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Intercept® i2he™
Amphetamine Oral Fluid Assay
FOR FORENSIC USE ONLY
For Use with Intercept® i2he™ Oral Fluid Collection Device
1001-0385 (65 mL Kit) INTENDED USE
The Intercept® i2he™ Amphetamine Oral Fluid Assay is intended for use in the qualitative determination of amphetamine in human oral fluid at a cutoff
concentration of 150 ng/mL in neat oral fluid. The specimen must be collected exclusively with the Intercept® i2he™ Oral Fluid Collection Device. The assay is calibrated against d-amphetamine and performed on clinical chemistry analyzers. This device is not intended for use for clinical diagnostic applications.
The Intercept® i2he™ Amphetamine Oral Fluid Assay provides only a preliminary analytical test result. A more specific alternative
method must be used to obtain a confirmed analytical result. Gas Chromatography/Mass Spectrometry (GC/MS) and Liquid
Chromatography-Tandem Mass Spectrometry (LC-MS/MS) are the preferred confirmatory methods.(1-3) Clinical consideration and
professional judgment should be applied to any drug of abuse test result particularly when preliminary positive results are used.
SUMMARY AND EXPLANATION OF THE TEST
Amphetamine is generally self-administered either by nasal inhalation or ingestion. The concentration of amphetamine is about three times higher in saliva
than in plasma and can be detected in saliva up to 50 hours.(4) Because of the high concentration of amphetamine in saliva and the effect of urine pH on the excretion of drug, saliva is a better medium for the determination of amphetamine.(5) Intercept® i2he™ Amphetamine Oral Fluid Assay uses recombinant DNA technology to produce a unique homogeneous enzyme immunoassay system.(6) The assay is based on the bacterial enzyme β-galactosidase, which has been genetically engineered into two inactive fragments. These fragments spontaneously re-associate to form fully active enzyme that, in the assay format, cleave a substrate, generating a color change that can be measured spectrophotometrically.
In the assay, analyte in the sample competes with analyte conjugated to one inactive fragment (enzyme donor) of β-galactosidase for antibody binding site. If analyte is present in the sample, it binds to antibody, leaving the inactive enzyme fragment free to form active enzyme. If the analyte is not present in the sample, antibody binds to analyte conjugated on the inactive fragment, inhibiting the re-association of inactive β-galactosidase fragments, and no active enzyme is formed. The amount of active enzyme formed and resultant absorbance change are directly proportional to the amount of analyte present in the REAGENTS
1. EA Reconstitution Buffer
Contains buffer salts, 1.5 mg/L mouse monoclonal anti-amphetamine antibody, stabilizer, and preservative.
1a EA Reagent
Contains 0.171 g/L Enzyme Acceptor (microbial), buffer salts and preservative.
2. ED Reconstitution Buffer
Contains buffer salts, stabilizers, and preservative.
2a ED Reagent
Contains 0.135 mg/L Enzyme Donor (microbial) conjugated to amphetamine derivative, 1.67 g/L chlorophenol red-β-D-galactopyranoside, stabilizers, detergent and preservative.
Additional Materials Required (sold separately):
1001-0394 Intercept® i2he™ Multi-Drug Oral Fluid Control Set A
1001-0395 Intercept® i2he™ Multi-Drug Oral Fluid Cutoff Calibrator Set A 1001-0396 Intercept® i2he™ Multi-Drug Oral Fluid Negative Calibrator Set A1001-0364 Intercept® i2he™ Oral Fluid Collection Device (50/Box) 1001-0365 Intercept® i2he™ Oral Fluid Collection Device (500/Box) PRECAUTIONS AND WARNINGS
1. This test is for forensic use only. 2. The reagents are harmful if swallowed.
3. Reagents used in the assay components contain ≤ 0.13% sodium azide. Avoid contact with skin and mucous membranes. Refer to SDS for additional precautions, handling instructions, and accidental exposure treatment.
4. In the case of accidental spill, clean and dispose of material according to your laboratory's SOP, local, and state regulations.
5. The reagents contain ≤ 0.2% bovine serum albumin (BSA). Avoid contact with skin and mucous membranes. Avoid inhalation. May cause skin or inhaled allergic reaction. Refer to SDS for additional precautions, handling instructions, and accidental exposure treatment.
6. In the case of damaged packaging on arrival, contact your technical support representative (refer to back page of this Package Insert).
REAGENT PREPARATION AND STORAGE
For preparation of the solutions, refer to the section below. Remove the kit from refrigerated storage (2-8°C) immediately prior to preparation of the
solutions.
Prepare the solutions in the following order to minimize possible contamination.
R2 ENZYME DONOR SOLUTION
Connect Bottle 2a (ED reagent) to Bottle 2 (ED Reconstitution Buffer) using one of the enclosed adapters. Mix by gentle inversion, ensuring that all the
lyophilized material from Bottle 2a is transferred into Bottle 2. Avoid the formation of foam. Detach Bottle 2a and adapter from Bottle 2 and discard. Cap Bottle 2 and let stand approximately 5 minutes at room temperature (21-25°C). Mix again. Record the reconstitution date on the bottle label. Place the bottle directly into the reagent compartment of the analyzer or into refrigerated storage (2-8°C) and let stand 30 minutes before use. R1 ENZYME ACCEPTOR SOLUTION
Connect Bottle 1a (EA reagent) to Bottle 1 (EA Reconstitution Buffer) using one of the enclosed adapters. Mix by gentle inversion, ensuring that all the
lyophilized material from Bottle 1a is transferred into Bottle 1. Avoid the formation of foam. Detach Bottle 1a and adapter from Bottle 1 and discard. Cap Bottle 1 and let stand approximately 5 minutes at room temperature (21-25°C). Mix again. Record the reconstitution date on the bottle label. Place the bottle directly into the reagent compartment of the analyzer or into refrigerated storage (2-8°C) and let stand 30 minutes before use.
NOTE 1: The components supplied in this kit are intended for use as an integral unit. Do not mix components from different lots.
NOTE 2: Avoid cross-contamination of reagents by matching reagent caps to the proper reagent bottle. The R2 solution (Enzyme Donor) should be yellow-
orange in color. A red or red-purple color indicates that the reagent has been contaminated and must be discarded.
NOTE 3: The R1 and R2 solutions must be at the reagent compartment storage temperature of the analyzer before performing the assay. Refer to the analyzer
specific application sheet for additional information.
Store reagents at 2-8°C. DO NOT FREEZE. For shelf life of the unopened components, refer to the box or bottle labels for the expiration date.
R1 Solution: 60 days refrigerated on analyzer or at 2-8°C.
R2 Solution: 60 days refrigerated on analyzer or at 2-8°C.
SPECIMEN COLLECTION AND HANDLING
Oral fluid samples are suitable for use in the Intercept® i2he™ Amphetamine Oral Fluid Assay. Collect oral fluid samples using the Intercept® i2he™ Oral
Fluid Collection Device. Care should be taken to preserve the chemical integrity of the oral fluid sample from the time it is collected until the time it is assayed by securely capping the samples. The samples can be stored at 4-37°C/39-98°F for up to 21 days. Storage at -20°C/-4°F is recommended for storage longer than 21 days.
NOTE: Handle oral fluid samples as if they were potentially infectious.
Samples within a pH range of 5-9 are suitable for testing with this assay.
Intercept® i2he™ SAMPLE PROCESSING PROCEDURE
Materials Required But Not Provided
1. Tubes suitable for centrifuging Intercept® i2he™ Specimen Vials.
2. Centrifuge capable of 600 – 800 x g.
1. Record the specimen identification number from the Intercept® i2he™ Specimen Vial onto the centrifuge tube.
2. Ensure that the specimen is within acceptable dating for testing, i.e., ≤ 21 days from the time of collection.
3. Hold the vial upright with the tip pointed up.
4. Move the pad away from the vial tip by gently tapping the vial.
5. Place a centrifuge tube over the vial.
6. Break the pointed tip of the vial off against the inside wall of the centrifuge tube.
7. Invert and centrifuge at 600 – 800 x g for 5 minutes.
8. Assay or store the resulting eluate at 2-8°C for up to 21 days or -10 to -20°C for longer than 21 days.
ASSAY PROCEDURE
The Intercept® i2he™ Oral Fluid Collection Device contains a preservative solution that dilutes the neat oral fluid sample. The calibrator and control levels
are set at diluted levels so that sample absorbance values can be compared directly to the absorbance value of the cutoff calibrator. The assay result is reported as a positive or negative result relative to the neat oral fluid cutoff of 150 ng/mL. The concentrations reported in this insert refer to the neat oral fluid concentration, unless otherwise noted.
NOTE: To correlate the Intercept® i2he™ result from the assay or the associated LC-MS/MS confirmation result to a neat oral fluid value, the result from the
Intercept® i2he™ sample should be multiplied by a factor of 3.
1. Pipet the processed oral fluid samples, calibrators, and controls into labeled cups and place the cups onto the analyzer.
2. Load reagents (reagent 1 and reagent 2) into the reagent compartment of the analyzer.
3. Program the run setup using 570 nm as a primary wavelength and 660 nm as the secondary wavelength. Refer to the parameter sheet for detailed instructions on how to program the analyzer.
4. Use the cutoff calibrator as a reference for distinguishing negative from positive samples.
QUALITY CONTROL
The Intercept® i2he™ Multi-Drug Oral Fluid Control Set A is designed to be used with the Intercept® i2he™ Amphetamine Oral Fluid Assay. Good laboratory
practice suggests the use of control specimens to ensure proper assay performance. Ensure that control results are within the established ranges determined by laboratory practices and guidelines. If control results fall outside of established ranges, assay results are invalid. All quality control requirements should be performed in conformance with local, state, and/or federal regulations or accreditation requirements.
INTERPRETATION OF RESULTS
A sample that exhibits a change in absorbance (∆A) value equal to or greater than the value obtained with the cutoff calibrator is considered positive. A
sample that exhibits a change in absorbance (∆A) value lower than the value obtained with the cutoff calibrator is considered negative.
EXTRACTION EFFICIENCY AND DRUG RECOVERY
Extraction efficiency of the preservative solution in recovering the drug from the collection device pad was determined by testing the recovery of the drug by
LC-MS/MS method. The oral fluid samples at the cutoff level and ± 50% control levels recovered within ± 20% from nominal values.
LIMITATIONS
A positive result from this assay indicates only the presence of amphetamine and does not necessarily correlate with the extent of physiological and
psychological effects.
It is possible that other substances and/or factors (e.g. technical or procedural), other than those investigated in the specificity study may interfere with the test and cause false results.
SPECIFIC PERFORMANCE CHARACTERISTICS
Typical performance results obtained on the Olympus AU 480 analyzer are shown below.
PRECISION AND CUTOFF CHARACTERIZATION
Negative neat oral fluid samples were collected and prepared by spiking amphetamine at negative, -75%, -50% and -25% below the cutoff, at the cutoff, and
+25%, +50%, +75% and 100% above the cutoff. All spiked neat oral fluid sample concentrations were confirmed by LC-MS/MS. The neat oral fluid samples were processed using the Intercept® i2he™ Oral Fluid Collection Device to obtain diluted oral fluid samples. The diluted oral fluid samples were confirmed by LC-MS/MS and tested in the Intercept® i2he™ Amphetamine Oral Fluid Assay in qualitative mode.
The randomized CLSI (EP5-A2) precision protocol was followed with six replicates of each sample for each run, 2 runs per day for five non-consecutive days, total N= 60/level. The results are summarized in the table below.
Intercept® i2he™
Tested Concentration
Neat Oral Fluid
Diluted Oral Fluid
Amphetamine Oral Fluid Assay
# Neg / # Pos
Intercept® i2he™
Tested Concentration
Neat Oral Fluid
Diluted Oral Fluid
Amphetamine Oral Fluid Assay
# Neg / # Pos
SPECIFICITY AND CROSS-REACTIVITY
Compounds used in over-the-counter cold medicines and other compounds that are structurally related to amphetamine were tested for cross-reactivity in
the assay. The cross-reactant solutions were prepared by adding the compounds to neat oral fluid samples at the concentration listed in the table below. The neat oral fluid samples were processed using the Intercept® i2he™ Oral Fluid Collection Device to obtain diluted oral fluid samples which were tested in the Intercept® i2he™ Amphetamine Oral Fluid Assay. The compounds listed below produced a negative result at the concentrations tested.
Intercept® i2he™
Tested Concentration In
Neat Oral Fluid (ng/mL)
Amphetamine Oral Fluid Assay
d, l-amphetamine Various common over-the-counter medications and structurally unrelated compounds were tested for cross-reactivity in the assay. The cross-reactant solutions were prepared by adding a known amount of each compound to neat oral fluid at the concentrations listed in the table below. The neat oral fluid samples were processed using the Intercept® i2he™ Oral Fluid Collection Device to obtain diluted oral fluid samples which were tested in the Intercept® i2he™ Amphetamine Oral Fluid Assay. All the compounds tested negative and did not show any cross-reactivity.
Intercept® i2he™
Tested Concentration In
Neat Oral Fluid (ng/mL)
Amphetamine Oral Fluid Assay
Acetylsalicylic Acid Intercept® i2he™
Tested Concentration In
Neat Oral Fluid (ng/mL)
Amphetamine Oral Fluid Assay
11-nor- ∆9 -THC-COOH ENDOGENOUS AND EXOGENOUS SUBSTANCES AND PH INTERFERENCE
The potential interference from several endogenous and exogenous substances, and pH on the detection accuracy of the samples containing amphetamine
at ± 50% of the cutoff concentration were tested in the assay. The interfering substances were added to neat oral fluid at the concentrations listed in the table below. The neat oral fluid samples were processed using the Intercept® i2he™ Oral Fluid Collection Device and tested in the Intercept® i2he™ Amphetamine Oral Fluid Assay. No interference was observed with the interfering substances and pH 5-9 samples at the ± 50% cutoff concentrations.
Intercept® i2he™ Amphetamine Oral Fluid Assay
Tested Concentration
Positive / Negative
In Neat Oral Fluid
Human Serum Albumin Intercept® i2he™ Amphetamine Oral Fluid Assay
Tested Concentration
Positive / Negative
In Neat Oral Fluid
Alcohol (Ethanol) Hydrogen Peroxide ADDITIONAL INTERFERENCE FROM OTHER FOOD AND DENTAL PRODUCTS
Potential interference from additional compounds was tested by collecting neat oral fluid from volunteers after use of the following substances: hard candy,
chewing gum, chewing tobacco, cigarettes, water and tooth whitening strips. The ± 50% controls in the presence of above interfering substances were detected accurately in the Intercept® i2he™ Amphetamine Oral Fluid Assay.
METHOD COMPARISON
Forty negative samples collected from rehabilitation clinics were supplemented by forty neat oral fluid samples spiked with d-amphetamine. The neat oral
fluid samples were processed using the Intercept® i2he™ Oral Fluid Collection Device. The diluted oral fluid samples were tested by both the Intercept® i2he™ Amphetamine Oral Fluid Assay and LC-MS/MS.
The overall concordance between the Intercept® i2he™ Amphetamine Oral Fluid Assay and LC-MS/MS using a cutoff of 150 ng/mL in neat oral fluid is 100%. The comparison of sample results by the Intercept® i2he™ Amphetamine Oral Fluid Assay to LC-MS/MS showed 100% sensitivity and 100% EXPLANATION OF SYMBOLS
Temperature Limitation Consult Instructions for Use Manufactured For (As in manufactured for OraSure Technologies, Inc. by Forensic Use Only 1. Cone, E. J., et al. (2002). Oral Fluid Testing for Drugs of Abuse: Positive Prevalence Rates by Intercept (TM) Immunoassay Screening and GC-MS- MS Confirmation and Suggested Cutoff Concentrations. Journal of Analytical Toxicology, 26, 541-547.
2. Maurer, H. H. (2005). Advances in Analytical Toxicology: The Current Role of Liquid Chromatography-Mass Spectrometry in Drug Quantification in Blood and Oral Fluid. Analytical and Bioanalytical Chemistry, 381, 110-118.
3. Lambert, W. E., et al. (2002). Simultaneous, quantitative determination of opiates, amphetamine, cocaine and Benzoylecgonine in oral fluid by liquid chromatography quadrupole-time of- flight mass spectrometry. Chromatogr B Analyt Technol Biomed Life Sci., 779, 321-30.
4. Azarnoff, D. L., Mortin, S. B., & Wan, S.H. (1978). Kinetics, salivary excretion of amphetamine isomers and effect of urinary pH. Clin. Pharmacol. Ther., 23, 589.
5. Beckett, A. H., Rowland, M., & Turner, P. (1965). Influence of urinary pH on excretion of amphetamine. Lancet, 1, 303.
6. Henderson, D. R., et al. (1986). CEDIA, A New homogeneous Immunoassay System. Clin. Chem., 32, 1637-1641.

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Intercept® i2he™ is a trademark of OraSure Technologies, Inc.

Source: http://www.orasure.com/docs/i2he-assay-PIs/Intercept-i2he-Amphetamine-Assay-PI.pdf