Methamphetamine Test

One step-Membrane Immunoassay for Qualitative Detection of Metbamphetamine and Its Metabolite in Human Urine

INTENDED USE 
The Methamphetamine test kit is an easy to use immunoassay intended for use in the qualitative analysis for Methamphetamine and its metabolite in human urine. It should not be used without proper supervision and is not intended for sale to the general public. The Methamphetamine test kit provides only a preliminary screening test result; a more specific alternate chemical method must be used in order to confirm the analytical result. Gas chromatography / Mass spectrometry (GC/MS) is the preferred confirmatory method (1). Clinical considerations and professional judgment should be applied to any drug of abuse analysis, particularly when positive results are indicated (2, 3).

SUMMARY AND EXPLANATION OF TEST
Methamphetamine is the most popular synthetic derivative of the amphetamines. These drugs are particularly potent central nervous system (CNS) stimulants. The most common amphetamines are d, 1-amphetamine, d-amphetamine, and methamphetamine. They are sympathomimetic agents, which at therapeutic doses, have been used as diet pills, to overcome narcolepsy, to treat attention deficit disorders in children, and during surgery to maintain blood pressure of patients under anesthesia. These qualities have spread its use to many population groups including students in universities (4). Acute higher doses, as when abused, lead to enhanced CNS stimulation, manifested euphoria, and decreased fatigue and anorexia. More acute responses are anxiety, confusion, paranoia, psychosis, seizures, and cardiac dysrhythmias. As a consequence, there is a strong tendency continue to use the amphetamines to maintain the high, but tolerance develops and increasingly larger doses are required to maintain the original levels of stimulation. Oral ingestion or intravenous amphetamine injection gives a rapid onset of action due to the rapid absorption after administration. Amphetamine is largely inactivated by the liver yielding metabolites which hydroxylate and deaminate the compounds, while some unchanged amphetamine is excreted in the urine (5). Methamphetamine is also excreted to some extent unchanged, but major metabolites of methamphetamine are amphetamine and an oxidized deaminated derivative (5). Thus, the presence of the parent compound in the urine indicates amphetamine or methamphetamine use.

Positive results should be confirmed by an appropriately sensitive and specific methodology using a different chemical principle. If performed under conditions that provide sufficient sensitivity, HPLC, GC and GC/MS may be adequate for some drugs of abuse. GC/MS is generally accepted as a rigorous confirmation technique for all drugs, since it provides the best level of confidence in the results.

PRINCIPLES OF PROCEDURE
The Methamphetamine test kit is a onestep membrane immunochromatographic test (6) based on antigen/antibody complexation and is used for the analysis of methamphetamine and its metabolite present in the test sample. The assay is based on the competition between the drug or drug metabolite in the sample and a drug conjugate immobilized on a porous membrane support for limited antibody sites on the colored microspheres. Test urine is delivered into a sample site located at one end of the porous membrane. When the drug is present in the urine test sample, the drug or drug metabolite competes for the limited antibody sites on the microspheres. When an adequate amount of drug is present, attachment of the colored microspheres to the probe site on the membrane occurs. Therefore, a positive urine sample will inhibit the formation of a precipitin line at the probe area. A reference or control line with a secondary antibody reaction is added to the membrane strip to indicate that the test is viable. This control line should always be present before making any test interpretation. Normally, a negative urine sample will produce two colored lines (the formation of a visible precipitin at the test zone in addition to the control line), and a positive urine sample will show only one line (the control line).

REAGENTS
Each Marijuana test is individually sealed in a foil pouch.

Each strip contains a membrane strip with a defined amount of colloidal gold conjugated with monoclonal (mouse) or polyclonal antibodies (sheep) in buffer solution. A secondary polyclonal antibody (goat anti-mouse lgG) is used to form the reference line. Also, an antigenic drug conjugate an animal serum are adsorbed onto the membrane. The entire membrane is then dried before assembly and is used in the dry form. All necessary reagents for performing the test are pre-formulated and included in each individual test strip. No additional reagents are required. One needs only to add the test sample.

MATERIALS PROVIDED
Each Methamphetamine test strip is individually sealed in a foil pouch.
Each pouch contains:
1.One Methamphetamine strip
2.Dessicant

Material required but not supplied:
Timer, sample container and disposable gloves. No other equipment or reagents are needed.

STORAGE
The components of this kit are stable until the marked expiration date when stored in the sealed pouch at 2 o-30 oC (36 o-86 oF).

• The test strip must be brought to room temperature prior to use.
• Do not allow test strips to be subjected to below freezing or above 30 oC temperatures.
• Urine samples if not used immediately after collection, should be stored refrigerated (2 o-8 oC).
• For storage longer than 3 days, the sample should be frozen ( < -20 oC).

WARNINGS AND PRECAUTIONS

1.Avoid cross-contamination of urine samples by using a new urine specimen cup and dropper for each urine sample.
2.If a test strip foil bag is torn or perforated, the test strip should not be used.
3.Do not use the assay beyond the expiration date as indicated on the pouch.
4.Open sealed pouch containing the test strip immediately prior to use. Prolonged exposure to ambient humidity will cause product deterioration.
5.Urine samples should not exhibit a strong positive test for protein since either heavy proteinuria or hematuria may adversely affect the performance of the test.
WARINING: Urine specimen and all materials coming in contact with them should be handled and disposed of as if infections and transmitting infection. Avoid contact with skin.

SPECIMEN COLLECTION AND HANDLING
Each Methamphetamine test kit is formulated for use with urine specimens. Fresh urine specimens should be used, and the urine does not require any special pretreatment. Urine samples should be collected such that testing may be performed as soon as possible, preferable during the same day as specimen collection. The sample taking out from a refrigerated storage must be allowed to warm up to room temperature before testing. Allow at least 30 minutes when the sample was first taken out from a refrigerated storage at 2 o-8 oC; give longer time if the sample needs to be thawed out first from frozen storage.

ASSAY PROCEDURE (CASSETTE)

1. SPECIMEN COLLECTION AND HANDLING

The Methamphetamine test kit is formulated for use with urine specimens. Fresh urine specimens should be used, and the urine does not require any special pretreatment.
Urine samples should be collected such that testing may be performed as soon as possible, preferable during the same day as specimen collection .The sample should be free form gross debris. Samples taking out from a refrigerated storage must be allowed to warm up to room temperature before testing.
Allow at least 30 minutes from when the sample was first taken out from a refrigerated storage at 2 o-8 oC, give longer time if the sample needs to be thawed out first from frozen storage

2.TEST PROCEDURE   1)Remove the test device from its foil wrapper by tearing along the slice. 2)Using the sample dispenser, withdraw the urine sample from the specimen cup and slowly dispense 3 drops (approximately 120uL ) into the circular sample well, being careful not to overfill the absorbent pad. ( see A ) 3)Read results within five minutes. DO NOT INTERPRET RESULT AFTER 10 MINUTES.

3. RESULT READING

1)Positive: Only one color band appearing in the "c" region (see B)

2)Negative: One band appearing in the "c" region, the other in the "T" region. (See C)

3)Invalid: No color bands appearing in the window at all, the test result is invalid. (See D)  

ASSAY PROCEDURE (DIPSTICK) 1.SPECIMEN COLLECTION AND HANDLING       (PLEASE REFER TO THE SAME SECTION OF CASSETTE) 2.TEST PROCEDURE

1) Remove the strip from the foil wrapper or the desiccated container (bring the container to the room temperature before opening to avoid condensation of moisture in container). Label the strip with patient or control identifications.   2) Immerse the strip into the urine with the arrow end pointing toward the urine. Do not cover the urine over the MAX (maximum) line. You may leave the strip in the urine or you may take the strip out after a minimum of 15 seconds in the urine and lay the strip flatly on a non-absorptive clean surface. (see E)

3)Read the result at 5 minutes.

DO NOT INTERPRET RESULT AFTER 10 MINUTES.

3.RESULT READING

1)Positive: One pink-purple colored band appears in the CONTROL area.

2)Negative: In addition the control band a clearly distinguishable pink-purple colored band also appears in the TEST area.

3) Inconclusive: If there is no distinct pink-purple colored band visible both in the TEST area and CONTROL area the test is inconclusive. It is recommended that in this case test be repeated or a fresh specimen be obtained and tested 48 hour later.  

LIMITATIONS OF PROCEDURE
This assay is designed for use with human urine only. A positive result from the methamphetamine test only indicates the presence of methamphetamine, or its metabolite and does not indicate or measure intoxication.
There is the possibility that other substances and/or factors not listed (e.g. technical or procedural errors) may interfere with the test and cause false results. See SPECIFICITY for lists of substances that will produce positive results and substances that do not interfere with test performance.

QUALITH CONTROL
Quality control specimens are available from commercial sources such as Bio-Rad Lyphochek urine toxicology controls which can be used for the periodic assessment of positive results obtained with the Methamphetamine test kit.

PERFORMANCE CHARACTERISTICS
1. SENSITIVITY
In contrast to GC/MS which detects a specific drug of drug metabolite, the Methamphetamine test kit immunoassay procedures can detect the parent methamphetamine molecule and methamphetamine-like substances. The Methamphetamine test kit has been shown to detect an average of 1000 ng/ml for methamphetamine in urine.

2. ACCURACY
The accuracy of the Methamphetamine test kit was evaluated in comparison to a commercially available immunoassay. One hundred twenty (120) urine samples were collected from presumed non-user volunteers and approximately seventy (70) positive samples from clinical laboratory have been tested by both methods. The results yielded 100% agreement on negative samples and 98% on positives.

3. SPECIFICITY
The following compounds will produce either positive (Table I) or negative (Table II) readings with the Methamphetamine test.

REFERENCES
1.Urine Testing for Drugs of Abuse, National Institute on Drug Abuse (NIDA) Research Monograph 73,1986
2.Mandatory Guidelines for Federal Workplace Drug Testing Programs, Fed. Register (1988), 53 (69).
3.MacBay, A.J.,Clin, Chem. (1987), 33,33B-40B.
4.Peroutka, S.N. Engl. J. Med. 317: 1542 (1987)
5.Baselt, R.C. and Cravey R.H. in Disposition of Toxic Drugs and Chemicals in Man, 3rd ed.
Biomedical Publ., Davis, CA., 1989, p. 249,622.
6.Sun, M. and Pfeiffer, R.R., “Analytical Test Device for Competition Assay for Drugs of Non-Protein  Antigens Using Immunochromatographic  Techniques” U.S. Patent # 5,238,652