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Drug Testing Methods and Clinical Interpretations of Test Results

DRUG TESTING METHODS AND CLINICAL INTERPRETATIONS OF TEST RESULTS

As interest increases in employment-related drug testing, the technologies and the interpretive skills of analysts continue to evolve. Although recent literature indicates that significant refinements and modifications to drug testing technology have been made, the complexity of drug effects is so great that many problems exist in interpretation of the test results. The most frequent problems that confront the toxicology laboratory relate to developing technology that can determine how much and when the drug was taken, how long after use the tests are capable of showing positive results, the causes and rates of false positive and false negatives, and how tests can be "beaten" by employees. These problems will be discussed and the various laboratory procedures that are used to combat these problems will be examined.

DRUG PROPERTIES: ABSORPTION, DISTRIBUTION, AND ELIMINATION PHASES

Detection of a drug depends largely on its absorption, distribution, and elimination properties. There are various routes of drug administration; oral (e.g., drinking Alcohol or swallowing pills), intravenous (e.g., Heroin injected into a vein) and inhalation (e.g., smoking Marijuana; snorting Co-Caine; sniffing Glue). Drugs taken orally are usually the slowest to be absorbed (i.e. the speed at which the drug reaches the brain and other body organs) whereas intravenous and inhalation routes result in the fastest absorption. Once the absorbed drug enters the blood stream it is rapidly distributed to the various tissues in the body. The amount of drug stored depends on the nature of the drug, the quantity, duration of ingestion, the tissue holding the drug and the frequency of use.

Some drugs are fat-soluble and are deposited in fat tissues. For example, δ9-Tetrahydrocannabinol (THC), the active ingredient in marijuana, is highly fat-soluble, resulting in rapid reductions in blood levels as the drug is being distributed to the various tissues. Blood levels of δ9-THC peak and start to decline in half the time it takes to smoke a marijuana "joint." Concentrations are known to fall by almost 90 per cent in the first hour. Depending on the amount of drug stored in the fat tissues, detection may be possible in the urine for many days after last use. There are cases where marijuana metabolites have been detected for as long as sixty days after last use, since small amounts from fat go back into blood and appear in the urine. Ethanol or ethyl alcohol (the beverage alcohol) is not fat-soluble but is distributed in the total body water. Since blood is mostly made up of water, the presence of alcohol is easier to detect than fat-soluble drugs like δ9-THC.

The "absorption" and "distribution" phases are followed by an "elimination" phase. The liver is the major detoxification centre in the body where the drugs are metabolized as blood circulates through this organ. The metabolites are then excreted into the urine through the kidneys. At the same time, drugs deposited in fat tissues are also slowly released into the blood stream and metabolized.

Drugs vary by their elimination half-life. An elimination half-life is the amount of time needed for the drug level to fall by 50 percent. Every half-life the drug level falls by 50 percent. Table 1 shows the impact of the half-life on the amount of drug left in the body. At the end of 7 half-lives over 99 percent of the drug will be eliminated from the body. (See Table 2 for drug half-lives). The half-life of a drug is heavily influenced by a variety of factors including the individual's age, sex, physical condition as well as clinical status. A compromised liver and concurrent presence of another disease or drug have the potential of enhancing the toxic effects of the drug by slowing down the elimination process. Under different clinical conditions, however, this process may be speeded up. Therefore, great variation can be found in the half-lives of the same drug.

Approximately six half-lives are required to eliminate 99 per cent of any drug. Because cocaine's half-life is relatively short, averaging one hour, only six hours are needed for elimination of 99 per cent of the drug. On the other hand, cocaine's metabolites have a longer half-life and can be detected for a considerably longer period of time through urine drug assays. Compared to cocaine, Phenobarbital has a much longer half-life of 80-120 hours, so that at least 480 hours (or 20 days) are required to eliminate 99 per cent of the drug. Since there is much variation in the half-life of different drugs and the absolute amount of drug present can be very small, it is crucial that the appropriate body fluid for analysis is selected for testing.

Ethanol is absorbed from the stomach by simple diffusion. Gastric absorption is fastest when strong drinks, distilled spirits containing 40 to 50 percent ethanol by volume are consumed. Dilute beverages, such as beer (4-5% ethanol) or wine (11-12% ethanol) are absorbed slowly. Alcohol is absorbed very rapidly from the small intestines. The essential action of food is to delay gastric emptying and thus slow the absorption process. Typically, studies have shown that peak BAC is reached between 30 minutes and 90 minutes of consumption; earlier on an empty stomach and later on a full stomach. Once absorbed, ethanol rapidly diffuses throughout the aqueous compartments of the body, going wherever water goes.

Amount of drug left in the body Amount of drug eliminated
Start 100% 100%
End of 1st half-life 50.0% 50.0%
End of 2nd half-life 25.0% 75.0%
End of 3rd half-life 12.5% 87.5%
End of 4th half-life 6.25% 93.75%
End of 5th half-life 3.125% 96.87%
End of 6th half-life 1.56% 98.44%
End of 7th half-life 0.78% 99.22%
Drug Half-life(t2) Detection period
Methamphetamine 12-34 hours 2-3 days
Amphetamine (metabolite of methamphetamine) 7-34 hours
Heroin 60-90 minutes In minutes
Morphine (metabolite of heroin) 1.3-6.7 hours Opiates positive for 2-4 days (EIA)
6-Mono-acetyl-morphine (MAM) 30 minutes Few hours
Phencyclidine (PCP) 7-16 hours 2-3 days
Cocaine 0.5-1.5 hours Few hours
Benzoylecgonine (metabolite of cocaine) 5-7 hours 3-5 days
δ9-Tetrahydrocannabinol 14-38 hours 90% fall in 1 hour (blood)
δ9-Tetrahydrocannabinoic acid (marijuana metabolite in urine) Depending on use, few days to many weeks
Benzodiazepines Few hours to days days to weeks, depending on half life
Diazepam 15-40 hours 2 weeks
Flunitrazepam (rohypnol) 9-25 hours 0.2% excreted unchanged!
Methadone 15-40 hours In chronic patients ~22-24 hours
Barbiturate (phenobarbital) 35-120 hours 1-2 weeks after last use
Alcohol (ethanol) Blood levels fall by an average of 4-5 mmol/L/hour (15-18 mg/100 mL)/hour 1.5 > 12 hours depending on the peak blood level. Urine typically positive for an additional 1-2 hour.
Gamma-hydroxybutyrate (GHB) 0.3-1.0 hour Less than 12 hours
*The detection period is very much dose-dependent. The larger the dose, the longer the period the drug/metabolite can be detected in the urine

Absorption, distribution into different tissues and elimination are dynamic processes and take place simultaneously. The rate of removal of ethanol from the body is the sum of the rates of excretion in urine, breath and sweat, and the rate of the metabolism in the liver and other tissues. In humans, alcohol metabolism follows a "zero" order kinetics, i.e., it is largely independent of alcohol concentration in the blood and its levels decline almost linearly over time. The implication of this is that BAC falls at a constant rate over time. In social drinkers it is from 0.015 to 0.018 percent (15 mg/ 100mL to 18 mg/100mL) per hour and in heavy drinkers it is typically between 0.018 and 0.025 percent (18mg/100mL to 25mg/100mL) per hour. In the alcoholic patient, the elimination rate is generally higher. In forensic calculations, a rate of 0.015 percent (15mg/100mL) per hour is usually used. In our studies we have found 0.018 percent (18mg/100mL) per hour to be the average rate of metabolism. The larger the dose of alcohol given, the longer the duration of the measurable blood alcohol concentration.

SELECTION OF DRUGS TO BE TESTED

A number of different criteria can be applied to the drug(s) or category of drugs that should be tested or monitored. Drug availability, clinical effects and robustness of the analytical method(s) used for analysis are probably the most important.

Availability.

Prescription patterns of psycho-active and other drugs vary from place to place and country to country. Abuse of Benzodiazepine nitrazepam is common in Europe but almost unknown in North America, since it is not sold here. The psychoactive chemical Cathinon (cathine), the active ingredient in the leaves of the Khat plant, is chewed in northeast Africa, is not a problem in North America. Codeine, an Opioid available in Canada as Over-The-Counter preparations, is sold only by prescription in the United States.

A wide availability of "legal" Stimulants poses an interesting problem since they are a common finding in accident victims. A study carried out by the U.S. National Transportation Safety Board from October 1987 to September 1988 showed that over-the-counter stimulantssuch as ephedrine, pseudoephedrine and phenylpropanolaminewere commonly found among drivers killed in heavy truck accidents. Amongst the eight States that participated in this safety study almost all Am-Phetamine use was in the California region. Similar findings are also reported from emergency rooms over the past five years as well as from admissions in a trauma unit due to motor-vehicle accidents. All this suggest that drug use varies not only from place to place but also region to region within a given country.

Thus, the selection of a drug to be tested and monitored, appropriate for one country and place, may not necessarily be appropriate for another country.

Clinical Effects.

Drugs that manifest abuse potential and impair behavior such that job performance can be affected are prime candidates for testing or monitoring in the workplace. Alcohol and cocaine are examples of this.

Analytical Methods.

A false positive finding can have a serious impact on the livelihood of the person being tested. Therefore, special attention needs to be paid to the testing methods. Ideally the analytical method should be specific for the drug being tested (i.e., no false positive), easy and inexpensive to perform. Confirmation methods should also be readily available. Availability of technical and scientific expertise to perform the tests is also essential.

Interpretation of the analytical results also needs to be carefully considered as even a normal diet can result in a positive drug identification. For example, poppy seed ingestion can result in a true positive analytical result (Opiates, like heroin, are derived from the poppy plant Papaver Soniferum) but it is a false positive for drug use. Some ethnic diets may also lead to these confounding problems, as when food containing poppy seeds is eaten during Ramadan.

What should be analyzed? Ideally the analysis should look for the parent drug rather than its metabolite, although this may not always be possible as some drugs are very rapidly metabolized (e.g., heroin metabolism to Morphine). Sensitivity of the analytical procedure should be dictated by the drugs' psychoactive pharmacological properties. If the drug is shown to be devoid of abuse potential then its detection beyond the time of pharmacological activity, although important in the clinical management of the patient, does not necessarily serve a useful purpose for a workplace drug screening programme.

The guidelines developed by the National Institute on Drug Abuse in April 1988, address five "illegal" drugs: marijuana, Phencyclidene, Amphetamine, cocaine and heroin. Rapid screening methods that allowed for "mass screening" were available at that time, as were the confirmation methods for these five drugs. Mood altering substances such as benzodiazepines, Barbiturates and some stimulants such as antihistamines are at present excluded from these regulations in the United States. This is probably due to the wide availability of these drugs as medications within the general population and the technological requirements for screening and monitoring of these drugs.

TYPES OF TESTING: BLOOD, URINE, AND HAIR SPECIMENS

Blood and urine are the most commonly used biological fluids in the analysis for drugs other than alcohol. Blood, obtained by an invasive procedure, is available only in small quantities and drug concentration levels in blood are typically low. Urine is the preferred sample of choice as it is available in larger volumes, contains the metabolite and requires less invasive procedures in its collection. Both sampling procedures, however, are limited in their ability as they only determine the absolute amount of drug present in the fluid being examined. This quantity is dependent upon the amount of the drug used, when it was last used, as well as the half-life of the drug.

Recently, hair samples have been used to detect drug use. A number of technical problems must be overcome before hair can be used as a definitive proof of drug use. Hair treatment and environmental absorption are but two of the many concerns and problems that have been cited. An advisory committee of the Society of Forensic Toxicology has recently reported that "The committee concluded that, because of these deficiencies, results of Hair Analysis alone do not constitute sufficient evidence of drug use for application in the workplace."

Various body fluids such as sweat, saliva, blood, urine and breath, have been used for alcohol analysis. Breath, though not a body fluid, is commonly used by law enforcement authorities. Although a number of variables can effect breath/blood ratio, a 2100:1 alveolar breath/blood conversion ratio has been used and accepted for use with Breathalyzers. Breath-testing equipment calibrated with a blood:breath conversion factor of 2100 consistently underestimate actual Blood Alcohol Concentrations (BAC). Accuracy of breath analysis results is subject to various instruments and biological factors. Potential errors in breath analysis can also be caused by the presence of residual alcohol in the mouth. Immediately after drinking there is enough alcohol vapour in the mouth to give artificially high concentrations on breath analysis. Generally this effect disappears twenty minutes after drinking but high values for as long as forty-five minutes have been reported.

As of the early 1990s, all existing technologies are limited in terms of determining how much or when the drug was consumed.

Blood and saliva concentrations reflect the current blood alcohol concentration, but generally a blood sample is used in hospitals to access the patient in the casualty wards. In programmes requiring monitoring of alcohol use, urine is probably the sample of choice. Urine alcohol concentration, which represents the average blood alcohol concentration between voiding, has the potential of being "positive" while the blood may be "negative."

MEASURING IMPAIRMENT

Except for alcohol, the degree to which a person is influenced or impaired by a drug at the time of the test cannot be determined from test results alone. Correlations between positive blood levels and degree of impairment are usually stronger than correlations between urine levels and degree of impairment; however, neither blood nor urine tests are sufficiently accurate to indicate impairment even at high levels of concentration. Human studies using marijuana and cocaine have shown that a "perceived high" is reached after the drug concentration has peaked in the blood. Generally, blood can only show positive results for a short time after drug consumption, whereas urine can be positive for a few days to weeks after last use. For example, metabolites of9-THC (active ingredient in marijuana) that are lipid-soluble can be detected in the urine from a few days to many weeks, depending on the drug-habit of the user. Excretion of the drug in urine and its concentrations are also affected by several factors, such as dilution and pH (acidity) of the urine. I have seen many cases where a strong, positive urine sample for Cannabinoids was found in the morning, a borderline positive in the afternoon, followed by a strong positive the next morning; I have also seen similar cases with respect to phenobarbital.

A positive urine test cannot reveal the form in which the drug was originally takenor when and how much was taken. For example, Crack-cocaine, impure cocaine powder or cocaine paste (which can be smoked, inhaled, injected or chewed) all give the same result in the urine test. The consumption of poppy seeds has been reported to give positive results for opiate use, because some seeds contain traces of opiates and some have been known to be contaminated with Opium derivatives. Similarly, consumption of herbal Coca tea has resulted in positive results for cocaine use. These diverse incidences illustrate the difficulties involved in measuring impairment using urine results.

The problem of interpreting urine-test results is one of the major bases of concern for restricting their use in the employment setting. Even the effectiveness of preemployment drug-screening tests, due to the difficulties in interpretation is being questioned. Based on a study of 2,229 pre-employment drug screening tests and follow-up, one group of researchers have come to the following conclusion "our findings raise the possibility that a preemployment drug screening may be decreasingly effective in predicting adverse outcomes associated with marijuana use after the first year of employment". They make a similar comment about cocaine.

There is no threshold for alcohol effects on performance or motor-vehicle-accident risk. Although the effects of alcohol on impairment and crash risk appear more dramatically above 80mg/100mL, a review of literature would suggest that impairment may be observed at levels as low as 15mg/100mL. It is not possible to specify a blood alcohol concentration level above which all drivers are dangerous and below which they are safe or at "normal" risk. An author of a major literature review on the behavioral effects of alcohol concluded that "that alcohol sensitivity can vary from time to time, person to person, and situation to situation, the setting of a "safe" BAC will always be arbitrary, being based on a low, but a non-zero, incidence of effects below that level" and "the most striking feature to emerge from any review of the effects of alcohol on behaviour is the marked lack of agreement between authors, amounting, in many instances, to direct contradiction. This is especially true for the effects of smaller dose."

"Legal" BAC levels differ in different countries. Some even have more than one legal limit over which the driver of a vehicle is considered as "impaired". Some European countries have 50mg/ 100ml others have 80mg/100ml as their legal limits. In the United States, the legal limits vary from 80mg/100mL to 100mg/100mL in different states, but employees who are regulated by the U. S. Department of Transportation have a BAC legal limit of 40mg/100mL. In Canada there are also two limits: 50mg/100mL and 80mg/100mL. BAC levels between 50mg/100mL and 80mg/100mL call for suspension of driving privileges but above 80mg/100mL are subject to criminal charges.

URINE TESTING METHODS

Urine is the most commonly used fluid for drug screening. The methods most commonly used in toxicology laboratories are: immunoassay, chromatographic and chromatography coupled with mass spectrometry. These methods vary considerably with respect to their sensitivity and reliability. Thin-layer chromatography is least expensive, gas chromatography coupled with mass spectrometry (GC/MS), which is considered as nearly perfect or "gold standard", is the most expensive. Table 2 summarizes the various methods.

Immunoassays (EIA, EMIT, FPIA, CEDIA and KIMS).

Immunoassay methods are used for preliminary screening (i.e., initial screening). Since these methods are based on an antibody-antigen reaction, small amounts of the drug or metabolite(s) can be detected. Antibodies specific to a particular drug are produced by injecting laboratory animals with the drug. These antibodies are then tagged with markers such as an enzyme (enzyme immunoassay, EIA), a radio isotope (radioimmunoassay, RIA) or a fluorescence (fluorescence polarization immunoassay, FPIA) label. Reagents containing these labelled antibodies can then be introduced into urine samples, and if the specific drug against which the antibody was made is present, a reaction will occur. RIA is the oldest immunoassay method used to detect drugs. The major drawback of this method is that it requires a separation step and generates radioactive waste. RIA also requires special equipment to measure radioactivity.

Typically, immunoassays are designed for a class of drugs. Thus, their specificity (the ability to detect the presence of a specific drug) is not very good, since substances that have similar chemical structures will "cross react" and give a false positive reaction. For example, the immunoassay method for cannabinoids was developed to detect the carboxylic acid metabolite of9-THC. Yet, there is a suggestion in the literature that some nonsteroidal anti-inflammatory drugs, such as ibuprofen (a nonprescription drug in the U. S. and Canada) and naproxyn give random or sporadic false positive results for cannabinoids. Cough-syrup codeine will also give a positive reaction for the morphine (a metabolic product of heroin use) immunoassay and many antihistamines that are available over-the-counter may yield positive reactions for amphetamines. While some reagent manufacturers claim to have overcome many of these cross-reactivity problems, confirmation by a nonimmunoassay method is very important.

Urine test kits, designed to detect drugs, have been available in North America for the past few years. More recently, single and multiple test immunoassay kits designed for home and on-site testing have also been introduced. These kits generally carry a cautionary disclaimer that positive test results must be confirmed by the reference GC/MS method. When used in the non-laboratory environment, they are prone to procedural inaccuracies, poor quality control, abuse and misinterpretations. Therefore, these kits should be used with great caution. The risk of labelling a person with a false positive is high without the accompanying confirmatory analysis. Table 3 summarizes the advantages and disadvantages of immunoassay testing.

Chromatographic methods.

Separation of a mixture is the main outcome of the chromatographic method. For illustrative purposes, if one were to put a drop of ink on a blotting paper and hold the tip of the paper in water, one would observe the water rise in the paper. After a period of time and under the right conditions, the single ink spot would separate into many different compounds (spots) of different colours (blue ink is a mixture of many dyes). This process, where a mixture of substances is separated in a stationary medium (filter paper), is called chromatography. The types of chromatographic processes used in the analysis of drugs include thin-layer, gas, and liquid chromatography as well as a combination of gas or liquid chromatography with mass spectrometry.

Of the several chromatographic methods, thin layer Chromatography (TLC) is the one most similar to the ink separation example mentioned above. This method requires extensive sample preparation and technical expertise on the part of the analyst, but it is inexpensive and very powerful if used properly. With the exception of Cannabis, which requires separate sample preparation, a large number of drugs (e.g., cocaine, amphetamine, codeine and morphine) can be screened at the same time. By combining different TLC systems, a high degree of specificity can be obtained, although the training of the analyst is crucial because of the subjectivity involved in interpreting the results. To identify positive TLC "spots," the technologist looks for the drugs and or its metabolite pattern, often by spraying with reagents that react to form different colors with different drugs. The trained technologist can comfortably identify more than forty different drugs.

1. Immunoassays
Enzyme immunoassay (EIA)
Enzyme-multiplied immunoassay technique (EMIT)
Fluorescence polarization immunoassay (FPIA)
Radio immunoassay (RIA)
Kinetic interaction of microparticles in solution (KIMS)
Cloned enzyme donor immunoassay (CEDIA)
Rapid slide tests (point-of-care testing)
2. Chromatographic Methods
Thin-layer chromatography (TLC)
Liquid chromatography (HPLC)
Gas chromatography (GC)
3. Chromatography/Mass Spectrometry
Gas chromatography/mass spectrometry (GC/MS)
Liquid chromatography/mass spectrometry (HPLC/MS)

Similar to TLC, gas chromatography (GC) requires extensive sample preparation. In GC, the sample to be analyzed is introduced via a syringe into a narrow bore (capillary) column which sits in an oven. The column, which typically contains a liquid adsorbed onto an inert surface, is flushed with a carrier gas such as helium or nitrogen. (GC is also sometimes referred to as gas-liquid chromatography (GLC). In a properly set up GC system, a mixture of substances introduced into the carrier gas is volatilized, and the individual components of the mixture migrate through the column at different speeds. Detection takes place at the end of the heated column and is generally a destructive process. Very often the substance to be analyzed is "derivatized" to make it volatile or change its chromatographic characteristics.

In contrast to GC, high pressure liquid chromatography (HPLC) a liquid under high pressure is used to flush the column rather than a gas. Typically, the column operates at room or slightly above room temperature. This method is generally used for substances that are difficult to volatilize (e.g., Steroids) or are heat labile (e.g., benzodiazepines).

Gas chromatography/mass spectrometry (GC/MS) is a combination of two sophisticated technologies. GC physically separates (chromatographs or purifies) the compound, and MS fragments it so that a fingerprint of the chemical (drug) can be obtained. Although sample preparation is extensive, when the methods are used together the combination is regarded as the "gold standard" by most authorities. This combination is sensitive i.e., can detect low levels, is specific, and can identify all types of drugs in any body fluid. Furthermore, assay sensitivity can be enhanced by treating the test substance with reagents. When coupled with MS, HPLC/MS is the method of choice for substances that are difficult to volatilize (e.g. steroids).

Given the higher costs associated with CG/MS, urine samples are usually tested in batches for broad classes of drugs by immunoassays and positive screens are later subjected to confirmation by this more expensive technique.

Table 4 gives a summary of the advantages and disadvantages of each method of chromatographic drug testing and Table 5 compares all the methods of testing. The initial minimal immunoassay and GC/MS (cut-off) levels for five drugs or classes of drugs as suggested by the U.S. National Institute of Drug Abuse, are listed in Table 6.

Procedures for alcohol testing.

Since the introduction of the micro method for alcohol analysis

Advantages
1. Screening tests can be done quickly because automation and batch processing are possible.
2. Technologists doing routine clinical chemistry testing can be easily trained.
3. Detection limits are low and can be tailored to meet the program screening requirements. For example, lower
detection thresholds can be raised to eliminate positives due to passive inhalation of marijuana smoke.
4. Immunoassays are relatively inexpensive, although the single-test kits can be very expensive when quality
assurance and quality control samples are included.
5. Immunoassays do not require a specialized laboratory. Most clinical laboratories have automated instruments
to do the procedures.
Disadvantages
1. Although the tests are useful for detecting classes of drugs, specificity for individual drugs is weak.
2. Since the antibody is generated from laboratory animals, there can be a lot-to-lot or batch-to-batch variation
in the antibody reagents.
3. Results must be confirmed by another nonimmunoassay method.
4. A radioactive isotope is used in RIA that requires compliance with special licensing procedures, use of gamma
counters to measure radioactivity, and disposal of the radioactive waste.
5. Only a single drug can be tested for at one time.
Advantages
All the chromatographic methods are specific and sensitive and can screen a large number of drugs at the same time.
TLC Negligible capital outlay is needed.
GC The procedure can be automated.
HPLC Of the chromatographic procedures, this has the easiest sample preparation requirements.
The procedure can be automated.
GC/MS This is the "gold standard" test.
Computerized identification of fingerprint patterns makes identification easy.
The procedure can be automated.
This is currently the preferred method for defense in the legal system.
Disadvantages
All chromatographic methods are labor-intensive and require highly trained staff. Although the chromatographic
methods are specific, confirmation is still desirable.
TLC Interpretation is subjective, hence, training and experience in interpretation capabilities of the
technologist are crucial.
HPLC or GC Equipment costs are high, ranging between $25,000 to $60,000, depending on the type of
detector and automation selected (1994 $)
GC/MS Equipment costs are the highest, ranging from $120,000 to $2000,000, depending on the
the degree of sophistication required (1994 $). Due to the complexity of the instrument, highly
trained operators and technologists are required.
Test Initial Test Confirmatory Test
THC metaboliteb 100 ng/mL 15 ng/mL
Cocaine metabolitesc 300 ng/mL 150 ng/mL
Opiate metabolitesd 2000 ng/mL
Morphine 300 ng/mL
Codeine 300 ng/mL
Phencyclidine (PCP) 25 ng/mL 25 ng/mL
Amphetamines 1000 ng/mL
Amphetamine 500 ng/mL
Methamphetamine 500 ng/mL
Alcohol 10 mg/100 mL 10 mg/100 mL
aApril 1988, National Institute of Drug Abuse (NIDA) Guidelines, SAMHSA 1998.
bTHC metabolite is 11-nor-delta-9 THC carboxylic acid.
cCocaine metabolite is benzoylecgonine.
d25 ng/mL if immunoassay is specific for free morphine.

in blood by Widmark in 1922, many new methods and modifications have been introduced. The distillation/oxidation methods are generally nonspecific for alcohol (ethanol), whereas biochemical methods (spectrophotometric) using alcohol dehydrogenase (ADH) obtained from yeast and the gas chromatographic method that are currently used are specific for ethanol. The radiative attenuation energy technique and those using alcohol oxidase method are non-specific and will detect not only ethanol but also other alcohols. The recently introduced alcohol dipstick based on the ADH enzyme system is not only specific for ethanol, but also sensitive and does not require instrumentation. It can be used for the detection of ethanol in all body fluids and can provide semi-quantitative results in ranges of pharmacological-toxicological interest. Alcohol dipsticks are being used in a number of laboratories as a screening device.

Breath can be analyzed by using a variety of instruments. Most of the instruments used today detect ethanol by using thermal conductivity, colorimetry, fuel cell, infrared or gas chromatography. Typically in most countries, local statutes define the instrument and method that can be used for evidenciary purposes. A variety of breathalyser instruments ranging in costs from $100 to $1000 are available to do the test. These instruments are compact and portable. Canadian law enforcement authorities use the breathalyser "Alert" which can give a "pass" or "fail" result as a roadside alcohol-screening device. The "failed" person is generally subjected to a "Borkenstein" breathalyser to measure the BAC before any charges are brought. Many devices are available to preserve the breath sample for later analysis if a breathalyser is not available immediately. In forensic laboratories, gas chromatography (North America) or biochemical procedures (many European countries) are used to analyze biological samples.

Blood samples that cannot be analyzed soon after collection should have sodium fluoride (NaF) added as a preservative. Alcohol dehydrogenase (ADH), the enzyme responsible for the oxidation of alcohol, is also present in the red blood cell and will slowly metabolise the alcohol, causing its concentration to drop if the preservative is not added. Large amounts of alcohol can be produced in-vitro in the urine samples of diabetic patients if samples are not processed immediately or properly preserved.

INTERPRETATIONS OF TEST RESULTS

False negatives.

A positive or negative result is highly dependent on the sensitivity of the drug detection method. A false negative occurs when the drug is present but is not found because the detection limit of the method used is too high or the absolute quantity of the drug in the specimen is too low.

Large amounts of fluids consumed prior to obtaining a sample for analysis can affect detection of drugs in urine samples. Under conditions of dilution, although the absolute amount of drug or metabolite excreted maybe the same over a period of time, the final concentration per millilitre will be reduced and may give a false negative result. Acidity levels in the urine may also affect the excretion of the drug into the urine. In some cases elimination is enhanced, whereas in other cases, the drug is reabsorbed.

Several measures can be used to decrease the likelihood of obtaining a false negative result. First, sensitivity of the method can be enhanced by analyzing for the drugs' metabolites. Heroin use, for example, is determined by the presence of its metabolite, morphine. Increasing the specimen volume used for analysis or treating it with chemicals can also make laboratory methods more sensitive. Studies have shown that a 5mg dose of Valium® is usually detected for three to four days; however, when these improved methods are utilized, sensitivity can be increased, such that, the same dose can be detected for up to 20 days. One important drawback of such high sensitivities is, that estimates of when the drug was taken are far less accurate.

False positives.

A false positive occurs when results show that the drug is present, when in fact it is not. False-positive tests are obtained if an interfering drug or substance is present in the biological fluid and it cross-reacts with the reagents. An example of this is Daypro (oxaprozin) will give a false positive for benzodiazepines. Other substances may have a metabolite that will give a positive reaction. An example of this is Selegiline, an antiparkinson drug, which has amphetamine as one of its metabolites. Although this would be analytically a true positive, it is a false positive from a drug abuse perspective. As discussed in the previous section on immunoassay, an initially positive test based on an

EMIT RIA TLC GC GC/MS
FPIA HPLC
Ease of sample preparation X X X
Less highly trained technologists required X X
Limited equipment required X X X
Low detection limits X X X X X
Adjustable lower threshold X X
Highly specific and sensitive X X X
Computerized identification possible X
Screen for several drugs at a time X X X
Procedure can be automated X X X X
Special atomic energy license required X
Confirmation of results required X X X X
Interpretation is subjective X

immunoassay technique should always be confirmed with an nonimmunoassay method. A confirmed positive finding only implies that the urine sample contains the detected drug and nothing more.

At times false positives are attributable to ingested substances such as allergy medications. Some authors have suggested that employees subject to drug screening refrain from using popular over-the-counter medications, such as Alka-Seltzer Plus and Sudafed, because they have caused false-positives. Some natural substances such as herbal teas and poppy seeds can also give positive responses to screens. These may be analytically true positives but need to be distinguished from those due to illegal drug use. In some instances, false-positives have been due to mistakes or sabotage of the chain of custody for urine samples.

COMMON ADULTERATION METHODS

The method of switching "clean" urine for "dirty" urine; resubmitting one's own or urine that is provided by someone else are the most common ways to fool the drug screening system. A number of entrepreneurs have attempted to bypass urine-specimen inspection by substituting clean urine. For example, a company in Florida sells lyophilized (freeze dried) clean urine samples through newspaper and magazine advertisements. Hiding condoms containing "clean" urine on the body or inside the vagina is another common trick.

Some have substituted apple juice and tea in samples for analysis. Patients are known to add everything from bleach, liquid soap, eye-drops, and many other household products, hoping that their drug use will be masked. Others may hide a masking substance under their fingernails and release it into the urine specimen. Another method is to poke a small hole into the container with a pin so that the sample leaks out by the time it reaches the laboratory.

Since addition of table salt (NaCl) or bleach to the urine is a common practice, many laboratories routinely test for Na and Cl in the urine. Liquid soap and crystalline drain cleaners that are strong alkaline products containing sodium hydroxide (NaOH) are also used to adulterate the urine sample. These contaminators can be detected by checking for high levels of pH in the urine sample. In-vivo alkalizing or acidifying the urine pH can also change the excretion pattern of some drugs including amphetamines, barbiturates and phencyclidine (PCP).

Water-loading (drinking large amounts of water prior to voiding) poses an interesting challenge to testing laboratories. Specific gravity has been used to detect dilution; however, the measurement range is limited so it is not yet useful. Creatinine levels on random urine samples appear to be a promising method for detection of water-loading. A number of adulteration methods are being advertised on the Internet. Invariably, one of the instructions for adulteration is to drink copious amounts of fluids to bring about in-vivo dilution or water loading. Some Internet sites even sell adulterants that can be added to the urine. Typically these products either try to oxidise the drug present or try to change the pH of the urine to interfere with the analytical method. Most of the laboratories involved in drug testing routinely test for the various adulterants. To detect resubmitted samples a "urine fingerprinting" method using dietary components has been described.

Drug users are very resourceful and their ingenuity should not be underestimated. To reduce the opportunities for specimen contamination, some workplaces require that employees provide a urine sample under direct supervision. Another technique used to detect any sample adulteration is to take the temperature of the sample. In a study, we took the temperature of urine samples when taken within one minute of voiding; it falls between 36.5°C and 34 degrees Celsius, reflecting the inner body core temperature. It is very difficult to achieve this narrow temperature range by hiding a condom filled with urine under the armpit or adding water from a tap or toilet bowl to the urine sample. It is important that the temperature of the specimen be measured immediately after the sample is taken, since it can drop rapidly.

LABORATORY PROCEDURAL AND SECURITY STANDARDS

It is important that the laboratory drug testing facility has qualified individuals who follow a specific set of laboratory procedures and meet recommended security standards.

SUMMARY AND CONCLUSION

In this paper, major issues related to drug testing are discussed. For example, drug-testing techniques measure drug presence but are not sophisticated enough to measure impairment from drug use. It is also very difficult to determine the route of drug administration, quantity, frequency, or when the drug was last taken.

Selection of the drug to be tested should depend on the local availability of the drug, its abuse potential, and clinical effects, as well as the available analytical technology and expertise in testing and interpretation of the laboratory results. The most sophisticated drug-testing approach, gas chromatography in combination with mass spectrometry, is considered as a gold standard and thus utilized in confirmatory testing. Typically GC/MS is preceded by a rapid immunoassay method to eliminate the majority of negative samples.

Despite the existence of sophisticated drug-testing methods, incorrect test results can still occur. These can be due to the presence of interfering substances or adulteration of the urine sample. Patients have been known to adulterate urine samples to avoid drug detection. A number of techniques can be employed to reduce the likelihood of obtaining erroneous results, as well as detect adulterated urine samples. [Parana "positive" drug finding can have a serious impact on the livelihood of an individual, therefore the performance of these tests should adhere to the strictest laboratory standards of performance. Only qualified and experienced individuals with proper laboratory equipment should perform these analyses. Standards of laboratory performance must meet local legal and forensic requirements. Access to the patient samples as well as laboratory records must be restricted in order to prevent tampering of samples and results. To maintain confidentiality and assure proper interpretation of results, the results must be communicated only to the physician reviewing the case/patient. Chain of custody and all documents pertaining to the urine sample must be maintained so that they can be examined in case of a legal challenge. Laboratory must have a complete record on quality control. Finally, specific initial and confirmatory testing requirements should be met.

Bhushan M. Kapur

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