HANDLE ALL URINE SPECIMENS AS IF THEY ARE POTENTIALLY INFECTIOUS. Catalog Number: 311UR-0025 25 mL Kit Catalog Number: 311UR-0100 100 mL Kit ASSAY PROCEDURE Catalog Number: 311UR-0500 500 mL Kit Analyzers capable of maintaining a constant temperature, pipetting samples and Catalog Number: 311UR-0060W 60 mL Kit reagents, mixing reagents, timing the reaction accurately and measuring enzymatic Intended Use: rates at 340 nm can be used to perform the assay. The assay has been validated for The Immunalysis Methamphetamine Urine Enzyme Immunoassay is a homogeneous use with the Olympus AU400e, the following Beckman Coulter analyzers: AU480, enzyme immunoassay with a dual cutoff of 500ng/mL and 1000ng/mL. The assay is AU680, AU2700, AU5400 and AU5800. intended for use in laboratories for the qualitative and semi-quantitative analysis of Methamphetamine in human urine with automated clinical chemistry analyzers. This assay Refer to the analyzer-specific Application Sheet which contains parameters for use. is calibrated against Methamphetamine. This in-vitro diagnostic device is for prescription QUALITY CONTROL AND CALIBRATION use only. Good laboratory practice suggests the use of control specimens to ensure assay The semi-quantitative mode is for purposes of enabling laboratories to determine an performance. Control results must fall within established ranges determined by your appropriate dilution of the specimen for confirmation by a confirmatory method such as laboratory. QC materials should be used in accordance with local, state and/or federal Gas Chromatography/ Mass Spectrometry (GC-MS) or permitting laboratories to establish regulations or accreditation requirements. quality control procedures. For a qualitative analysis use either the 500ng/mL or 1000ng/mL calibrator as a cutoff The Immunalysis Methamphetamine Urine Enzyme Immunoassay Kit provides only level to distinguish “positive” and “negative” specimens. For semi-quantitative analysis a preliminary analytical test result. A more specific alternate chemical method must use all calibrators. be used in order to obtain a confirmed analytical result. GC-MS or Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the preferred RESULTS AND EXPECTED VALUES confirmatory method. Clinical consideration and professional judgment should be Qualitative Results applied to any drug of abuse test result, particularly when preliminary positive A specimen that exhibits a change in absorbance ( ∆A) value, equal to or greater than results are used. the value obtained with the cutoff calibrator is considered positive. A sample that EXPLANATION OF THE TEST exhibits a change in absorbance ( ∆A) value, lower than the value obtained with the [1] Methamphetamine (USAN) also known as metamfetamine (INN), meth, ice, speed, crystal, cutoff calibrator is considered negative. glass, tik, N-methylamphetamine, methylamphetamine, and desoxyephedrine, is a Semi-quantitative Results psychostimulant of the phenethylamine and amphetamine class of psychoactive drugs. A rough estimate of drug concentration in the specimens can be obtained by running a Methamphetamine increases alertness, concentration, energy, and in high doses, can induce standard curve with all calibrators and calculating samples of the standard curve. [2][3] euphoria, enhance self-esteem and increase libido. Methamphetamine has a high potential for abuse and addiction, activating the psychological reward system by triggering a cascading release LIMITATIONS of dopamine in the brain. Methamphetamine is metabolized in the liver with the main metabolites • The assay is designed for use with human urine only. being amphetamine (active) and 4-hydroxymethamphetamine (pholedrine). It is excreted by the • Boric Acid at 1% w/v may cause false negative results. Boric Acid is not kidneys, with the rate of excretion into the urine heavily influenced by urinary pH. Between 30- recommended as a preservative for urine. 54% of an oral dose is excreted in urine as unchanged methamphetamine and 10-23% as • Other substances and factors not listed may interfere with the test and cause false unchanged amphetamine. Following an intravenous dose, 45% is excreted as unchanged parent [4] results. drug and 7% amphetamine. The half-life of methamphetamine is variable with a mean value of • [5] Interpretation of results must take into account that urine concentrations can vary between 9 and 12 hours. extensively with fluid intake and other biological variables. Immunoassays that PRINCIPLES OF THE PROCEDURE produce a single result in the presence of a drug and its metabolites cannot fully This assay uses Methamphetamine recombinant antibody. The assay is based on the quantitate the concentration of individual components. competition of Methamphetamine labeled enzyme glucose-6-phosphate dehydrogenase (G6PDH) and the free drug in the urine sample for the fixed amount of antibody binding SPECIFIC PERFORMANCE CHARACTERISTICS sites. In the absence of the free drug in the sample, the antibody binds the drug enzyme Performance results obtained on the Olympus AU400e analyzer are shown below. conjugate and enzyme activity is inhibited. This creates a dose response relationship Additionally, the following analyzers have been validated for use with the between drug concentration in the urine and enzyme activity. The enzyme G6PDH activity Methamphetamine Urine Enzyme Immunoassay. is determined at 340 nm spectrophotometrically by the conversion of NAD to NADH. Manufacturer Analyzer REAGENTS Beckman Coulter AU480 Beckman Coulter AU680 Immunalysis Methamphetamine Urine Enzyme Immunoassay Kit Beckman Coulter AU2700 Beckman Coulter AU5400 Antibody/Substrate Reagent (RA) - This contains recombinant antibodies to Beckman Coulter AU5800 Methamphetamine, glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide Refer to the analyzer-specific Application Sheet for information on analyzer settings (NAD) in Tris buffer with Sodium Azide as a preservative. and parameters. Enzyme Conjugate Reagent (RE) - This contains glucose-6-phosphate dehydrogenase Semi-Quantitative Precision was determined by testing calibrators and controls for 20 (G6PDH) labeled with Methamphetamine in Tris buffer with Sodium Azide as a days, 2 runs per day in replicates of 2 (N=80). preservative. Table 1: Semi-Quantitative Precision Additional Materials Required (sold separately): 500ng/mL Cutoff Catalog No.: Concentration (ng/mL) Result Total Result 10000 -15 or Immunalysis Multi -Drug Calibrators Level 1 Calibrator containing 150ng/mL 125ng/mL 10000-25 of Benzoylecgonine, 500ng/mL of Methamphetamine, 100ng/mL of NEG 80 Negative Morphine, 12.5ng/mL of PCP and 100ng/mL of Oxazepam 250ng/mL NEG 80 Negative 10001 -15 or Immunalysis Multi -Drug Calibrators Level 2 Calibrator containing 300ng/mL 375ng/mL (Control LOW) NEG 80 Negative 10001-25 of Benzoylecgonine, 1000ng/mL of Methamphetamine, 300ng/mL of 500ng/mL Calibrator N/A 35 Negative/45 Positive Morphine, 25g/mL of PCP and 200ng/mL of Oxazepam 625ng/mL (Control HIGH) POS 80 Positive 10002 -15 or Immunalysis Multi -Drug Calibrators Level 3 Calib rator containing 500ng/mL 750ng/mL POS 80 Positive 10002-25 of Benzoylecgonine, 1500ng/mL of Methamphetamine, 500ng/mL of 875ng/mL POS 80 Positive Morphine, 50ng/mL of PCP and 500ng/mL of Oxazepam 1000ng/mL POS 80 Positive 10003 -15 or Immunalysis Multi -Drug Calibrators Level 4 Calibrator containing 1000ng/mL 1000ng/mL Cutoff 10003-25 of Benzoylecgonine, 2000ng/mL of Methamphetamine, 1000ng/mL of Morphine, 100ng/mL of PCP and 1000ng/mL of Oxazepam Concentration (ng/mL) Result Total Result 3000 -15 o r 250ng/mL NEG 80 Negative Immunalysis Multi-Drug Control Set 1 containing LOW and HIGH Control 1 3000-25 500ng/mL NEG 80 Negative 3001 -15 or Immunalysis Multi -Drug Control Set 2 containing LOW and HIGH Control 2 750ng/mL (Control LOW) NEG 80 Negative 3001-25 1000ng/mL Calibrator N/A 37 Negative/43 Positive 1250ng/mL (Control HIGH) POS 80 Positive WARNING 1500ng/mL POS 80 Positive 1) H303 The reagents may be harmful if swallowed. 1750ng/mL POS 80 Positive 2) H290 Reagents contain Sodium Azide as a preservative. When disposing of these 2000ng/mL POS 80 Positive reagents flush with large volumes of water to prevent azide build-up which could react with See Application Sheet for calibration frequency recommendation. copper and lead plumbing to form potentially explosive metal azides. 3) Do not use reagents beyond their expiration dates. Qualitative Precision was determined by testing calibrators and controls for 20 Preparation and Storage days, 2 runs per day in replicates of 2 (N=80). The Immunalysis Methamphetamine Urine Enzyme Immunoassay Kit reagents are Table 2: Qualitative Precision provided ready to use and may be used directly from the refrigerator. 500ng/mL Cutoff Reagent bottles should remain closed when not in use with screw caps tightly Concentration (ng/mL) Result Total Result closed. The expiration date of the kit is stated on the label. The expiration date reflects the 125ng/mL NEG 80 Negative stability claim of 18 months stored at 2 – 8°C. The reagents are stable opened when 250ng/mL NEG 80 Negative stored on-board the instrument for 28 days at 2 – 8°C 375ng/mL (Control LOW) NEG 80 Negative SPECIMEN COLLECTION - Storage and Handling Instructions 500ng/mL Calibrator Urine samples should be collected in industry standard urine collection containers. Ensure N/A 41 Negative/39 Positive that the samples are free of gross debris. Highly turbid specimens should be centrifuged 625ng/mL (Control HIGH) POS 80 Positive before analysis. Adulteration of urine specimens can cause erroneous results. Urine 750ng/mL POS 80 Positive specimens stored at ambient temperature up to 30°C up to 14 days may be used. 875ng/mL POS 80 Positive Samples should be well mixed before assay. 1000ng/mL POS 80 Positive

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1000ng/mL Cutoff Analytes Conc (ng/mL) % Cross -React. Concentration (ng/mL) Result Total Result Diphenhydramine 1,000,000 <0.050 250ng/mL NEG 80 Negative Phenypropanolamine 1,000,000 < 0.0005 500ng/mL NEG 80 Negative Table 10: Cross Reactivity with Structurally Related Compounds – 1000ng/mL cutoff 750ng/mL (Control LOW) NEG 80 Negative Analytes Conc (ng/mL) % Cross -React. 1000ng/mL Calibrator N/A 44 Negative/36 Positive (+) Methamphetamine 1,000 100.00 (-) Methamphetamine 200,000 0.44 1250ng/mL (Control HIGH) POS 80 Positive (+) Amphetamine 60,000 2.50 1500ng/mL POS 80 Positive (-) Amphetamine 1,000,000 <0.10 1750ng/mL POS 80 Positive Methylenedioxyamphetamine (MDA) 40,000 2.50 2000ng/mL POS 80 Positive Methoxyamphetamine (PMA) 40,000 3.33 See Application Sheet for calibration frequency recommendation. Methylenedioxymethamphetamine (MDMA) 1,000 71.43 MDEA 5,000 14.29 Accuracy Fenfluramine 10,000 6.06 A total of 80 urine specimens were previously confirmed by outside labs and analyzed by (+) Pseudoephedrine 200,000 0.67 Immunalysis Methamphetamine Urine Enzyme Immunoassay.The HEIA cutoffs were (-) Pseudoephedrine 1,000,000 0.13 500ng/mL and 1000ng/mL of Methamphetamine to distinguish positive results from (-) Ephedrine 200,000 0.57 negative results. (+) Ephedrine 1,000,000 <0.10 Qualitative – 500ng/mL cutoff and 1000ng/mL cutoff Phentermine 1,000,000 <0.10 40 specimens were negative by both methods. 40 specimens were positive by both Tyramine 1,000,000 <0.10 methods. Results are shown in Table 3. Phenylephrine 1,000,000 <0.10 Table 3 Diphenhydramine 1,000,000 <0.10 LC -MS /MS Phenypropanolamine 1,000,000 <0.10 (+) (-) Interference Immunalysis Methamphetamine (+) 40 0 The potential interference of unrelated drugs, endogenous substances, boric acid, pH and Urine Enzyme Immunoassay (-) 0 40 specific gravity on recovery of Methamphetamine using the Immunalysis Semi-Quantitative – 500ng/mL cutoff Methamphetamine Urine Enzyme Immunoassay was assessed by spiking known amounts 39 specimens were negative by both methods. 40 specimens were positive by both of each potentially interfering substances into the LOW (375 ng/mL and 750 ng/mL) and methods. 1 specimens was positive by assay and negative by LC-MS/MS. Results are HIGH (625 ng/mL and 1250 ng/mL) controls for the 500 ng/mL and 1000 ng/mL cutoff. shown in Table 4. Discordant results are shown in Table 5. Unrelated Drugs Table 4 No interference was observed by the addition of concentrations up to 100,000ng/mL of the LC -MS /MS following potentially interfering substances. (+) (-) 4-Bromo-2,5,Dimethoxyphenethylamine, 6-Acetylmorphine, 7-Aminoclonazepam, Immunalysis Methamphetamine (+) 40 1 Acetaminophen, Acetylsalicylic Acid, Alprazolam, Amitriptyline, Amobarbital, Benzoylecgonine, Urine Enzyme Immunoassay (-) 0 39 Benzylpiperazine, Bromazepam, Buprenorphine, Bupropion, Butabarbital, Caffeine, Carbamazepine, Chlordiazepoxide, Chlorpromazine, cis-Tramadol, Clobazam, Clomipramine, Table 5 Clonazepam, Cocaine, Codeine, Cyclobenzaprine, N-Demethyltapentadol, Delta-9-THC, Semi -Quantitative Results 500ng Cutoff LC-MS/MS Desipramine, Dextromethorphan, Diazepam, Dihydrocodeine, Doxepin, EDDP, Ethyl β-D- In -House ID Value Result Confirmation glucuronide, Ethylmorphine, Fentanyl, Flunitrazepam, Fluoxetine, Flurazepam, Heroin, 16559 544.9 Positive 494 Hexobarbital, Hydrocodone, Hydromorphone, 11-hydroxy-delta-9-THC, Ibuprofen, Imipramine, Semi-Quantitative – 1000ng/mL cutoff Ketamine, Levorphanol Tartrate, Lidocaine, Lorazepam, LSD, Maprotiline, Meperidine, 40 specimens were negative by both methods. 39 specimens were positive by both Meprobamate, Methadone, Methaquolone, Methylphenidate, Morphine, Morphine-3β- β methods. 1 specimen was negative by assay and positive by LC-MS/MS. Results are glucuronide, Morphine-6 -glucuronide, Nalorphine, Naloxone, Naltrexone, Nitrazepam, Norbuprenorphine, Norcodeine, Nordiazepam, Normorphine, Norpropoxyphene, Nortriptyline, shown in Table 6. Discordant results are shown in Table 7. Oxazepam, Oxycodone, Oxymorphone, PCP, Pentazocine, Pentobarbital, Phenobarbital, Table 6 Phentermine, Phenytoin, Prazepam, Propranolol, Protriptyline, Ranitidine, Ritalinic Acid, LC -MS /MS Secobarbital, Sufentanil Citrate, Temazepam, 11-nor-9 carboxy THC, Thioridazine, Triazolam, (+) (-) Trifluoromethylphenyl-piperazine, Trimipramine, Venlafaxine Immunalysis Methamphetamine (+) 39 0 Acetaminophen, Acetylsalicylic Acid, Benzoylecgonine and Caffeine were added up to Urine Enzyme Immunoassay (-) 1 40 500,000ng/mL. Endogenous Substances, pH and Specific Gravity Table 7 No Interference was observed by the addition of the following compounds, up to the Semi -Quantitative Results 1000ng Cutoff LC-MS/MS In -House ID concentrations listed below. Confirmation Value Result Table 11: Endogenous Substances, pH and Specific Gravity 16597 998.5 Negative 1017 Analyte Concentration Analytical Recovery Acetone 1.0g/dL Negative human urine was spiked with a high concentration of Methamphetamine and Ascorbic Acid 1.5g/dL serially diluted to the concentrations below. For each level the drug concentration was Bilirubin 0.002g/dL calculated and the average concentration obtained by the Immunalysis Methamphetamine Creatinine 0.5g/dL Urine Enzyme Immunoassay is shown in Table 8 below. Ethanol 1.0g/dL Table 8: Analytical Recovery Galactose 0.01g/dL Expected Concentration (ng/mL) Mean Concentration (ng/mL) Recovery (%) γ-Globulin 0.5g/dL 200 272.7 136.4 Glucose 2.0g/dL 400 436.8 109.2 Hemoglobin 0.115g/dL Human Serum Albumin 0.5g/dL 600 674.6 112.4 Oxalic Acid 0.1g/dL 800 830.0 103.8 Riboflavin 0.0075g/dL 1000 1107.6 110.8 Sodium Azide 1% w/v 1200 1247.0 103.9 Sodium Chloride 6.0g/dL 1400 1481.2 105.8 Sodium Fluoride 1% w/v 1600 1711.5 107.0 Urea 6.0g/dL 1800 1917.4 106.5 pH 3, 4, 5, 6, 7, 8, 9, 10 and 11 2000 2080.7 104.0 Specific Gravity 1.000, 1.002, 1.005, 1.010, 1.015 1.020, 1.025 and 1.030 2200 2226.4 101.2 Boric Acid at 1% w/v may cause false negative results. Boric Acid is not recommended Specificity as a preservative for urine. The specificity of the Immunalysis Methamphetamine Urine EIA was determined by generating inhibition curves for each of the compounds listed below. The results are REFERENCES summarized below in Table 9 and Table 10 and are expressed as the minimum 1) "Methamphetamine". Drug profiles . European Monitoring Centre for Drugs and Drug concentration of metabolite or compound required to produce a response approximately Addiction (EMCDDA). 16 August 2010. http://www.emcdda.europa.eu/publications/drug- equivalent to the cutoff of the assay: profiles/methamphetamine. Table 9: Cross Reactivity with Structurally Related Compounds – 500ng/mL cutoff 2) Mack, Avram H.; Frances, Richard J.; Miller, Sheldon I. (2005). Clinical Textbook of Addictive Disorders, Third Edition . New York: The Guilford Press. p. 207. ISBN 1-59385- Analytes Conc (ng/mL) % Cross -React. 174-X. (+) Methamphetamine 500 100.00 3) B. K. Logan. Methamphetamine - Effects on Human Performance and Behavior . Forensic (-) Methamphetamine 90,000 0.56 Science Review, Vol. 14, no. 1/2 (2002), p. 142. (+) Amphetamine 20,000 2.50 4) National Highway Traffic Safety Administration (NHTSA) - Methamphetamine (And (-) Amphetamine 900,000 0.06 Amphetamine). Methylenedioxyamphetamine (MDA) 18,000 2.78 5) Schep LJ, Slaughter RJ, Beasley DM (August 2010). "The clinical toxicology of Methoxyamphetamine (PMA) 15,000 3.33 metamfetamine". Clinical Toxicology (Philadelphia, Pa.) 48 (7): 675–94. Methylenedioxymethamphetamine (MDMA) 800 62.50 doi:10.3109/15563650.2010.516752. ISSN 1556-3650. PMID 20849327. MDEA 3,000 16.67 Fenfluramine 7,000 7.14 European Authorized Representative: (+) Pseudoephedrine 75,000 0.67 CEpartner4U , Esdoornlaan 13, 3951DB Maarn. (-) Pseudoephedrine 300,000 0.17 The Netherlands. www.cepartner4u.eu (-) Ephedrine 65,000 0.77 (+) Ephedrine 1,000,000 <0.05 IMMUNALYSIS CORPORATION Phentermine 500,000 0.10 829 Towne Center Drive Revision: AB Tyramine 850,000 0.06 Pomona, CA 91767 Revision Date: 08/2017 Phenylephrine 800,000 0.06 (909) 482-0840

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