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^) 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 immu-noassay 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 of S9-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 non-immunoassay 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 im-munoassay 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 chromato-graphic 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

TABLE 3

Common Drug-Testing Methods 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 chroma-tography (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., benzodiaze-pines).

Gas chromatography/mass spectrometry(GC/ MS) is a combination of two sophisticated technol-

TABLE 4

Advantages and Disadvantages of Immunoassays ogies. 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.

TABLE 5

Summary of Chromatographic Methods

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.

HPLC or GC

GC/MS

Interpretation is subjective, hence, training and experience in interpretation capabilities of the technologist are crucial.

Equipment costs are high, ranging between $25,000 to $60,000, depending on the type of detector and automation selected (1994 $)

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.

TABLE 6

Cut-off Levels for Initial and Confirmatory Tests"

TABLE 6

Cut-off Levels for Initial and Confirmatory Tests"

Test

Initial Test

Confirmatory Test

THC metaboliteb

100 ng/mL

15 ng/mL

Cocaine metabolites0

300 ng/mL

150 ng/mL

Opiate metabolites'1

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

"April 1988, National Institute of Drug Abuse (NIDA) Guidelines, SAMHSA 1998.

bTHC metabolite is 11-nor-delta-9 THC carboxylic acid.

"Cocaine 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 (spectrophotometry) using alcohol dehydrogenase (ADH) obtained from yeast and the gas chro-matographic 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, col-orimetry, 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 chroma-tography (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.

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