Amphetamines II (AMPII) is an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines in human urine on COBAS INTEGRA systems at cutoff concentrations of 300 ng/mL, 500 ng/mL and 1000 ng/mL when calibrated with a-methamphetamine. Semiquantitative test results may be obtained that permit laboratories to assess assay performance as part of a quality control program. Semiquantitative assays are intended to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas chromatography/mass spectrometry (GC/MS).

Amphetamines II provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. Gas chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method.1 Clinical consideration and professional judgement should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

Device Description

The Amphetamines II test is an immunoassay for use on automated clinical chemistry analyzers. The device consists of two wet reagents; a soluble drug-conjugate, and an antibody-bound microparticle solution. During the assay, in the absence of sample drug in urine, soluble drug-conjugates bind to antibody-bound microparticles, causing the formation of particle aggregates. When a urine sample contains the drug in question, this drug competes with the drug derivative conjugate for microparticle-bound antibody. Antibody bound to sample drug is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The rate of absorbance change is proportional to the concentration of drug in the sample. Calibrators, ranging in concentration from 0-5000 ng/mL depending on cutoff and test mode, are run with the assay. Concentrations of controls and unknowns are calculated from the standard curve in semi-quantitative mode. Results for controls or calibrators are determined as preliminary positive or negative relative to the cutoff in qualitative mode.

C.f.a.s. DAT Qualitative Clinical, C.f.a.s. DAT Qualitative Plus, C.f.a.s. DAT Qualitative Plus Clinical, Preciset DAT Plus I Calibrators, and Preciset DAT Plus II Calibrators are ready to use, multianalyte calibrators prepared by the quantitative addition of drug or drug metabolite to drug-free human urine.

Control Set DAT I, II, and III, and Control Set DAT Clinical are ready to use multianalyte controls prepared by the quantitative addition of drug or drug metabolite to drug-free urine.

AI/ML Overview

Here's an analysis of the provided text regarding the Amphetamines II Assay, structured to address your specific points:

The provided document describes the Amphetamines II Assay for Integra Family of Analyzers, an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines in human urine. The device's performance is primarily evaluated through precision and accuracy studies against Gas Chromatography/Mass Spectrometry (GC/MS).

1. Table of Acceptance Criteria and Reported Device Performance

The general acceptance criteria for this type of immunoassay are implicit in the performance studies: accurate detection of amphetamines/methamphetamines in urine relative to established cutoffs, with good precision and minimal interference from other substances. The detailed criteria would typically be established internally by the manufacturer and submitted to regulatory bodies.

Here's a summary of the stated performance, interpreted against general expectations for such assays:

Acceptance Criterion (Implicit)	Reported Device Performance
Precision	Qualitative Mode:- 300 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50% of cutoff. 83/84 Negative at -25% of cutoff. 27/84 Negative, 57/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.- 500 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50% of cutoff. 84/84 Negative at -25% of cutoff. 28/84 Negative, 56/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.- 1000 ng/mL Cutoff (MAMP): 84/84 Negative at zero, -75%, -50%, -25% of cutoff. 13/84 Negative, 71/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.- 300 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 83/84 Negative at -25% of cutoff. 2/84 Negative, 82/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.- 500 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 82/84 Negative, 2/84 Positive at -25% of cutoff. 6/84 Negative, 78/84 Positive at cutoff. 84/84 Positive at +25%, +50%, +75%, +100% of cutoff.- 1000 ng/mL Cutoff (AMP): 84/84 Negative at zero, -75%, -50% of cutoff. 82/84 Negative, 2/84 Positive at -25% of cutoff. 7/84 Negative, 77/84 Positive at cutoff. 1/84 Negative, 83/84 Positive at +25% of cutoff. 84/84 Positive at +50%, +75%, +100% of cutoff.Semiquantitative Mode (SD, CV% ranges reported):- For MAMP at 300, 500, 1000 ng/mL cutoffs: - Repeatability (within-run) CV% for zero to +100% of cutoff generally good (e.g., 5.7%-95.4% for 300 ng/mL, with highest CVs at very low concentrations). - Intermediate Precision (total) CV% for zero to +100% of cutoff also generally good (e.g., 6.5%-123.8% for 300 ng/mL, with highest CVs at very low concentrations).- For AMP at 300, 500, 1000 ng/mL cutoffs: - Repeatability (within-run) CV% generally good (e.g., 5.3%-110.0% for 300 ng/mL). - Intermediate Precision (total) CV% also generally good (e.g., 6.5%-135.7% for 300 ng/mL).
Accuracy (Agreement with GC/MS)	MAMP (d-methamphetamine):- Qualitative & Semiquantitative Assays: - 100% agreement for High Positive by GC/MS (greater than +50% of cutoff) across all cutoffs (300, 500, 1000 ng/mL). - 100% agreement for Near Cutoff Positive by GC/MS (between cutoff and +50%) across all cutoffs. - For Low Negatives (below cutoff) / Near Cutoff Negative (between -50% and cutoff): - 300 ng/mL Cutoff: 90% (Qualitative & Semiquantitative). - 500 ng/mL Cutoff: 91% (Qualitative & Semiquantitative). - 1000 ng/mL Cutoff: 90% (Qualitative & Semiquantitative).AMP (d-amphetamine):- Qualitative & Semiquantitative Assays: - 100% agreement for High Positive by GC/MS (greater than +50% of cutoff) across all cutoffs. - 100% agreement for Near Cutoff Positive by GC/MS (between cutoff and +50%) across all cutoffs. - For Low Negatives (below cutoff) / Near Cutoff Negative (between -50% and cutoff): - 300 ng/mL Cutoff: 92.5% (Qualitative & Semiquantitative). - 500 ng/mL Cutoff: 92.5% (Qualitative & Semiquantitative). - 1000 ng/mL Cutoff: 97.5% (Qualitative), 95% (Semiquantitative).
Analytical Specificity / Cross-reactivity	A wide range of structurally similar compounds (e.g., MDMA, MDA, MDEA, BDB, l-Methamphetamine, l-Amphetamine) show varying degrees of cross-reactivity, with some "designer drugs" showing higher cross-reactivity than others (e.g., MDMA at 264% at 300 ng/mL MAMP equivalent). Most other common drugs/substances (e.g., acetaminophen, ibuprofen, caffeine, opiates, benzodiazepines) show either no significant interference or proper recovery of spiked samples at specified high concentrations (100,000 ng/mL).
Interference from Common Substances	No interference observed from high concentrations of common urine components (e.g., Acetone, Ascorbic Acid, Bilirubin, Creatinine, Ethanol, Glucose, Hemoglobin, Human serum albumin, Oxalic Acid, Sodium Chloride, Urea) for both qualitative and semiquantitative modes across all cutoffs when spiked with d-methamphetamine or d-amphetamine at -25% and +25% of cutoff.
pH and Specific Gravity Interference	No control cross-overs for specific gravities from 1.001 to 1.034. ≤ 5% cross-overs for pH ranging from 4.5 to 8.0.

2. Sample Size Used for the Test Set and Data Provenance

Precision Test Set Sample Size:
- For each cutoff (300, 500, 1000 ng/mL) and each drug (d-methamphetamine, d-amphetamine): 9 concentrations were tested.
- Each concentration was tested in 2 replicates per run, 2 runs per day for 21 days.
- Total N = 84 determinations per concentration level. (e.g., 9 concentrations * 84 determinations = 756 total determinations per drug per cutoff for precision).
- Data Provenance: "9 samples obtained from a human urine sample pool" for initial spiking. The source of this urine pool is not specified (e.g., country of origin). This was a retrospective study measuring inherent characteristics of the assay.
Accuracy Test Set Sample Size:
- Negative Samples: 36 urine samples confirmed negative by GC/MS.
- Positive Samples: 36 samples confirmed positive by GC/MS (containing d-methamphetamine or d-amphetamine).
- Near Cutoff Samples: For each cutoff, 4 negative near cutoff samples and 4 positive near cutoff samples were assayed.
- 4 additional unaltered samples (d-methamphetamine, 85-204 ng/mL) evaluated with 500 ng/mL cutoff.
- Data Provenance: "samples obtained from a clinical laboratory" and "unaltered near cutoff samples." Geographic origin is not specified. This was a retrospective study comparing the device to a gold standard.
Analytical Specificity Test Set Sample Size: Not explicitly stated as a count of individual samples, but inhibition curves were generated for each of the many tested compounds.
Interference Test Set Sample Size: Not explicitly stated as a count of individual samples, but various compounds were added to urine pools at specified concentrations. This appears to be a laboratory retrospective study.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

The ground truth for the test set (accuracy study) was established by Gas Chromatography/Mass Spectrometry (GC/MS).
This method is explicitly stated as "the preferred confirmatory method" and the device's traceability is stated as "Traceability: This method has been standardized against GC/MS."
There is no mention of human expert involvement in establishing the ground truth; it relies on the analytical capabilities of GC/MS. Therefore, qualifications of human experts for ground truth are not applicable in this context.

4. Adjudication Method for the Test Set

Adjudication methods like 2+1 or 3+1 typically apply to studies where human readers interpret data, and discrepancies need to be resolved.
In this context, the device's results are compared directly to the quantitative results from GC/MS. There is no human interpretation of the device's output or a need for expert adjudication of "truth" beyond the GC/MS result itself. Therefore, no adjudication method as described (e.g., 2+1) was used or is applicable.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, an MRMC comparative effectiveness study was not done.
This device is an automated immunoassay for analytical detection, not a system designed to assist human readers in a diagnostic task (e.g., interpreting medical images). Therefore, a study comparing human readers with and without AI assistance is not relevant to this device.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) Was Done

Yes, this entire submission details standalone (algorithm only) performance.
The Amphetamines II Assay is an automated in vitro diagnostic test run on COBAS INTEGRA systems. Its performance data (precision, accuracy, specificity, interference) are all based on the analytical capabilities of the instrument and reagents without human intervention in the result determination process. Human involvement would be limited to operating the analyzer, reviewing quality control, and interpreting the final preliminary positive/negative result in a clinical context (which the device explicitly states should be followed by GC/MS confirmation and professional judgment).

7. The Type of Ground Truth Used

The primary ground truth used for the accuracy study was Gas Chromatography/Mass Spectrometry (GC/MS) results. This is a highly accurate and specific analytical method, often considered the "gold standard" for drug confirmation in toxicology.
For precision and some interference studies, spiked urine samples with known concentrations of d-methamphetamine or d-amphetamine were used, where the known concentration is the ground truth.

8. The Sample Size for the Training Set

The document does not mention a training set sample size.
This is an immunoassay, not a machine learning or AI-based diagnostic tool that typically requires a large training set. The "training" of such a system involves the development and optimization of the reagents and assay parameters by the manufacturer, rather than a data-driven training process in the sense of machine learning.

9. How the Ground Truth for the Training Set Was Established

As a traditional immunoassay, there is no explicit "training set" in the machine learning sense.
The development of the assay's reagents, antibody specificity, and reaction conditions would have been guided by extensive biochemical research and experimentation to ensure specific binding and accurate signal generation across the desired concentration range. The "ground truth" during this development (or "training") phase would be the known concentrations of target analytes and interfering substances used to optimize the assay's performance characteristics. This is an iterative process of development and validation, rather than a single training set with established ground truth.

Summary

{0}------------------------------------------------

510(k) Summary: Amphetamines II Assay for Integra Family of Analyzers

According to the requirements of 21 CFR 807.92, the following Introduction information provides sufficient detail to understand the basis for a determination of substantial equivalence.

Submitter Roche Diagnostics Name, Address, 9115 Hague Rd. Contact Indianapolis, IN 46250 317-521-3742

JUL 2 8 2010

Contact Person: Michelle Neff

Kogzblogy

Proprietary name: Amphetamines II Device Name ·

Common name: Amphetamine metabolite test system

Classification name: Enzyme Immunoassay, Amphetamine

Product Code: DKZ

{1}------------------------------------------------

Device Description

Control Set DAT I, II, and III, and Control Set DAT Clinical are ready to use multianalyte controls prepared by the quantitative addition of drug or drug metabolite to drug-free urine.

{2}------------------------------------------------

Amphetamines II (AMPII) is an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines on COBAS INTEGRA systems in human urine at cutoff concentrations of 300 ng/mL, 500 ng/mL and 1000 ng/mL when calibrated with d-methamphetamine. Semiquantitative test results may be obtained that permit laboratories to assess assay performance as part of a quality control program. . Semiquantitative assays are intended to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas chromatography/mass spectrometry (GC/MS).

Amphetamines II provides only a preliminary analytical test result. A more specific alternate chemical method must be used in obtain a a confirmed a analytical aresult. Gas order to chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method.1 Clinical consideration and professional judgement should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

{3}------------------------------------------------

Comparison to the Predicate Device

The ONLINE Amphetamines II assay is substantially equivalent to other products in commercial distribution intended for similar use. Most notably, we claim substantial equivalence to the currently marketed Amphetamines II assay (K083764).

The ONLINE Amphetamines II assay contains an in vitro diagnostic reagent system intended for use on the Roche Integra analyzers for the semi-quantitative and qualitative detection of amphetamines in human urine at cutoff concentrations of 300, 500, and 1000 ng/mL.

The Roche ONLINE Amphetamines II assay utilizes a modified KIMS technology relative to the currently marketed Abuscreen OnLine Amphetamines assay. Differences between this application and the cleared assay include:

1. use of amphetamine and methamphetamine, and monoclonal antibodies attached to microparticles in solution
1. a soluble drug-polymer conjugate
1. increased sensitivity to "designer drugs"and their metabolites, and
1. addition of 300 and 500 ng/mL cutoff concentrations.

The recommended calibrators to be used with the proposed ONLINE Amphetamines II assay are the Preciset DAT Plus I, Preciset DAT Plus II and Cfas DAT Qualitative Plus Calibrators (K060645).

The recommended controls to be used with the proposed ONLINE Amphetamines II assay are the Control Set DAT I, Control Set DAT II, Control Set DAT III (K080183).

{4}------------------------------------------------

Comparison of Technological Characteristics
Feature	Amphetamines IIAssay, Integra 800	Predicate Device: Amphetamines IIAssay, Hitachi 917 (K083764)
Methodology	Same	KIMS, Kinetic interaction ofmicroparticles in solution
Sample Type	Same	Urine
Intended Use	Same	Qualitative and semi-quantitativedetection of amphetamine andmethamphetamine
Reagent	Same	1. Conjugate Working Solution: Conjugatedamphetamine and methamphetaminederivatives in buffer with bovine serumalbumin(BSA) and 0.09% sodium azide.2. Antibody/Microparticle Working Solution:Microparticles attached to amphetamine andmethamphetamine antibodies (mousemonoclonal) in buffer with bovine serumalbumin (BSA) and 0.09% sodium azide.
Cutoff	Same	300, 500, 1000 ng/mL
Calibrator/ControlMatrix	Same	Human urine

. . . . . .

. . . .

{5}------------------------------------------------

AMPII

Amphetamines II

Order information
COBAS INTEGRA	200 Tests	Cat. No. 04512936 190
ONLINE DAT		System-ID 07 6835 9
Amphetamines II
Preciset DAT Plus I	6 × 5 mL	Cat. No. 03304671 190
calibrators
CAL 1-6
Preciset DAT Plus II	6 × 5 mL	Cat. No. 03304680 190
calibrators
CAL 1-6
C.f.a.s. DAT Qualitative	6 x 5 mL	Cat. No. 03304698 190
Plus
C.f.a.s. DAT Qualitative	3 x 5 mL	Cat. No. 04590856 190
Plus Clinical
Control Set DAT II		Cat. No. 03312968 190
(for 300 ng/mL assay)
PreciPos DAT Set II	2 x 10 mL
PreciNeg DAT Set II	2 x 10 mL
Control Set DAT I		Cat. No. 03312950 190
(for 500 ng/mL assay)
PreciPos DAT Set I	2 x 10 mL
PreciNeg DAT Set I	2 x 10 mL
Control Set DAT Clinical		Cat. No. 04500873 190
(for 500 ng/mL assay)
PreciPos DAT Clinical	2 x 10 mL
PreciNeg DAT Clinical	2 x 10 mL
Control Set DAT III		Cat. No. 03312976 190
(for 1000 ng/mL assay)
PreciPos DAT Set III	2 x 10 mL
PreciNeg DAT Set III	2 x 10 mL

System information

Test AM3S2: 0-358: for semiquantitative assay, 300 ng/mL Test AM5S2: 0-359: for semiquantitative assay, 500 ng/mL Test AM1S2: 0-360: for semiquantitative assay, 1000 ng/mL Test AM3Q2: 0-330: for qualitative assay, 300 ng/mL Test AM5Q2: 0-340: for qualitative assay, 500 ng/mL Test AM1Q2: 0-350: for qualitative assay, 1000 ng/mL Test AM5QC: 0-341: for qualitative assay, 500 ng/mL; using C.f.a.s. DAT Qualitative Plus Clinical

{6}------------------------------------------------

Intended use

Amphetamines II (AMPII) is an in vitro diagnostic test for the qualitative and semiquantitative detection of amphetamines and methamphetamines in human urine on COBAS INTEGRA systems at cutoff concentrations of 300 ng/mL, 500 ng/mL and 1000 ng/mL when calibrated with d-methamphetamine. Semiquantitative test results may be obtained that permit laboratories to assess assay performance as part of a quality control program. Semiquantitative assays are intended to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as gas chromatography/mass spectrometry (GC/MS).

Amphetamines II provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. GC/MS is the preferred confirmatory method.1 Clinical consideration and professional judgement should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

Summarv

The amphetamines are known as the sympathomimetic amines as they mimic the effects of stimulation of the sympathetic nervous system. These small molecules, based on Bphenvlethylamine, structurally resemble the bodies own catecholamines. A wide variety have been created via substitutions anywhere on the structure. The amphetamines are potent central nervous stimulants. As such they can increase wakefulness, physical activity, and decrease appetite. The amphetamines have some limited indications and approval for use in ADHD, narcolepsy, and obesity. However, because these CNS stimulants convey a sense of self-confidence, well being, and euphoria, they are highly addictive, widely abused, and consequently controlled substances.2 Abuse can lead to medical, psychological, and social consequences. Adverse health effects include memory loss, aggression, psychotic behavior, heart damage, malnutrition, and severe dental problems.2 Amphetamine may be self-administered either orally or by intravenous injection in amounts of up to 2000 mg daily by tolerant addicts. It is a metabolite of a number of other drugs including methamphetamine. Normally about 30 % is excreted unchanged in the 24 hour urine, but this may change to as much as 74 % in acid urine and may decrease to 1 % in alkaline urine.4 Amphetamines II is calibrated with d-methamphetamine and therefore the sensitivity towards amphetamines is different than d-methamphetamine, as indicated in the "Analytical specificity" section.

Test principle

The assay is based on the kinetic interaction of microparticles in a solution (KIMS) 5 as measured by changes in light transmission. In the absence of amphetamine or methamphetamine, soluble drug conjugates bind to antibody-bound microparticles, causing the formation of particle aggregates. As the aggregation reaction proceeds in the absence of amphetamine or methamphetamine, the absorbance increases.

When a urine sample contains amphetamine or methamphetamine, this drug competes with the drug derivative conjugate for microparticle-bound antibody. Antibody bound to amphetamine or methamphetamine is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The presence of amphetamine diminishes the increasing absorbance in proportion to the concentration of drug in the sample. Amphetamine or methamphetamine content is determined relative to the value obtained for a known cutoff concentration of d-methamphetamine.7

{7}------------------------------------------------

Reagents - working solutions

Conjugated amphetamine and methamphetamine derivatives; buffer; bovine serum R1 albumin: 0.09 % sodium azide
Microparticles attached to amphetamine and methamphetamine antibodies (mouse SR monoclonal); buffer; bovine serum albumin; 0.09 % sodium azide

Precautions and warnings

Pay attention to all precautions and warnings listed in this Method Manual, Chapter 1, Introduction.

Reagent handling

COBAS INTEGRA 800 analyzers

The reagent is automatically mixed for 1 minute after cobas c pack puncture and for half a minute during Begin of Day.

Storage and stability

Shelf life at 2 to 8 °C

See expiration date on cobas c pack label

COBAS INTEGRA 800 analyzers

On-board in use at 8 °C

84 days

The on-board in use stability period begins at the time of cobas c pack puncture. Do not freeze reagents. Reagents that have been frozen should be discarded.

Specimen collection and preparation

Only the specimens listed below were tested and found acceptable.

Urine: Collect urine samples in clean glass or plastic containers. Fresh urine samples do not require special handling or pretreatment, but an effort should be made to keep pipetted samples free of gross debris. Samples should be within the normal physiological pH range of 5 to 8. No additives or preservatives are required. It is recommended that urine specimens be stored at 2 to 8 °C and tested within 5 days of collection . Centrifuge highly turbid specimens before testing. Adulteration or dilution of the sample can cause erroneous results. If adulteration is suspected, another sample should be collected. Specimen validity testing is required for specimens collected under the Mandatory Guidelines for Federal Workplace Drug Testing Programs.2 Specimens containing human-sourced materials should be handled as if potentially infectious using safe laboratory procedures such as those outlined in Biosafety in Microbiological and Biomedical Laboratories (HHS Publication Number [CDC] 93-8395).

Caution

Specimen dilutions should only be used as an estimation for GC/MS and are not intended for patient values. Dilution procedures, when used, should be validated.

Materials provided

See "Reagents - working solutions" section for reagents.

Assay

{8}------------------------------------------------

For optimum performance of the assay follow the directions given in this document for the analyzer concerned. Refer to the appropriate operator's manual for analyzer-specific assay instructions.

Application for urine

COBAS INTEGRA 800 test definition

	Semiquantitative	Qualitative
Measuring mode	Absorbance	Absorbance
Abs. calculation mode	Endpoint	Endpoint
Reaction mode	R1-S-SR	R1-S-SR
Reaction direction	Increase	Increase
Reaction start	SR	SR
Wavelength A	659 nm	659 nm
Test range
AM3S2	0-2000 ng/mL	0-2000
AM5S2, AM1S2	0-5000 ng/mL
with postdilution
AM3S2	0-20000 ng/mL
AM5S2, AM1S2	0-50000 ng/mL
Postdilution factor	10 recommended b	No
Calc. first/last	46/86	46/86
Unit	ng/mL
b) For use when estimating concentration in preparation for GC/MS analysis.

Pipetting parameters
AM3S2, AM3Q2		Diluent (H2O)
R1	60 µL	20 µL
Sample	10 µL	5 µL
SR	47 µL	3 µL
Total volume	145 µL
		Diluent (H2O)
AM5S2, AM5Q2, AM5QC
R1	60 µL	20 µL
Sample	7.5 µL	5 µL
SR	47 µL	3 µL
Total volume	142.5 µL
AM1S2, AM1Q2		Diluent (H2O)
R1	60 µL	20 µL
Sample	5 µL	5 µL
SR	47 µL	3 µL
Total volume	140 µL

{9}------------------------------------------------

Calibration
Calibrators	Semiquantitative applications
AM3S2, 0-358	Preciset DAT Plus II calibrators
	6 Calibrators (300 cutoff, DATS8, 07 6796 °)
	0, 150, 300, 600, 1000, 2000 ng/mL
	d-methamphetamine
AM5S2, 0-359;AM1S2, 0-360	Preciset DAT Plus I calibrators
	6 Calibrators (500 and 1000 cutoffs, DATS2, 07 6764 6 °)
	0, 250, 500, 1000, 3000, 5000 ng/mL
	d-methamphetamine
	Qualitative applications
AM3Q2, 0-330	0 ng/mL (Preciset DAT Plus II calibrators, CAL 1) or deionizedwater and 300 ng/mL (Preciset DAT Plus II calibrators, CAL 3)
	2 Calibrators (300 cutoff, DATQ3, 07 6770 0°)
	For qualitative applications, the cutoff value is assigned as 1000
AM5Q2, 0-340	0 ng/mL (Preciset DAT Plus I calibrators, CAL 1) or deionizedwater and 500 ng/mL (C.f.a.s. DAT Qualitative Plus)
	2 Calibrators (500 cutoff, DATQ1, 07 6744 1°)
	For qualitative applications, the cutoff value is assigned as 1000
AM5QC, 0-341	0 ng/mL (Preciset DAT Plus I or II calibrators, CAL 1) ordeionized water and 500 ng/mL (C.f.a.s. DAT Qualitative PlusClinical)
	2 Calibrators (500 cutoff, DATQ5, 07 6880 4°)
	For qualitative applications, the cutoff value is assigned as 1000
AM1Q2, 0-350	0 ng/mL (Preciset DAT Plus I calibrators, CAL 1) or deionizedwater and 1000 ng/mL (Preciset DAT Plus I calibrators, CAL 4)
	2 Calibrators (1000 cutoff, DATQ2, 07 6768 9°)

c) Short name and ID used on the COBAS INTEGRA systems.

Calibration mode	Semiquantitative applications
COBAS INTEGRA 800 analyzers	Logit/log 4
	Qualitative applications
	Linear regression
Calibration replicate	Duplicate recommended
Calibration interval
COBAS INTEGRA 800 analyzers	Each lot, every 4 weeks, and as required following qualitycontrol procedures

A calibration curve is generated using the calibrators. Calibrators must be placed from the highest concentration first to the lowest last on the CAL/QC rack. This curve is retained in memory by the COBAS INTEGRA systems and recalled for later use.

· Traceability: This method has been standardized against GC/MS.

Quality control

{10}------------------------------------------------

Quality control

300 ng/mL cutoff AM3S2 and AM3Q2: Control Set DAT II PreciPos DAT Set II (DAT2P, 07 6771 9 ") PreciNeg DAT Set II (DAT2N, 07 6772 7 9)

500 ng/mL cutoff AM5S2 and AM5Q2: Control Set DAT I PreciPos DAT Set I (DATIP, 07 6753 0 ª) PreciNeg DAT Set I (DATIN, 07 6754 9 d)

AM5QC:

Control Set DAT Clinical PreciPos DAT Clinical (DATCP, 07 6879 0 °) PreciNeg DAT Clinical (DATCN. 07 6878 2 ")

1000 ng/mL cutoff AMIS2 and AMIQ2: Control Set DAT III PreciPos DAT Set III (DAT3P, 07 6773 5 ") PreciNeg DAT Set III (DAT3N, 07 6774 3 ª) User defined Recommended

Control sequence Control after calibration

d) Short name and ID used on the COBAS INTEGRA systems.

For quality control, use control materials as listed in the Order information section. Other suitable control material can be used in addition.

Drug concentrations of the controls have been verified by GC/MS.

The control intervals and limits should be adapted to each laboratory's individual requirements. Values obtained should fall within the defined limits.

Each laboratory should establish corrective measures to be taken if values fall outside the limits. Follow the applicable government regulations and local guidelines for quality control.

Results

COBAS INTEGRA systems report results with the following test flags: Semiquantitative result reporting AM3S2 (300 ng/mL cutoff)

{11}------------------------------------------------

Flag	COBAS INTEGRA	Value range
No flag	Negative	< 300 ng/mL
<TEST RNG	Negative	< 0 ng/mL
>TEST RNG	Positive	> 2000 ng/mL
POS 300	Positive	≥ 300 ng/mL

Value ranges listed above are based on a cutoff value of 300 ng/mL.

Semiquantitative result reporting

AM5S2 (500 ng/mL cutoff)

Flag	COBAS INTEGRA	Value range
No flag	Negative	< 500 ng/mL
<TEST RNG	Negative	< 0 ng/mL
>TEST RNG	Positive	> 5000 ng/mL
POS 500	Positive	≥ 500 ng/mL

Value ranges listed above are based on a cutoff value of 500 ng/mL.

Semiquantitative result reporting

AM1S2 (1000 ng/mL cutoff)

Flag	COBAS INTEGRA	Value range
No flag	Negative	< 1000 ng/mL
<TEST RNG	Negative	< 0 ng/mL
>TEST RNG	Positive	> 5000 ng/mL
POS 1000	Positive	≥ 1000 ng/mL

Value ranges listed above are based on a cutoff value of 1000 ng/mL.

Qualitative result reporting

AM3Q2 (300 ng/mL cutoff)

Flag	COBAS INTEGRA	Value range
No flag	Negative	< 1000
<TEST RNG	Negative	< 0
>TEST RNG	Positive	> 2000
POS 1000	Positive	≥ 1000

Value ranges above are based on assigning the cutoff of 300 ng/mL a value of 1000.

Qualitative result reporting

AM5Q2, AM5QC (500 ng/mL cutoff)
Flag	COBAS INTEGRA	Value range
No flag	Negative	< 1000

{12}------------------------------------------------

<test rng<br="">дарыптананының таптының атаптынанының таптанының қалыптынын аталанын атаптанының тұрғанаттан таптынан атаптын атаптын атаптын атаптын аттыр	Negative	< (
>TEST RNG	Positive	> 2000
POS 1000ан жаңған жұмысындарының тұрғынан айтынша айынша айынша айынша атынын аталымының айтынша б	Positive	≥ 1000

Value ranges above are based on assigning the cutoff of 500 ng/mL a value of 1000.

Qualitative result reporti

AM1Q2 (1000 ng/mL cutoff)
Flag	COBAS INTEGRA	Value range
No flag	Negative	< 1000
<TEST RNG	Negative	< 0
>TEST RNG	Positive	> 2000
POS 1000	Positive	$\ge$ 1000

Value ranges above are based on assigning the cutoff of 1000 ng/mL a value of 1000.

Semiquantitative result reporting

The semiquantitation of preliminary positive results should only be used by laboratories to determine an appropriate dilution of the specimen for confirmation by a confirmatory method such as GC/MS. It also permits the laboratory to establish quality control procedures and assess control performance.

Note: When using the post-dilution function (1:10 dilution), to ensure the sample was not overdiluted, the diluted result must be at least half the analyte cutoff value times 10. If the diluted result falls below half the analyte cutoff value times 10, repeat the sample with a smaller dilution. A dilution that produces a result closest to the analyte cutoff is the most accurate estimation. To estimate the positive sample's concentration, multiply the result by the appropriate dilution factor. Dilutions should only be used as an estimation for GC/MS.

Limitations10

See the Analytical specificity section of this method sheet for information on substances tested for cross-reactivity in this assay. There is the possibility that other substances and/or factors may interfere with the test and cause erroneous results (e.g., technical or procedural errors).

A preliminary positive result with this assay indicates the presence of amphetamine or methamphetamine in urine. It does not measure the level of intoxication.

{13}------------------------------------------------

ACTION REQUIRED

Special wash programming: The use of special wash steps is mandatory when certain test combinations are run together on COBAS INTEGRA 800 analyzer. Refer to the Method Manual, Introduction, Extra Wash Cycles for further instructions.

Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.

Expected values

No drug should be present in individuals that have not ingested amphetamines.

Specific performance data

Representative performance data on the COBAS INTEGRA 800 analyzer are given below. Results obtained in individual laboratories may differ.

Precision

A d-methamphetamine (MAMP) solution (1 mg/mL) was added to 9 samples obtained from a human urine sample pool to achieve concentrations at approximately - 100 %, - 75 %, - 50 %, -25 %, +/- 0 %, + 25 %, + 50 %, + 75 %, and + 100 % of the cutoff value. These samples were tested for precision in qualitative and semiquantitative modes. Following a CLSI (EPS-A2) precision protocol, samples were tested in 2 replicates per run, 2 runs per day for 21 days, total N = 84.

Qualitative - 300 ng/mL Cutoff
Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
MAMP	zero drug	84	84 Neg / 0 Pos
MAMP	- 75 %	84	84 Neg / 0 Pos
MAMP	- 50 %	84	84 Neg / 0 Pos
MAMP	- 25 %	84	83 Neg / 1 Pos
MAMP	cutoff	84	27 Neg / 57 Pos
MAMP	+ 25 %	84	0 Neg / 84 Pos
MAMP	+ 50 %	84	0 Neg / 84 Pos
MAMP	+ 75 %	84	0 Neg / 84 Pos
MAMP	+ 100 %	84	0 Neg / 84 Pos

{14}------------------------------------------------

Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
MAMP	zero drug	84	84 Neg / 0 Pos
MAMP	- 75 %	84	84 Neg / 0 Pos
MAMP	- 50 %	84	84 Neg / 0 Pos
MAMP	- 25 %	84	84 Neg / 0 Pos
MAMP	cutoff	84	28 Neg / 56 Pos
MAMP	+ 25 %	84	0 Neg / 84 Pos
MAMP	+ 50 %	84	0 Neg / 84 Pos
MAMP	+ 75 %	84	0 Neg / 84 Pos
MAMP	+ 100 %	84	0 Neg / 84 Pos

Qualitative - 500 ng/mL Cutoff

Qualitative - 1000 ng/mL Cutoff
Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
MAMP	zero drug	84	84 Neg / 0 Pos
MAMP	- 75 %	84	84 Neg / 0 Pos
MAMP	- 50 %	84	84 Neg / 0 Pos
MAMP	- 25 %	84	84 Neg / 0 Pos
MAMP	cutoff	84	13 Neg / 71 Pos
MAMP	+ 25 %	84	0 Neg / 84 Pos
MAMP	+ 50 %	84	0 Neg / 84 Pos
MAMP	+ 75 %	84	0 Neg / 84 Pos
MAMP	+ 100 %	84	0 Neg / 84 Pos

{15}------------------------------------------------

Drug	SampleConc.	Results# Neg /# Pos	Repeatability*		IntermediatePrecision**
			SD, ng/mL	CV,%	SD, ng/mL	CV,%
MAMP	zero drug	84 / 0	9.4	95.4	12.2	123.8
MAMP	- 75 %	84 / 0	12.9	15.5	15.7	18.8
MAMP	- 50 %	84 / 0	14.3	9.0	17.7	11.2
MAMP	- 25 %	84 / 0	13.2	5.4	19.8	8.2
MAMP	cutoff	34 / 50	23.2	7.6	23.5	7.7
MAMP	+ 25 %	0 / 84	20.9	5.2	24.4	6.1
MAMP	+ 50 %	0 / 84	22.5	5.0	29.5	6.6
MAMP	+ 75 %	0 / 84	27.4	5.0	36.5	6.6
MAMP	+ 100 %	0 / 84	35.7	5.7	40.8	6.5

Semiquantitative - 300 ng/mL Cutoff

Semiquantitative - 500 ng/mL Cutoff

Drug	SampleConc.	Results# Neg /# Pos	Repeatability*		IntermediatePrecision**
			SD, ng/mL	CV,%	SD, ng/mL	CV,%
MAMP	zero drug	84 / 0	9.6	117.3	11.0	134.1
MAMP	- 75 %	84 / 0	14.6	10.5	20.6	14.9
MAMP	- 50 %	84 / 0	20.0	7.9	21.9	8.6
MAMP	- 25 %	84 / 0	24.1	6.2	27.2	7.0
MAMP	cutoff	39 / 45	28.2	5.6	39.4	7.8
MAMP	+ 25 %	0 / 84	31.5	4.9	48.5	7.5
MAMP	+ 50 %	0 / 84	34.5	4.8	49.5	6.8
MAMP	+ 75 %	0 / 84	41.6	5.0	62.3	7.5
MAMP	+ 100 %	0 / 84	46.7	4.6	72.4	7.1

{16}------------------------------------------------

Drug	SampleConc.	Results# Neg /# Pos	Repeatability*		IntermediatePrecision**
			SD, ng/mL	CV,%	SD, ng/mL	CV,%
MAMP	zero drug	84 / 0	20.2	97.2	24.9	119.5
MAMP	- 75 %	84 / 0	25.3	9.3	30.7	11.2
MAMP	- 50 %	84 / 0	33.3	6.4	39.4	7.5
MAMP	- 25 %	84 / 0	34.3	4.6	52.1	7.0
MAMP	cutoff	25 / 59	56.9	5.5	71.3	6.9
MAMP	+ 25 %	0 / 84	56.6	4.2	79.3	5.9
MAMP	+ 50 %	0 / 84	63.6	4.0	81.3	5.2
MAMP	+ 75 %	0 / 84	60.3	3.5	101.6	5.9
MAMP	+ 100 %	0 / 84	108.1	5.1	126.6	6.0

Semiquantitative - 1000 ng/mL Cutoff

*repeatability = within-run precision

**intermediate precision = total precision / between run precision / between day precision

The same precision experiment was repeated utilizing d-amphetamine (AMP) as the target analyte instead of d-methamphetamine. The following tables show the results obtained on a COBAS INTEGRA 800 analyzer.

Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
AMP	zero drug	84	84 Neg / 0 Pos
AMP	- 75 %	84	84 Neg / 0 Pos
AMP	- 50 %	84	84 Neg / 0 Pos
AMP	- 25 %	84	83 Neg / 1 Pos
AMP	cutoff	84	2 Neg / 82 Pos
AMP	+ 25 %	84	0 Neg / 84 Pos
AMP	+ 50 %	84	0 Neg / 84 Pos
AMP	+ 75 %	84	0 Neg / 84 Pos
AMP	+ 100 %	84	0 Neg / 84 Pos

Qualitative - 300 ng/mL Cutoff

{17}------------------------------------------------

Qualitative - 500 ng/mL Cutoff

Qualitative - 500 ng/mL Cutoff
Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
AMP	zero drug	84	84 Neg / 0 Pos
AMP	- 75 %	84	84 Neg / 0 Pos
AMP	- 50 %	84	84 Neg / 0 Pos
AMP	- 25 %	84	82 Neg / 2 Pos
AMP	cutoff	84	6 Neg / 78 Pos
AMP	+ 25 %	84	0 Neg / 84 Pos
AMP	+ 50 %	84	0 Neg / 84 Pos
AMP	+ 75 %	84	0 Neg / 84 Pos
AMP	+ 100 %	84	0 Neg / 84 Pos

Drug	Concentration ofSample	Number ofDeterminations	Results# Neg / # Pos
AMP	zero drug	84	84 Neg / 0 Pos
AMP	- 75 %	84	84 Neg / 0 Pos
AMP	- 50 %	84	84 Neg / 0 Pos
AMP	- 25 %	84	82 Neg / 2 Pos
AMP	cutoff	84	7 Neg / 77 Pos
AMP	+ 25 %	84	1 Neg / 83 Pos
AMP	+ 50 %	84	0 Neg / 84 Pos
AMP	+ 75 %	84	0 Neg / 84 Pos
AMP	+ 100 %	84	0 Neg / 84 Pos

・

{18}------------------------------------------------

Drug	SampleConc.	Results# Neg /# Pos	Repeatability*		IntermediatePrecision**
			SD, ng/mL	CV,%	SD, ng/mL	CV,%
AMP	zero drug	84 / 0	8.6	110.0	10.6	135.7
AMP	- 75 %	84 / 0	10.4	16.8	13.6	21.9
AMP	- 50 %	84 / 0	14.6	10.0	19.9	13.6
AMP	- 25 %	84 / 0	15.7	6.6	20.6	8.7
AMP	cutoff	4 / 80	25.8	7.4	28.5	8.2
AMP	+ 25 %	0 / 84	30.7	7.1	32.2	7.4
AMP	+ 50 %	0 / 84	31.1	6.1	37.6	7.3
AMP	+ 75 %	0 / 84	29.1	4.9	41.2	7.0
AMP	+ 100 %	0 / 84	36.2	5.3	44.6	6.5
Drug	SampleConc.	Results# Neg# Pos	Repeatability*		IntermediatePrecision **
			SD, ng/mL	CV,%	SD, ng/mL	CV,%
AMP	zero drug	84 / 0	12.6	117.0	13.2	122.5
AMP	- 75 %	84 / 0	21.4	16.9	25.2	20.0
AMP	- 50 %	84 / 0	18.2	6.9	25.6	9.8
AMP	- 25 %	83 / 1	25.6	5.7	29.7	6.6
AMP	cutoff	0 / 84	29.1	4.7	38.3	6.2
AMP	+ 25 %	0-/ 84	42.0	ર. ર	51.7	6.8
AMP	+ 50 %	0 / 84	40.3	4.6	49.9	5.7
AMP	+ 75 %	0 / 84	58.7	5.9	59.7	6.0
AMP	+ 100 %	0 / 84	79.0	7.0	86.3	7 6

Semiquantitative - 300 ng/mL Cutoff

100 - 100 - 100 -

{19}------------------------------------------------

Drug	SampleConc.	Results# Neg /# Pos	Repeatability*		IntermediatePrecision **
			SD, ng/mL	CV,0%	SD, ng/mL	CV.0/0
AMP	zero drug	84 / 0	ાં 5.9	】】【 l	19.0	132.6
AMP	- 75 %	84 / 0	21.9	9.2	29.8	12.5
AMP	- 50 %	84 / 0	30.3	5.4	40.9	7.3
AMP	- 25 %	84 / 0	44.3	5.2	રરે :0	6.4
AMP	cutoff	6 / 78	રૂડે તે	4.8	84.3	7.6
AMP	+ 25 %	0 / 84	72.5	ર્ટ ર્	87 ર	6.7
AMP	+ ૨૦ %	0 / 84	65.7	4.4	તેરું ર	ર્ણ રે
AMP	+ 75 %	0 / 84	74.3	4.5	105.2	64
AMP	+ 100 %	0 / 84	106.7	ર તેવે	131.4	7.3

Semiquantitative - 1000 no/mL Cutoff

*repeatability = within-run precision

** intermediate precision = total precision / between run precision / between day precision

Accuracy

The accuracy of this assay was determined against d-methamphetamine or d-amphetamine GC/MS results. The evaluated cutoff concentrations for the GC/MS testing were the same as the assay screening cutoffs. For both d-methamphetamine, 36 urine samples, obtained from a clinical laboratory were confirmed to be negative by GC/MS and were evaluated with Amphetamines II. 100 % of these normal urines were negative with both the semiguantitative and qualitative assay relative to the 300 ng/mL, and 1000 ng/mL cutoffs. 4 additional unaltered samples, containing d-methamphetamine between 85-204 ng/mL (GC/MS confirmed), were tested with the 500 ng/mL cutoff and gave negative results.

36 samples obtained from a clinical laboratory, where they screened positive with a commercially available enzyme immunoassay and were subsequently confirmed positive by GC/MS to contain d-methamphetamine or d-amphetamine, were evaluated with Amphetamines II. 100% of these samples were positive relative to the 300 ng/mL, and 1000 ng/mL, cutoffs.

In addition, several unaltered near cutoff samples were run for each cutoff. These samples fell in the near cutoff negative range (between - 50 % and cutoff) and the near cutoff positive range (between cutoff and + 50 %). For each cutoff, 4 negative near cutoff samples and 4 positive near cutoff samples were assayed.

Data from the accuracy studies described above were combined with data generated from the unaltered near cutoff urine samples. The following results were obtained with the Amphetamines II assay on the COBAS INTEGRA 800 analyzer relative to the GC/MS values for both d-methamphetamine and d-amphetamine.

{20}------------------------------------------------

Amphetamines II Qualitative Assay Results (MAMP)

RocheONLINEDAT AMPIIassay	Low Neg	Near CutoffNegative byGC/MS(between -50 % andcutoff)	Near CutoffPositive byGC/MS(between cutoffand + 50 %)	High Positiveby GC/MS(greater than+ 50 %)	PercentAgreement withGC/MS (MAMP)
300 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	36	0	0	0	90 %
500 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	40	0	0	0	91 %
1000 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	36	0	0	0	90 %

Amphetamines II Semiquantitative Assay Results (MAMP)

RocheONLINEDAT AMPΙΙassay	Low Neg	Near CutoffNegative byGC/MS(between -50 % andcutoff)	Near CutoffPositive byGC/MS(between cutoffand + 50 %)	High Positiveby GC/MS(greater than+ 50 %)	PercentAgreement withGC/MS (MAMP)
300 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	36	0	0	0	90 %
500 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	40	0	0	0	91 %
1000 ng/mL Cutoff
Positive	0	4	4	36	100 %
Negative	36	0	0	0	90 %

{21}------------------------------------------------

Accuracy samples were categorized based upon the d-methamphetamine GC/MS concentration only. The table below identifies those samples with a d-methamphetamine concentration below the cutoff, in which the observed result on a COBAS INTEGRA 800 analyzer was positive. The expected results column identifies the result expected with the Amphetamines II assay based upon the d-methamphetamine (MAMP) value relative to the cutoff.

Cutoff Value(ng/mL)	Roche ONLINEDAT AMP IIOBSERVEDResult	Roche ONLINEDAT AMP IIEXPECTEDResult	GC/MS(ng/mL)	Drug / Metabolite
300 (SQ, Q)	Positive	Positive	173181	MAMPAMP
300 (SQ, Q)	Positive	Positive	278101	MAMPAMP
300 (SQ, Q)	Positive	Positive	171220	MAMPAMP
300 (SQ, Q)	Positive	Positive	291145	MAMPAMP
500 (SQ, Q)	Positive	Positive	488466	MAMPAMP
500 (SQ, Q)	Positive	Negative	325171	MAMPAMP
500 (SQ, Q)	Positive	Negative	291145	MAMPAMP
500 (SQ, Q)	Positive	Positive	472650	MAMPAMP
1000 (SQ, Q)	Positive	Positive	706443	MAMPAMP
1000 (SQ, Q)	Positive	Positive	540693	MAMPAMP
1000 (SQ, Q)	Positive	Positive	769395	MAMPAMP
1000 (SQ, Q)	Positive	Positive	572432	MAMPAMP

GC/MS Summary of Discrepant Results (MAMP)

{22}------------------------------------------------

Amphetamines II Qualitative Assay Results (AMP)

. 1

RocheONLINEDAT AMPIIassay	Low Neg	Near CutoffNegative byGC/MS(between -50 % andcutoff)	Near CutoffPositive byGC/MS(between cutoffand + 50 %)	High Positiveby GC/MS(greater than+ 50 %)	PercentAgreement withGC/MS (AMP)
300 ng/mL Cutoff
Positive	0	3	4	36	100 %
Negative	36	1	0	0	92.5 %
500 ng/mL Cutoff
Positive	0	3	4	36	100 %
Negative	36	1	0	0	92.5 %
1000 ng/mL Cutoff
Positive	0	1	4	36	100 %
Negative	36	3	0	0	97.5 %

. .

Amphetamines II Semiquantitative Assay Results (AMP)

. .

RocheONLINEDAT AMPIIassay	Low Neg	Near CutoffNegative byGC/MS(between -50 % andcutoff)	Near CutoffPositive byGC/MS(between cutoffand + 50 %)	High Positiveby GC/MS(greater than+ 50 %)	PercentAgreement withGC/MS (AMP)
300 ng/mL Cutoff
Positive	0	3	4	36	100 %
Negative	36	1	0	0	92.5 %
500 ng/mL Cutoff
Positive	0	3	4	36	100 %
Negative	36	1	0	0	92.5 %
1000 ng/mL Cutoff
Positive	0	2	4	36	100 %
Negative	36	2	0	0	95 %

{23}------------------------------------------------

Accuracy samples were categorized based upon the d-amphetamine GC/MS concentration only. The table below identifies those samples with a d-amphetamine concentration below the cutoff, in which the observed result on a COBAS INTEGRA 800 analyzer was positive. The expected results column identifies the result expected with the Amphetamines II assay based upon the damphetamine (AMP) value relative to the cutoff.

Cutoff Value(ng/mL)	Roche ONLINEDAT AMP IIOBSERVEDResult	Roche ONLINEDAT AMP IIEXPECTEDResult	GC/MS(ng/mL)	Drug / Metabolite
300 (SQ, Q)	Positive	Positive	157363	AMPMAMP
300 (SQ, Q)	Positive	Positive	181173	AMPMAMP
300 (SQ, Q)	Positive	Positive	220171	AMPMAMP
500 (SQ, Q)	Positive	Positive	438121	AMPMAMP
500 (SQ, Q)	Positive	Positive	4571152	AMPMAMP
500 (SQ, Q)	Positive	Positive	443706	AMPMAMP
1000 (SQ)	Positive	Negative	920	AMP
1000 (SQ, Q)	Positive	Positive	8371163	AMPMAMP

GC/MS Summary of Discrepant Results (AMP)

Analytical specificity

The specificity of Amphetamines II, on the COBAS INTEGRA 800 analyzer, for various phenethylamines and structurally similar compounds was determined by generating inhibition curves for each of the compounds listed for both semiquantitative modes and determining the approximate quantity of each compound that is equivalent in assay reactivity to the 300 ng/mL, 500 ng/mL, and 1000 ng/mL d-methamphetamine assay cutoff. The tables below show the semiquantitative results of the study for each assay cutoff. The same samples were run in the qualitative mode and all recovered appropriately negative or positive, based on the calculated cross-reactivity.

{24}------------------------------------------------

Compound	ng/mLEquivalent to300 ng/mLMAMP	Approx. PercentCross-reactivity
± MDMAc	114	264
± MDAf	249	121
± MDEAh	285	105
d -Amphetamine	311	96
d -Methamphetamine	327	92
± MBDB HClg	339	88
± BDB HCli	648	46
l -Methamphetamine	2754	11
l -Amphetamine	6443	5
Phendimetrazine	47473	0.63
Phentermine	66385	0.45
Tyramine	86271	0.35
d -Pseudoephedrine	87578	0.34
l -Ephedrine	94792	0.32
d,l -Phenylpropanolamine HCl	280374	0.11
d -Ephedrine	329670	0.09

and the country of the county of the county of

{25}------------------------------------------------

Compound	ng/mLEquivalent to500 ng/mLMAMP	Approx. PercentCross-reactivity
± MDMAc	173	289
± MDAf	433	115
d -Methamphetamine	444	113
d -Amphetamine	460	109
± MDEAh	494	101
± MBDB HClg	713	70
± BDB HCli	1209	41
l -Methamphetamine	4098	12
l -Amphetamine	11174	4
Phendimetrazine	72500	0.69
Phentermine	118483	0.42
d -Pseudoephedrine	132275	0.38
Tyramine	141243	0.35
l -Ephedrine	154321	0.32
d,l -Phenylpropanolamine HCl	390625	0.13
d -Ephedrine	413223	0.12

and the comments of the comments of the comments of the comments of

{26}------------------------------------------------

Compound	ng/mLEquivalent to1000 ng/mLMAMP	Approx. PercentCross-reactivity
± MDMAc	446	224
± MDAf	785	127
d -Methamphetamine	970	103
d -Amphetamine	1024	98
± MBDB HClg	1194	84
± MDEAh	1203	83
± BDB HCli	2262	44
l -Methamphetamine	9008	11
l -Amphetamine	23445	4
Phendimetrazine	156740	0.64
d -Pseudoephedrine	269542	0.37
Phentermine	294118	0.34
l -Ephedrine	317460	0.32
Tyramine	323625	0.31
d -Ephedrine	793651	0.13
d, l -Phenylpropanolamine HCl	1111111	0.09

e) d,l-3,4-Methylenedioxymethamphetamine

f) d,I-3,4-Methylenedioxyamphetamine

g ) d J-N-Methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine hydrochloride

h) d, l-3,4-Methylenedioxyethylamphetamine

i) d,/-3,4-Methylenedioxyphenyl-2-butanamine hydrochloride

{27}------------------------------------------------

Interference and cross-reactivity with unrelated drugs

The following compounds were added at the listed concentrations to a human urine pool spiked with either d-amphetamine or d-methamphetamine at approximately the negative and positive control concentrations for each cutoff (+/- 25 % of assay cutoff). For each compound, the control level samples recovered properly for the 300 ng/mL, 500 ng/mL, and 1000 ng/mL cutoff in both semiquantitative and qualitative modes.

		SemiquantitativeAll Cutoffs		QualitativeAll Cutoffs
Compound	Conc. (ng/mL)	Low Ctrl	High Ctrl	Low Ctrl	High Ctrl
Acetaminophen	100000	NEG	POS	NEG	POS
Acetylsalicylic acid	100000	NEG	POS	NEG	POS
Amitriptyline	100000	NEG	POS	NEG	POS
Aspartame	40000	NEG	POS	NEG	POS
Benzocaine	100000	NEG	POS	NEG	POS
Benzoylecgonine	100000	NEG	POS	NEG	POS
Caffeine	100000	NEG	POS	NEG	POS
Cannabidiol	100000	NEG	POS	NEG	POS
Cocaine	100000	NEG	POS	NEG	POS
Codeine	100000	NEG	POS	NEG	POS
Desipramine HCl	100000	NEG	POS	NEG	POS
Dextromethorphan	100000	NEG	POS	NEG	POS
Dextropropoxyphene	100000	NEG	POS	NEG	POS
Diazepam	100000	NEG	POS	NEG	POS
Digoxin	100000	NEG	POS	NEG	POS
Diphenhydramine	100000	NEG	POS	NEG	POS
Diphenylhydantoin	100000	NEG	POS	NEG	POS
Doxepin	100000	NEG	POS	NEG	POS
Ecgonine	100000	NEG	POS	NEG	POS
Ecgonine methyl ester	100000	NEG	POS	NEG	POS
Erythromycin	100000	NEG	POS	NEG	POS
Furosemide	100000	NEG	POS	NEG	POS
Guaiacol glycerol ether	100000	NEG	POS	NEG	POS
Hydrochlorothiazide	100000	NEG	POS	NEG	POS
Ibuprofen	100000	NEG	POS	NEG	POS
Ketamine	100000	NEG	POS	NEG	POS
Levothyroxine	100000	NEG	POS	NEG	POS
LSD	2500	NEG	POS	NEG	POS
Meperidine	100000	NEG	POS	NEG	POS
Methadone	100000	NEG	POS	NEG	POS
Methaqualone	75000	NEG	POS	NEG	POS
Morphine	100000	NEG	POS	NEG	POS
Naloxone	100000	NEG	POS	NEG	POS
Naltrexone	100000	NEG	POS	NEG	POS
Naproxen	100000	NEG	POS	NEG	POS
Niacinamide	100000	NEG	POS	NEG	POS
Nicotine	100000	NEG	POS	NEG	POS
Nifedipine	100000	NEG	POS	NEG	POS
Nordiazepam	100000	NEG	POS	NEG	POS
Omeprazole	100000	NEG	POS	NEG	POS
Oxazepam	100000	NEG	POS	NEG	POS
Penicillin G	100000	NEG	POS	NEG	POS
Phencyclidine	40000	NEG	POS	NEG	POS
Phenobarbital	100000	NEG	POS	NEG	POS
Quinine	100000	NEG	POS	NEG	POS
Secobarbital	100000	NEG	POS	NEG	POS
Tetracycline	100000	NEG	POS	NEG	POS
Δ9-THC	10000	NEG	POS	NEG	POS

{28}------------------------------------------------

The compounds were additionally added to aliquots of pooled drug-free human urine at a concentration of 100000 ng/mL. None of these compounds gave values in the assay that were equal to or greater than 0.19 % cross-reactivity and no results were greater than the assay cutoffs (300 ng/mL, 500 ng/mL, and 1000 ng/mL), with the following exception.

The cross-reactivity for LSD was tested at a concentration of 2500 ng/mL. The results obtained were 0.32 %, and 0.71 %, for the 300 ng/mL and 1000 ng/mL assay cutoffs respectively.

Interference with other substances

Interfering substances were added to urine containing d-methamphetamine (MAMP) at - 25 % and + 25 % of the cutoff level at the concentration listed below. The same substances were additionally added to urine containing d-amphetamine (AMP) at - 25 % and + 25 % of the cutoff level at the concentration listed below. All samples were tested and the following results were obtained on a COBAS INTEGRA 800 analyzer. The value in the table indicates the level at which no interference was found for samples containing either d-methamphetamine or d-amphetamine.

Qualitative		300 ng/mL Cutoff		500 ng/mL Cutoff		1000 ng/mL Cutoff
Compound	Cmpd.Conc.	NegLevel	PosLevel	NegLevel	PosLevel	NegLevel	PosLevel
Acetone	7.9 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Ascorbic Acid	10 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
ConjugatedBilirubin	0.1 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Creatinine	5 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Ethanol	7.9 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Glucose	12 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Hemoglobin	1 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Human serumalbumin	3 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Oxalic Acid	2 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
Sodium Chloride	23 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos

{29}------------------------------------------------

Urea	60 mg/mL	Neg	Pos	Neg	Pos	Neg	Pos
------	----------	-----	-----	-----	-----	-----	-----

The same experiment was performed in the semiquantitative mode for each cutoff. All negative and positive controls recovered properly, within 80 % - 120 %, in the presence of the interfering substance.

An additional protocol was executed in which samples containing either AMP or MAMP at control levels (± 25 % of cutoff) with specific gravities ranging from 1.001 to 1.034 were tested. As with the other interferences, there were no control cross-overs on any of the 3 assay cutoffs at either extreme specific gravity level.

Samples having a pH ranging from 4.5 to 8.0 and containing either AMP or MAMP at control levels (+ 25 % of cutoff) were also tested. There were ≤ 5 % cross-overs on any of the 3 assay cutoffs.

Any modification of the instrument as set forth in this labeling requires validation by the laboratory.

References

1. Karch SB, ed. Drug Abuse Handbook. Boca Raton, FL: CRC Press LLC 1998.
1. Borenstein M. Central Nervous System Stimulants. In: Troy D. ed. Remington: The Science and Practice of Pharmacy. 21st ed. Baltimore, MD: Lippincott Williams & Wilkins Co 2005:1551.
1. NIDA Research Report Methamphetamine Abuse and Addiction: NIH Publication No. 06-4210. National Institute on Drug Abuse 6001 Executive Blvd., Room 5213, Bethesda, MD 20892-9561.
1. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man. 7th ed. Foster City, CA: Biomedical Publications 2004:67.
1. Armbruster DA, Schwarzhoff RH, Pierce BL, Hubster EC. Method comparison of EMIT II and ONLINE with RIA for drug screening. J Forensic Sci 1993;38:1326-1341.
1. Armbruster DA, Schwarzhoff RH, Hubster EC, Liserio MK. Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening. Clin Chem 1993;39:2137-2146.
1. Rouse S, Motter K, McNally A, et al. An Abuscreen OnLine Immunoassay for the Detection of Amphetamine in Urine on the COBAS MIRA Automated Analyzer. Clin Chem 1991;37(6):995. Abstract.
1. Toxicology and Drug Testing in the Clinical Laboratory; Approved Guideline. 2nd ed. (C52-A2). Wayne, PA: Clinical and Laboratory Standards Institute 2007;27:33.
1. Mandatory Guidelines for Federal Workplace Drug Testing Programs (Revised Specimen Validity Testing). Fed Regist 2004;69:19643-19673.
1. Data on file at Roche Diagnostics.

COBAS INTEGRA, COBAS C, and PRECISET are trademarks of Roche. Other brands are trademarks of their respective holders.

C 2010, Roche Diagnostics

{30}------------------------------------------------

Image /page/30/Picture/1 description: The image shows the logo for the Department of Health & Human Services USA. The logo consists of a circular border with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" arranged around the top half of the circle. Inside the circle is a stylized emblem featuring a symbol that resembles an abstract eagle or bird-like figure with three distinct, curved lines forming the body and wings.

Food and Drug Administration 10903 New Hampshire Avenue Document Mail Center - WO66-0609 Silver Spring, MD 20993-0002

Jan 2 8 2010

Roche Diagnostics c/o Mrs. Michelle Neff 9115 Hague Road Indianapolis, IN 46250

Re: K093664

Trade/Device Name: Amphetamines II Assay Regulation Number: 21 CFR §862.3100 Regulation Name: Amphetamine Test System Regulatory Class: Class II Product Code: DKZ, LAF Dated: July 2, 2010 Received: July 6, 2010

Dear Mrs. Neff:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include reguirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

{31}------------------------------------------------

Page 2

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at (301) 796-5760. For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or ( 301 ) 796-5680 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Signature

Courtney C. Harper, Ph.D. Director Division of Chemistry and Toxicology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

{32}------------------------------------------------

Indication for Use

510(k) Number (if known):

K093664

JUL 2 8 2010

Device Name: Amphetamines II Indication For Use:

Prescription Use XXX (21 CFR Part 801 Subpart D) And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K093664

Regulation Number and Section

§ 862.3100 Amphetamine test system.

(a)
Identification. An amphetamine test system is a device intended to measure amphetamine, a central nervous system stimulating drug, in plasma and urine. Measurements obtained by this device are used in the diagnosis and treatment of amphetamine use or overdose and in monitoring levels of amphetamine to ensure appropriate therapy.(b)
Classification. Class II (special controls). An amphetamine 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).