Drug tests are analytical procedures that may be performed on blood, urine, or gastric fluid for the purpose of identifying an unknown drug or measuring the concentration of a specific drug.
Purpose
Drug tests are usually performed for three reasons. 1) To identify an abuse drug. The majority of drug abuse involves one or more of the following substances and these comprise a typical drug of abuse panel: amphetamines, cannabinoids, cocaine, ethanol, opiates (morphine and codeine compounds), and phencyclidine (PCP). Over 85% of drug abuse cases involve those drugs or one of the following: barbiturates, benzodiazepines, methadone, propoxyhene, LSD, methaqualone, and anti-depressants. 2) To identify a drug which may have been ingested or administered in a toxic or lethal dose either accidentally or on purpose. In addition to poisons such as pesticides and heavy metals such as arsenic, drugs are often implicated in accidental overdose and suicide situations. The three most commonly encountered drugs seen in overdose situations are ethanol, salicylate (aspirin), and acetaminophen. 3) To determine whether the amount of a drug in the blood is within therapeutic limits. This process, called therapeutic drug monitoring (TDM), is used to insure that the dose and dose interval of the drug are sufficient to maintain a therapeutic blood concentration throughout drug therapy without risk of toxicity. TDM is also performed to verify that a patient is complying with the physician's orders.
Drug abuse testing
Drug screening may be performed on urine, blood serum or plasma, or gastric fluid, but urine is the sample of choice for symptomatic cases because drugs and their metabolites concentrate in the urine. Clinical or emergency department settings require the use of a screening method because the identity of the drug is not usually known. Drug screening methods may be designed to detect a class of related drugs. For example, a drug test for amphetamines may detect methamphetamine, dexamphetamine, methylenedeoxymethamphetamine (Ectasy), and phenylpropanolamine. The latter drug is a decongestant that sometimes cross reacts with the antibodies used in the amphetamine assay (analysis). Although drug screening may be sufficient to treat the patient, medicolegal implications are usually involved and this necessitates the need for positive sample identification and confirmatory drug testing. The confirmatory test need not be more specific than the screening test, but must utilize a different method of detection. This obviates the chance of a false positive test result caused by an interfering substance unless the interferent affects both methods. Drug screening programs are also used in occupational settings as a condition of employment, and extensively by the criminal justice system for criminal investigations and monitoring persons who have been convicted of drug related offenses. These situations require stringent adherence to procedures for documenting chain-of-custody of the specimen and confirmatory testing. Federal drug testing worksites must follow the Department of Transportation (DOT) chain-of-custody procedures for collection and transport of urine samples for drug testing. Labor-atories certified by the U.S. Substance Abuse and Mental Health Services Administration (SAMHSA) must use the gas chromatography with mass spectroscopy (GC-MS) method to confirm a positive drug screening test. This method is the gold standard for drug identification because it determines the mass spectrum of the drug which is a fingerprint of its chemical composition.
Specimen collection and transport
Urine specimens should be collected in a room with separate areas for workspace and toilet. The sink should be located in the workspace area. The patient or client must be positively identified via two forms of photoidentification or a passport. A form such as a DOT Custody Control Form should be used for chain-of-custody documentation. This form should include labels for the collection bottle and bag, and signature lines for all persons who will receive the specimen. At minimum the client must be observed entering and leaving the toilet area and should be instructed to remove outer garments and empty his or her pockets. The toilet should contain a bluing agent and the client should be instructed not to flush the toilet. The collection container should be unwrapped in the client's presence and affixed with a temperature measuring strip. The sample should be examined by the collector for adulteration and rejected if not within perscribed limits for volume (at least 30 mL) and temperature (90-100°F). An acceptable sample is labeled across the lid and side so the seal will be broken if the lid is removed. The laboratory should perform a test for urinary creatinine, pH, or specific gravity to check specimen integrity.
Blood samples are collected by venipuncture using standard precautions for reducing exposure to blood-borne pathogens. It is not necessary to restrict fluids or food prior to collection. Blood should be collected in tubes containing no additive. Risks of venipuncture include bruising of the skin or bleeding into the skin.
The EMIT principle
The most commonly used drug screening method is immunoassay. There are several immunoassay methods available including the enzyme multiplied immunoassay technique (EMIT), solid phase immunoassay fluorescence polarization immunoassay (FPIA), and cloned enzyme donor immunoassay (CEDIA). In addition, thin layer chromatography is sometimes used as a screening test. This method is more time consuming than immunoassay but is more comprehensive. The EMIT method is the most commonly used platform for drug of abuse screening. All EMIT assays follow the same scheme regardless of the drug being tested. EMIT assays measure enzyme activity. In a typical EMIT assay, urine is mixed with an antibody specific for the drug (e.g., methadone) and an enzyme-conjugated form of the drug. The enzyme used in EMIT testing is glucose-6-phosphate dehydrogenase. If not bound by the antibody, the enzyme will catalyze the oxidation of glucose-6-phosphate in the reagent forming 6-phosphogluconate and NADH. The production of NADH causes an increase in the absorption of 340 nm light. If no drug is present in the urine sample, all of the antibody will bind to the enzyme-conjugated drug. The antibody will block the catalytic site of the enzyme preventing the formation of NADH. If the drug is present in the urine sample, it will bind to some of the antibody, reducing the amount of antibody available to bind to the enzyme-conjugated drug. Therefore, the activity of the enzyme will be proportional to the concentration of drug in the urine sample.
In order to give maximum sensitivity the concentration of antibody is less than the concentration of the enzyme-conjugated drug. The labeled drug competes with any drug in the urine for binding sites on the antibody. If no drug is present in the sample, there will still be some unbound enzyme-conjugated drug that will produce NADH. However, the rate of enzyme activity will be less than that of the cutoff calibrator solution.
The enzyme activity of the sample is compared to that of a cutoff calibrator. The concentration of drug in the calibrator is set to a level recommended by SAMHSA for a positive test result. If the patient's sample result is greater than that of the cutoff calibrator, the drug test is presumed to be positive. Results below the cutoff calibrator are interpreted as negative. Activity less than the cutoff can result from endogenous drug present at very low levels in the absence of substance abuse. For example, poppy seeds used in baking contain minute amounts of opiates that cause some reactivity with enzyme immunoassays for opiates. Generally, the level of opiate detected will be below the cutoff value. Typically, the low calibrator and positive and negative urine control samples are assayed at least once per day along with the urine specimens.
EMIT is approved by the FDA only for urine specimens. Samples should be collected in clean plastic containers and refrigerated if not run within one hour. They can be refrigerated for up to three days or frozen, if longer storage is required. The pH of the sample must be between five and eight.
Confirmatory drug testing
Confirmation of a positive drug test by immunoassay is performed by a chromatographic method. Confirmatory methods include gas chromatography (GC), gas chromatography with mass spectroscopy detection (GC-MS), thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). The methods most commonly employed in clinical practice are GC and TLC. In forensic laboratories and SAMHSA approved toxicology laboratories confirmation is done using GC-MS. All chromatography techniques require extraction of the drug from the biological fluid. This is accomplished by adjusting the pH of the sample to minimize ionization of the drug and addition of an organic solvent. The nonionized drug molecules will be more soluble in the organic phase and can be separated from water-soluble interfering substances. Extraction also serves to concentrate the drugs. In general, a pH of nine promotes extraction of alkaline and neutral drugs, and a pH of 4.5 promotes extraction of neutral or acidic drugs. Most abuse drugs with the exception of barbiturates and some benzodiazepines are extracted at an alkaline p H. Chromatography is a method used to separate molecules of similar structure. The process of separation depends upon nature of the chromatographic medium. Separation can result from partitioning (solubility differences), adsorption, size exclusion, ion exchange, and affinity bonding.
Gas chromatography
Gas chromatography is performed using a glass column packed with a liquid separation medium such as polyethylene glycol or an open glass capillary that is coated with a liquid polymer separation medium. GC measures only those substances that are volatile or can be separated into volatile compounds. GC separates molecules primarily on the basis of solubility. The sample is introduced into the instrument injection port and is vaporized by a high temperature. The vapors are carried by an inert gas (usually nitrogen) into a temperature-controlled column where they separate based upon their boiling point. Molecules of low boiling move faster through the column and elute first. When the drugs leave the column they are most often detected by a process called flame ionization. A small hydrogen-air flame is used to excite the molecule, causing release of an outer shell electron. This produces a current that is proportional to the concentration of the molecules. The instrument produces a recorder tracing of a peak when a compound is detected. The peak height or area is proportional to the drug concentration. The time between introduction of the sample and the appearance of the peak is called the retention time. Under standardized conditions, the retention times of unknown substances can be compared to those of drug standards to identify the drug in the sample. GC is the reference method for measuring ethanol in blood and is sufficiently sensitive and specific to identify most drugs extracted from biological samples.
GC-MS is a form of gas chromatography. The detector used is a mass spectrometer. This device usually uses an electron beam to break the eluted drug into ion fragments. The ions are kept apart by application of a vacuum and are separated according to their mass to charge (m/z) ratio in the mass analyzer, which is usually a quadrupole mass filter. This device produces alternating direct current voltage and radio frequency waves that attract and repel the ions. Ions of different m/z ratios move at different rates as the frequencies change and leave the filter at different times. The ions are detected by a dynode as they leave the filter. When the ion strikes the dynode it causes the element to release a shower of electrons. This current is used to produce a peak corresponding in height to the concentration of the ion. A recorder tracing of all of the ion fragments constitute the mass spectrum of the drug. As no two drugs have the identical mass spectrum this method conclusively identifies the drug. For drug identification the GC-MS is used in a mode called total ion chromatography, which displays the complete mass spectrum of the eluate and allows comparison to a computerized library of drug standards. For quantitative analysis the selected ion monitoring (SIM) mode is used. This mode measures the principal ions of the drug and can more accurately quantify the drug at lower concentrations.
When testing for abuse substances the timing of specimen collection is very important because drugs are metabolized and eliminated at different rates. Dosage, length of use, and individual differences in absorption, metabolism, and elimination cause the window of detection to vary. Approximate detection times are shown below for some commonly abused drugs:
The same dose and dosing schedule for a drug can be therapeutic for some patients, and subtherapeutic or toxic for others. Age, gender, smoking, genetics, protein binding, concurrent medications, and renal and hepatic function cause variation in drug absorption, distribution, and clearance, which affects blood levels of the drug. The study of the behavior of a drug in the body is called pharmacokinetics. Pharmacokinetics describes the relationship between drug dose and blood concentrations. When two or more measurements are made after the drug reaches steady state, the results can be used to determine the dose and dosing interval needed to achieve the desired blood level. Tests for therapeutic drugs are performed for four reasons. 1) To determine whether the dose and dosing interval are able to maintain the desired blood level of the drug. 2) To permit empirical adjustment of the dose when the drug level falls outside the therapeutic range. 3) To verify that the patient is complying with the prescribed treatment. 4) To evaluate the magnitude of an intentional or accidental drug overdose.
In practice, only those drugs that have toxic potential near the therapeutic range need to be monitored. Drugs that should be monitored include many anticonvulsants, aminoglycosideantibiotics, antiasthmatics, antiarrhythmics, antineoplastics, antidepressants, and immunosuppressive drugs used in organ transplantation. As orally ingested drugs are metabolized and eliminated between doses, blood levels are time dependent. Shortly following absorption and distribution of the drug in the body, the blood level will peak. As the drug is metabolized and eliminated the blood level will fall until replaced by the next oral dose. When serial measurements of drug are plotted, the result is a dose response curve made up of repeating peaks and troughs. Accurate timing of sample collection is required to properly interpret blood drug test results. For most drugs, there is not a great difference between peak and trough blood drug levels, and measurement of trough drug concentration is sufficient to evaluate the patient. In cases where the trough-peak range is large, both trough and peak levels need to be considered. This is the case when monitoring aminoglycoside antibiotics. When measuring trough blood levels of a drug the sample should be collected just before the next dose is given. Collection time for peak blood levels depends upon the drug and route of administration. For aminoglycoside antibiotics peak levels are usually drawn 30 minutes following an IV (intravenous) dose and 60 minutes following an IM (intramuscular) dose.
KEY TERMS
Immunoassay—A method for measuring biological substances such as drugs, proteins, and hormones. It utilizes antibodies specific for the substance being tested.
Enzyme—A protein that accelerates the rate of a biochemical reaction.
Enzyme immunoassay—A procedure employing an enzyme bound to an antigen or antibody. The antibody binds to the antigen of interest and the enzymatic reaction measures the concentration of the antigen.
Chromatography—A technique used to separate closely related biological molecules which exploits one or more of the following differences: solubility, molecular size, adsorption, ion exchange, affinity bonding.
Gas chromatography—A chromatographic method that utilizes a gas for the carrier or mobile phase and a liquid for the stationary phase.
Gas chromatography with mass spectroscopy—A method that employs a gas chromatograph to separate the molecules and a mass spectrometer to identify and quantify the separated compounds.
High performance liquid chromatography—A chromatographic method that utilizes a liquid mobile phase or carrier and a liquid stationary phase. Sample is forced through the stationary phase by a high pressure pump.
Thin layer chromatography—A chromatographic method that uses a thin layer of silica gel and a liquid mobile phase which migrates upward through the silica gel by capillary action. Molecules are carried by the mobile phase and separate on the basis of solubility.
Therapeutic drug monitoring—The measurement of a drug in blood serum or plasma in order to determine the adequacy of dosing and prevent drug toxicity.
Trough level—A drug assay performed on a sample collected before the next dose is absorbed.
Peak level—A drug assay performed on a sample following complete absorption and distribution.
Therapeutic drug measurements
Both immunoassay and chromatographic methods are used to quantify therapeutic drugs. EMIT, CEDIA, and FPIA assays are the most commonly used immunoassays. Gas chromatography and high-performance liquid chromatography are the most commonly used chromatographic methods.
FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA). This method measures the plane polarized fluorescence of fluorscein-labeled antigen without the need for an enzyme conjugate. Fluorscein conjugated to a drug competes with the drug in the sample for a limited number of antibody molcecules. Plane-polarized UV light is transmitted through the sample. Both the unbound and antibody-bound, fluorscein-labeled drug absorb the UV light and the fluorescein becomes excited. The unbound labeled drug is rotating rapidly and emits light that is unpolarized. The labeled drug that is bound by antibody rotates more slowly and will emit light that is plane polarized. The detector responds to plane-polarized light only because a polarizing filter is placed between the cuvet and detector. Antibody binds to more fluorescein labeled drug when there is less drug in the patient's serum. This slows down its rotation giving a greater plane polarized signal. Therefore, plane-polarized fluorescent intensity is inversely proportional to the drug concentration in the patient's sample.
CLONED ENZYME DONOR IMMUNOASSAY (CEDIA). A technique related to EMIT is CEDIA (cloned enzyme donor immunoassay). The method uses a drug conjugated to a fragment of the enzyme [.beta]-galactosidase which is called the enzyme donor (ED). The ED reagent also contains the substrate, cholorophenol red-[.beta]-D-galactopyranose. This is mixed with urine or serum and a second reagent containing a monoclonal antibody against the drug and a second fragment of [.beta]-galactosidase called the enzyme acceptor (EA). If drug is present in the sample it neutralizes the antibody. The two enzyme fragments associate forming an active enzyme which splits the substrate, liberating the red dye. Absorbance is directly proportional to drug concentration. If drug is not present, the antibody binds to the ED fragment preventing formation of active enzyme.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC). HPLC is a chromatography method that uses liquid mobile and stationary phases. The mobile phase is usually a buffer to which a polarity modifier such as acetonitrile has been added. The stationary phase consists of a stainless steel column of silica gel that is bonded to a nonpolar liquid. The most common column packing for clinical use is octadecylsilane (C18). The stationary phase is less polar than the mobile phase, and this causes compounds which are lower in polarity to be retained longer than more polar molecules. The column is packed very tightly to give thousands of surfaces upon which partitioning can occur. Therefore, a pump is used to move the mobile phase through the column. To obtain a flow rate of 1.5 2.0 mL/min it is not uncommon to develop a pressure of 1,200-2,500 pounds per square inch at the start of the column. The separated molecules enter an optical flowcell after eluting from the column. UV (ultra-violet) light is passed through the flowcell and a photo-multiplier tube or photodiode array detects the transmitted light. When a drug enters the flowcell it will absorb a portion of the incident UV light causing an increase in absorbance (optical density). This signal is applied to a chart recorder, which produces a peak that is proportional in height and area to the concentration of the drug. HPLC is time consuming and is usually reserved for assays of drugs for which there is no available immunoassay. It has the advantage of being able to separate drug metabolites from parent compounds and separate compounds that are nonvolatile (for example, anabolic steroids).
Results
Results for drug of abuse tests should be negative (ie. below the low calibrator cutoff). Results for therapeutic drugs should fall within the published therapeutic range for the drug and treatment. Some typical therapeutic limits for commonly measured drugs are shown below:
Therapeutic drug tests are ordered by physicians. Blood and urine samples may be collected by a nurse or phlebotomist. In the case of drug of abuse testing performed on behalf of an employer or government agency, drug testing is supervised by a medical officer appointed by the institution. Urine samples are collected and transported by nontesting personnel who should have special training in chain-of-custody procedures. Drug testing is performed by a clinical laboratory scientist, CLS (NCA) or medical technologist, MT (ASCP) or by a clinical laboratory technician, CLT (NCA) or medical laboratory technician, MLT (ASCP). Clinical toxicologists, clinical chemists, and clinical pharmacologists may be responsible for interpreting therapeutic drug tests and recommending doseage adjustments to the physician. Psychiatrists, psychologists, nurses, and social workers who are trained in drug abuse treatment are involved in evaluation, treatment, and counseling of drug abusers.
BOOKS
Burtis, C.A. and E.R. Ashwood (eds). Tietz Fundamentals of Clinical Chemistry, 5th ed. Philadelphia: W.B. Saunders, 2001.
Henry, J.B. (ed). Clinical Diagnosis and Management by Laboratory Methods, 20th ed. Philadelphia: W.B. Saunders, 2001.
Kaplan, L.A. and A.J. Pesce (eds). Clinical Chemistry Theory Analysis and Correlation, 3rd ed. St. Louis: C.V. Mosby, 1999.
