Testing information

TEST PRINCIPLE
Most drug testing devices are based on the principle of competitive immunochemical reaction between a chemically labeled drug (drug-protein conjugate) and the drug or drug metabolites which may be present in the urine sample for the limited antibody binding sites. The test contains a nitrocellulose membrane strip precoated with drug-protein conjugate (or antibody) in the test region and a pad containing colored antibody (or drug-protein)-colloidal gold conjugate.

During the test, the urine sample is allowed to migrate upward and dehydrate the antibody (or drug protein)- colloidal gold conjugate. The mixture then migrates along the membrane chromatographically by the capillary action to the immobilized drug-protein (or antibody) band on the test region.

When drug is absent in the urine, the colored antibody (or drug-protein)-colloidal gold conjugate and immobilized drug-protein (or antibody) bind specifically to form a visible line in the test region as the antibody complexes with the drug-protein.

When drug is present in the urine, it will compete with drug-protein for the limited antibody sites. The line on the test region will become less intense with increasing drug concentration.

When a sufficient concentration of drug is present in the urine, it will fill the limited antibody binding sites. This will prevent attachment of the colored antibody (or drug-protein) – colloidal gold conjugate to the drug-protein (or antibody) on the test region.

Therefore, the presence of the line on the test region indicates a negative result for the drug and the absence of the test line on the test region indicates a positive result for the drug. A visible line generated by a different antigen/antibody reaction is also present at the control region of the test strip. This line should always appear, regardless of the presence of drugs or metabolites in the urine sample. This means that a negative urine sample will produce both test line and control line, and a positive urine sample will generate only control line.

The presence of control line serves as a built-in control, which demonstrates that the test is performed properly.

Some Drugs of Abuse devices use a built in specimen validity test which is based on the color response of chemical indicators for the presence of adulterants.

Creatinine (Cr), nitrite (Ni), pH, bleach/oxidant (Bl), and specific gravity (S.G.) are tested to determine the integrity of urine samples.

  • Cr: Creatinine reacts with a creatinine indicator in an alkaline medium to forms a purplish-brown color complex. The color intensity is directly proportional to the concentration of creatinine. Urine samples with creatinine concentrations of less than 20 mg/ml are dilute, which may be indicative of adulteration.

  • Ni: Nitrite reacts with the reagent’s aromatic amine to form a diazonium salt which couples with an indicator to yield a pink-red/purple color complex. Urine samples containing nitrite at levels greater than 15 mg/dl are considered adulterated.

  • pH: The pH determination of urine sample is based on color change of indicator in different acidic or basic medium. The normal urine pH ranges from 4 to 9. Urine pH below 4 or above 9 indicates adulteration of the specimen with acids or alkalines.

  • Bl: Bleach or other oxidizing agents react with an oxidant indicator to form a color complex. Observation of a blue-green, brown, or orange color indicates adulteration with bleach or other oxidizing agents.

  • S.G.: The S.G. test is based on the pKa change of certain pretreated polyelectrolytes in relation to the ionic concentration. In the presence of an indicator, the colors changes from dark blue to blue-green in urine of low ionic concentration to green and yellow green in urine of higher ionic concentration. Urine specific gravity below 1.005 or above 1.025 is considered abnormal, which may indicate adulteration.

WARNINGS AND PRECAUTIONS

  • For professional in vitro diagnostic use only
  • Urine specimens may be potentially infectious. Proper handling and disposal methods should be established.
  • Avoid cross-contamination of urine samples by using a new specimen collection container for each urine sample.
  • Test device should remain sealed until ready for use.
  • Do not use the test kit after the expiration date.
  • A positive test result does not always mean an individual has taken the drug illegally as the drug can be administered legally.
  • Do not store and or expose reagent kits at temperature greater than 30°C.
  • Do not freeze.

SPECIMEN COLLECTION AND HANDLING
Fresh urine does not require any special handling or pretreatment. A fresh urine sample should be collected in the container provided. Alternately, a clean, dry plastic or glass container may be used for specimen collection. If the specimen will not be tested after the specimen collection, the specimen may be refrigerated at 2-8°C up to 2 days or frozen at -20°C for longer period of time. Specimens that have been refrigerated must be equilibrated to room temperature prior to testing. Specimens previously frozen must be thawed and mixed thoroughly prior to testing.
Urine specimens and all materials coming in contact with them should be handled and disposed as if capable of transmitting infection. Avoid contact with skin by wearing gloves and proper laboratory attire.
ASSAY PROCEDURE

Preparation: If specimen, control, or test devices have been stored at refrigerated temperatures, allow them to warm to room temperature before testing.

Do not open test device pouch until ready to perform the test.

Drug Chart

Test Calibrator Cut-off (ng/ml)
AMP d-Amphetamine 1000
BAR Secobarbital 300
BUP Buprenorphine 10
BZO Oxazepam 300
COC150 Benzoylecgonine 150
COC300 Benzoylecgonine 300
MDMA 3,4-methylenedioxymethamphetamine 500
mAMP / MET500 d-Methamphetamine 500
mAMP / MET1000 d-Methamphetamine 1000
MTD Methadone 300
OPI300 Morphine 300
OPI2000 Morphine 2000
OXY Oxycodone 100
PCP Phencyclidine 25
PPX Propoxyphene 300
TCA Nortriptyline 1000
THC 11-nor-?9-THC-9-COOH 50

The configurations of a drug test device may consist of any combination of the drugs listed above with or without specimen validity test. The specimen validity test provides information regarding the integrity of urine sample in the drugs of abuse test through the semi-quantitative determination of creatinine, nitrite, pH, bleach/oxidant, and specific gravity in human urine.

The Drugs of Abuse test is used to obtain a visual, qualitative result and is intended for professional use only. This assay provides only a preliminary result. Clinical consideration and professional judgment must be applied to any drug of abuse test result, particularly in evaluating a preliminary positive result. In order to obtain a confirmed analytical result, a more specific alternate chemical method is needed. Gas Chromatography/Mass Spectroscopy (GC/MS) is the preferred confirmation method.
INTERPRETATION OF RESULTS

Specimen Validity Tests: Specimen validity test results are obtained by directly comparing the color of each test pad with the color block of Adulteration Color Comparison Chart. Potentially adulterated urine sample will produce abnormal color response. Unadulterated urine sample will produce normal color response.

Adulteration chart

DRUGS OF ABUSE TESTS
Negative (-): Colored lines appear in both Control Region (C) and Test Region (1, 2, or T). The line in the control region is the control line, which is used to indicate proper performance of the device. The line in the test region is the drug probe line. The test line may have varying intensity either weaker or stronger in color than that of the control line. A negative result for a drug indicates that the concentration of that drug in urine is below the cutoff level.
Positive (+): Colored line appears in the control region. No line appears in the test region. The complete absence of a test line indicates a positive result for that drug. A preliminary positive result for a drug indicates that the concentration of that drug in urine is at or above the cutoff level.
Invalid: No colored line appears in the control region. If the control line does not form, the test result is inconclusive and should be repeated.

QUALITY CONTROL

An internal procedural control is included in the test device. A line must form in the Control band region regardless of the presence or absence of drugs or metabolites.

The presence of the line in the Control region indicates that the proper sample volume has been used and that the reagents are migrating properly.

If the line in the Control region does not form, the test is considered invalid. To ensure proper kit performance, it is recommended that the test devices be tested once a week with external controls.

External controls are available from commercial sources. It is important to make sure that the control values are within established limits. If the values of external control do not fall within established limits, the test results are invalid.

Additional controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations.

LIMITATIONS OF PROCEDURE

  • The assay is designed for use with human urine only.

  • A positive result with any of the tests indicates only the presence of a drug/metabolite and does not indicate or measure intoxication.

  • There is a possibility that technical or procedural error as well other substances as factors not listed may interfere with the test and cause false results.

  • If adulteration is suspected, the test should be repeated with new sample.

EXPLANATION OF DRUGS

Open Me

Amphetamine

Methamphetamine, amphetamine, and metabolites are potent central nervous system stimulants. Acute higher doses induce euphoria, alertness, and sense of increased energy and power. More acute responses produce anxiety, paranoia, psychotic behavior, and cardiac dysrhythmias. Methamphetamine is excreted in urine as amphetamine and oxidized as deaminated and hydroxylated derivatives. However, methamphetamine is also excreted to some extent unchanged. Thus the presence of the parent compound in the urine indicates methamphetamine use.

Barbiturates

Barbiturates are classified as central nervous system depressants. These compounds produce a state of intoxication that is similar to alcohol intoxication. Symptoms include slurred speech, loss of motor coordination and impaired judgment. Depending on the dose, frequency, and duration of use, one can rapidly develop tolerance, physical dependence and psychological dependence on barbiturates. Barbiturates are taken orally, or by intravenous and intramuscular injections. They are excreted in urine as parent compound as well as metabolites.

Benzodiazepine

Benzodiazepines are central nervous system (CNS) depressants commonly prescribed for the short-term treatment of anxiety and insomnia. In general, benzodiazepines act as hypnotics in high doses, as anxiolytics in moderate doses and as sedatives in low doses. The use of benzodiazepines can result in drowsiness and confusion. Psychological and physical dependence on benzodiazepines can develop if high doses of the drug are given over a prolonged period. Benzodiazepines are taken orally or by intramuscular or intravenous injection, and are extensively oxidized in the liver to metabolites. Parent compounds, as well as metabolites are excreted in the urine.

Buprenorphine

Buprenorphineis a synthetic thebaine derivative that has both analgesic and opioid antagonist properties. As an analgesic, it is about 25 to 40 times more potent than morphine. Symptoms of over dosage include confusion, dizziness, pinpoint pupils, hallucinations, hypotension, respiratory difficulty, seizures and coma. Burpenorphine is metabolized in man primarily by N-dealkylation and conjugation to form norbuprenorphine, which is pharmacologically active, and conjugates of buprenorphine and norbuprenorphine. Within 144 hours of a single intramuscular dose of drug, 95% is eliminated, with 68% in the feces and 27% in the urine. Buprenorphine and its metabolites in urine may be detected as a result of buprenorphine, norbuprenorphine, Buprenorphine-3-beta-D-glucuronide, and Norbuprenorphine-3-beta-D-glucuronide.

Cocaine

Cocaine is a potent central nervous system stimulant and a local anesthetic found in the leaves of the coca plant. The psychological effects induced by using cocaine are euphoria, confidence and sense of increased energy. These psychological effects are accompanied by increased heart rate, dilation of the pupils, fever, tremors and sweating. Cocaine is excreted in the urine primarily as benzoylecgonine in a short period of time. Benzoylecgonine has a biological half-life of 5 to 8 hours, which is much longer than that of cocaine (0.5 to 1.5 hour), and can be generally detected for 24 to 60 hours after cocaine use or exposure.

3,4-methylenedioxymethamphetamine

(MDMA) is classified as both a stimulant and a hallucinogen. Like methamphetamine, adverse effects of MDMA/Ecstasy use include jaw clenching, teeth grinding, dilated pupils, perspiring, anxiety, blurred vision, vomiting, and increased blood pressure and heart rate. Overdose of 3,4- methylenedioxymethamphetamine may cause heart failure or extreme heart stroke. 3,4-methylenedioxymethamphetamine is taken orally in tablets or capsules and excreted in urine as parent compound as well as metabolites.

Methadone

Methadone is a synthetic analgesic drug originally used for the treatment of narcotic addiction. The psychological effects induced by using methadone are analgesia, sedation, and respiratory depression. Overdose of methadone may cause coma or even death. Methadone is taken orally or intravenously and is metabolized in the liver and has a biological half-life of 15-60 hours.

Opiates

Opiates, such as heroin, morphine, and codeine, are central nervous system (CNS) depressants. The use of opiates at high doses produces euphoria and release from anxiety. Physical dependence is apparent in users and leads to depressed coordination, disrupted decision making, decreased respiration, hypothermia and coma. Heroin is quickly metabolized to morphine, morphine glucuronide and 6-acetylmorphine. Thus, the presence of morphine (or the metabolite, morphine glucuronide) in the urine indicates heroin, morphine, and/or codeine use.

Oxycodone

Oxycodone is a semi-synthetic opioid with a structural similarity to codeine. It produces potent euphoria, analgesic and sedative effects, and has a dependence liability similar to morphine. Oxycodone is most often administered orally and is metabolized by demethylation to noroxycodone and oxymorphone followed by glucuronidation and excreted in urine. The window of detection for oxycodone in urine is expected to be similar to that of other opioids such as morphine.

Phencyclidine

Phencyclidine, commonly known as “angel dust” and “crystal cyclone”, is an arylcyclohexylamine that is originally used as an anesthetic agent and a veterinary tranquilizer. The drug is abused by oral or nasal ingestion, smoking, or intravenous injection. It produces hallucinations, lethargy, disorientation, loss of coordination, trance-like ecstatic states, a sense of euphoria and visual distortions. It is well absorbed following all routes of administration. Unchanged PCP is excreted in urine in moderate amounts (10% of the dose).

Propoxyphene

Propoxyphene is a mildly effective narcotic analgesic that has been in clinical use. It is less potent than codeine, and bears a close structural relationship to methadone. Propoxyphene is available in oral formulations either as the hydrochloride (32 or 65mg) or as the napsylate salt (50 or 100 mg), and is often found in combination with aspirin or acetaminophen. Overdosage with propoxyphene can result in stupor, coma, convulsions, respiratory depression, cardiac arrhythmias, hypotension, pulmonary edema and circulatory collapse. Propoxyphene is metabolized primarily via Ndemethylation to norpropoxyphene. The amounts of metabolites excreted in the 20 hour urine following a 130 mg single oral dose of propoxyphene hydrochloride were: 1.1% propoxyphene, 13.2% norpropoxyphene and 0.7% dinorpropoxyphene.

Tetrahydrocannabinol

(THC) is generally accepted to be the principle active component in marijuana. When ingested or smoked, it produces euphoric effects. Abusers exhibit central nervous system effects, altered mood and sensory perceptions, loss of coordination, impaired short term memory, anxiety, paranoia, depression, confusion, hallucinations and increased heart rate. When marijuana is ingested, the drug is metabolized by the liver, the primary metabolite of marijuana excreted in the urine is 11-nor- ? -9-tetrahydrocannabinol-9-carboxylic acid. Therefore, the presence of detected cannabinoids including the primary carboxyl metabolite in the urine indicates marijuana/cannabis use.

Tricyclic antidepressants

(TCAs) have been prescribed for depression and compulsive disorders. Because of the possibility of causing serious cardiac complications, TCAs can be lethal if misused at high doses. TCAs are taken orally or sometimes by injection. TCAs are metabolized in the liver. Both TCAs and their metabolites are excreted in urine mostly in the form of metabolites for up to ten days. The length of time following drug use of which a positive result may occur is dependent upon several factors, including the frequency and amount of drug, metabolic rate, excretion rate, drug half-life, and the drug user’s age, weight, activity and diet.

Adulterants

Adulteration of urine samples may cause erroneous results in drugs of abuse test by either interfering with the drug screening test and/or destroying the drugs in the urine. Dilution of urine with water is probably the simplest urine adulteration method. Bleach, vinegar, Visine, sodium bicarbonate, sodium nitrite, Drano, soft drinks and hydrogen peroxide are the examples of compounds used to adulterate the urine sample. It is important to insure the integrity of urine samples in drugs of abuse test.