negative (concentration between 50% below the cutoff and the cutoff concentration for oxazepam) | Near Cutoff Positive (concentration between 50% above the cutoff and the cutoff concentration for oxazepam) | High Positive (concentration > 50% above the cutoff concentration for oxazepam) | Percent Agreement with GCMS for oxazepam (based on cross reactivity profile) | | --- | --- | --- | --- | --- | --- | --- | | 200 ng/mL cutoff Benzodiazepine Assay ↓ | | | | | | | | Positive | 0 | 0 | 5 | 8 | 34 | 100 | | Negative | 32 | 0 | 1 | 0 | 0 | 86.8 | Daytona Qualitative | GCMS for oxazepam (based on cross reactivity profile) → | Negative | Low Negative by GC/MS (concentration < 50% below the cutoff concentration for oxazepam) | Near Cutoff Negative (concentration between 50% below the cutoff and the cutoff concentration for oxazepam) | Near Cutoff Positive (concentration between 50% above the cutoff and the cutoff concentration for oxazepam) | High Positive (concentration > 50% above the cutoff concentration for oxazepam) | Percent Agreement with GCMS for oxazepam (based on cross reactivity profile) | | --- | --- | --- | --- | --- | --- | --- | | 200 ng/mL cutoff Benzodiazepine Assay ↓ | | | | | | | | Positive | 0 | 0 | 7 | 6 | 34 | 100 | | Negative | 32 | 0 | 1 | 0 | 0 | 82.5 | {10} Imola – Semi-quantitative | GCMS for oxazepam (based on cross reactivity profile) → | Negative | Low Negative by GC/MS (concentration < 50% below the cutoff concentration for oxazepam) | Near Cutoff Negative (concentration between 50% below the cutoff and the cutoff concentration for oxazepam) | Near Cutoff Positive (concentration between 50% above the cutoff and the cutoff concentration for oxazepam) | High Positive (concentration > 50% above the cutoff concentration for oxazepam) | Percent Agreement with GCMS for oxazepam (based on cross reactivity profile) | | --- | --- | --- | --- | --- | --- | --- | | 200 ng/mL cutoff Benzodiazepine Assay ↓ | | | | | | | | Positive | 0 | 0 | 5 | 10 | 32 | 100 | | Negative | 32 | 0 | 1 | 0 | 0 | 89.4 | Imola – Qualitative | GCMS for oxazepam (based on cross reactivity profile) → | Negative | Low Negative by GC/MS (concentration < 50% below the cutoff concentration for oxazepam) | Near Cutoff Negative (concentration between 50% below the cutoff and the cutoff concentration for oxazepam) | Near Cutoff Positive (concentration between 50% above the cutoff and the cutoff concentration for oxazepam) | High Positive (concentration > 50% above the cutoff concentration for oxazepam) | Percent Agreement with GCMS for oxazepam (based on cross reactivity profile) | | --- | --- | --- | --- | --- | --- | --- | | 200 ng/mL cutoff Benzodiazepine Assay ↓ | | | | | | | | Positive | 0 | 0 | 3 | 11 | 31 | 100 | | Negative | 32 | 0 | 3 | 0 | 0 | 92.1 | GC/MS Summary of Discrepant Results: Daytona – Semi-quantitative | Cut-off value (ng/mL) for oxazepam | Randox Benz Assay (POS/NEG) | Drug/Metabolite GC/MS value (ng/mL) based on cross reactivity profile | | --- | --- | --- | | 200 | POS | 169 (Nordiazepam) | {11} 12 | | POS | 169 (Nordiazepam) | | --- | --- | --- | | | POS | 160 (Oxazepam) | | | POS | 180 (Oxazepam) | | | POS | 190 (Oxazepam) | Daytona – Qualitative | Cut-off value (ng/mL) for oxazepam | Randox Benz Assay (POS/NEG) | Drug/Metabolite GC/MS value (ng/mL) based on cross reactivity profile | | --- | --- | --- | | 200 | POS | 140.43 (Nordiazepam) | | | POS | 140.43 (Nordiazepam) | | | POS | 198.09 (Nordiazepam) | | | POS | 199.02 (Nordiazepam) | | | POS | 160 (Oxazepam) | | | POS | 180 (Oxazepam) | | | POS | 190 (Oxazepam) | Imola – Semi-quantitative | Cut-off value (ng/mL) for oxazepam | Randox Benz Assay (POS/NEG) | Drug/Metabolite GC/MS value (ng/mL) based on cross reactivity profile | | --- | --- | --- | | 200 | POS | 178 (Nordiazepam) | | | POS | 178 (Nordiazepam) | | | POS | 150 (Oxazepam) | | | POS | 160 (Oxazepam) | | | POS | 180 (Oxazepam) | Imola – Qualitative | Cut-off value (ng/mL) for oxazepam | Randox Benz Assay (POS/NEG) | Drug/Metabolite GC/MS value (ng/mL) based on cross reactivity profile | | --- | --- | --- | | 200 | POS | 162 (Nordiazepam) | | | POS | 162 (Nordiazepam) | | | POS | 180 (Oxazepam) | The tables above show that in this study qualitatively discrepant results were observed only for near-cutoff samples (+/- 50% of the cutoff concentration.) b. Matrix comparison: Not applicable. The test is only for urine specimens. 3. Clinical studies: a. Clinical Sensitivity: {12} Not applicable. Not reviewed for this device type. b. Clinical specificity: Not applicable. Not reviewed for this device type. c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. 4. Clinical cut-off: Not applicable; the device is for determining presumptive positive or negative based on the analytical cutoff of 200 ng/mL. 5. Expected values/Reference range: Not applicable. The test is not intended for quantifying benzodiazepine; the semi-quantitative mode is for purposes of (1) enabling laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as GCMS and (2) permitting laboratories to establish quality control procedures. 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. 13