Drug testing methods.pdf

7/27/2019 Drug testing methods.pdf

1/35

Drug-testing methods and clinical interpretations oftest results

B. M. KAPUR Director of Clinical Laboratory, Addiction Research

Foundation, Toronto, Qntario, Canada

ABSTRACT

In the present paper, major issues related to drug testing arediscussed. For example, drug-testing techniques measure thepresence of a drug but are not sophisticated enough to measureimpairment from drug use. Moreover, it is difficult to determine theroute of drug administration, quantity or frequency, as well as whenthe drug was taken, on the basis of the laboratory results.

Selection of the drug to be tested should depend on the localavailability of the drug, its abuse potential and clinical effects, aswell as on the availability of analytical technology and expertise intesting and in interpreting laboratory results. The mostsophisticated drug-testing approach is gas chromatography coupledwith mass spectrometry (GC/MS), which is regarded as a "goldstandard"; it is used in confiramtory testing. Typically, GC/MS ispreceded by a rapid immunoassay method to eliminate the majorityof the "negative" samples.

Despite the existence of sophisticated drug-testing methods, it isstill possible to obtaining incorrect test results and to detectadulterated urine samples.

A "positive" drug finding can have a serious impact on the livelihoodof an individual, therefore, persons conducting such tests shouldadhere to the strictest standards of laboratory performance. Onlyqualified and experienced individuals with proper laboratoryequipment should perform these analyses. The standards oflaboratory performance ,must meet local legal and forensic

requirements. Access to patient samples and laboratory recordsmust be restricted in order to prevent the tampering of samples andresults. In order to maintain confidentiality, the results must becommunicated only to the medical review officer. Chain-of-custodydocuments and all file so that they can be examined in case of alegal challenge. The laboratory must have a complete record onquality control. Finally, specific initial and confirmatory testingrequirements should be met.

Introduction


2/35

As interest increases in employment-related drug testing, thetechnologies and the interpretive skills of analysts continue toevolve. Although recent literature indicates that significantrefinements and modifications have been made in drug-testingtechnology, the complexity of drug effects is so great that many

problems exist in the interpretation of test results. The mostfrequent problems that confront the toxicology laboratory areassociated with developing technology that can determine howmuch and when a drug was taken, how long after use the tests arecapable of showing positive results, the causes and rates of falsepositives and false negatives, and how tests can be "beaten" byemployees. In the present paper, these problems are discussed andthe various laboratory procedures used to combat the problems areexamined.

I. Drug properties: absorption, distribution and eliminationphases

Detection of a drug depends largely on its absorption, distributionand elimination properties. There are various routes of drugadministration; oral drinking, e.g. alcohol), intravenous (injectinginto a vein, e.g. heroin) and inhalation (smoking, e.g. marijuana;snorting, e.g. cocaine; and sniffing, e.g. glue). Drugs taken orallyare usually the slowest to be absorbed (i.e. by the brain and otherbody organs), whereas the intravenous and inhalation routes result

in the fastest absorption. Once the drug enters the bloodstream it israpidly distributed to the various tissues in the body. The amount ofdrug stored depends on the nature of the drug, the quantity, theduration of ingestion, the tissue holding the drug and the frequencyof use.

Some drugs are fat-soluble and are deposited in fat tissues. Forexample, 9-tetrahydrocannabinol (THC), the active ingredient inmarijuana, is highly fat-soluble, resulting in a rapid reduction inlevels of THC in the blood as the drug is distributed to the various

tissues [1] . Some studies have shown that THC levels peak andstart to decline in half the time it takes to smoke a marijuana"joint". Concentrations are known to fall by almost 90 per cent inthe first hour [1] , [2] suggesting that a higher degree ofsophistication in laboratory analysis is needed to detect fat-solubledrugs. Depending on the amount of drug stored, however, fat-soluble drugs. Depending on the amount of drug stored, however,detection in the urine may be possible for as long as 60 days afterlast use [3] . Ethanol or ethyl alcohol is the beverage alcohol that isconsumed by people. Alcohol* is not fat-soluble and is distributed in

the total body water [4] . Since blood is mostly made up of water,the presence of alcohol is more easily detectable than fat-soluble
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f01#f01http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f01#f01http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f02#f02http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f03#f03http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f04#f04http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f04#f04http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f03#f03http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f02#f02http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f01#f01http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f01#f01


3/35

drugs like THC. The absorption and distribution phases are followedby an elimination phase. The liver is the major detoxification centrein the body; drugs are metabolized as blood circulates through theorgan. The metabolites are then excreted into the urine through thekidneys. At the same time, drugs deposited in fat tissues are also

slowly released into the bloodstream and metabolized.

Drugs vary by their elimination half -lives, which is the timerequired for the blood levels to decline by 50 per cent (see table 1).The half -life of a drug is heavily influenced by a variety of factors,including the individual's age, sex, physical condition and clinicalstatus. A compromised liver and the concurrent presence of anotherdisease or drug have the potential of enhancing the toxic effects ofthe drug by slowing down the elimination process. Under differentclinical conditions, however, the process may be speeded up.

Therefore, great variation may be found in the half -lives of thesame drug.

Approximately six half -lives are required to eliminate 99 per cent ofany drug. Because the half-life of cocaine is relatively short,averaging one hour [5] , only six hours are needed for theelimination of 99 per cent of the drug. Cocaine metabolites have alonger half -life and can be detected for a considerably longer periodof time through urine drug assays. Compared with cocaine,phenobarbital has a much longer half - life (80-120 hours), so that

at least 480 hours, or 20 days, are required to eliminate 99 per centof the drug. Since there is much variation in the half -lives ofdifferent drugs and the absolute amount of drug present can bevery small, it is crucial that the appropriate body fluid for analysis isselected for testing.

Elimination of ethanol follows a different pattern. Its levels declinealmost linearly over time. The average elimination rate is between15 mg/100 ml and 20 mg/100 ml (0.015-0.02 per cent) per hour,although rates of between 10 mg/100 ml and 30 mg/100 ml (0.01-

0.03 per cent) per hour have also been observed. In the alcoholicpatient, the elimination rate is generally higher. In forensiccalculations, a rate of 15 mg/100 ml (0.015 per cent) per hour isusually used.

Table 1.

Drug half-lives and approximate urine detection periods

DrugHalf-lifea/

Detectionperiod

Methamphetamine 12-34hours

2-3 days
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f05#f05http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f05#f05


4/35

Amphetamine (metabolite of

methamphetamine)7-34hours

Heroin

60-90

minutes Minutes

Morphine (metabolite of

heroin)1.3-6.7hours

Phencyclidine. (PCP)7-16hours

2-3 days

Cocaine0.5-1.5hours

A few hours

Benzoyltegonine (metabolite

of cocaine) 5-7 hours 3-5 days

?-9-Tetrahydrocannabinol14-38hours

90 per centfall in 1 hour(blood)

?-9-TetrahydrocannabinolicDepending onuse, any-

acid (marijuana metabolite inwherebetween afew days

urine)and manyweeks

Alcohol (ethanol)Bloodlevels fallby

1.5-12 hours,depending on

anaverageof 15-

the peakblood level;urine

18 mg/100ml/hour

is typicallypositive foran

additional 1-2hours

Source-R. C. Baselt, Disposition of ToxicDrugs and Chemicals in Man, 2nd ed.(Davis, California, Biomedical Publications,1982)..

a/ The detection period is dose-


5/35

dependent. The larger the dose, thelonger the period that the drug or,metabolite can be detected in the urine.

II. Selection of drugs to be testedA number of different criteria can be applied to the drug(s) orcategory of drugs that-should be tested or monitored. Drugavailability, clinical effects and robustness of the analyticalmethod(s) used for analysis are probably the most important.

A. Availability

Prescription patterns of psychoactive and other drugs vary fromplace to place and from Country to Country [6] , [7] . The abuse ofthe benzodiazepine nitrazepam is common in Europe but almostunknown in Canada and the United States of America, as it is notavailable on markets in North America. The psychoactive chemicalcathine is the active ingredient in the leaves of the khat (Cathaedulis) plant, which are chewed in north-eastern Africa; the practicedoes not appear to have crossed over to the European or the NorthAmerican continent. In Canada, the narcotic drug codeine isavailable as over-the-counter, non- prescription medication,whereas in the United States it is available only with a physician'sprescription.

The wide availability of "legal" stimulants poses an interestingproblem since they are commonly found in accident victims. A studycarried out in the United States by. the National TransportationSafety Board [8] from October 1987 to September 1988 showedthat over-the- counter stimulants - antihistamines.such asephedrine, pseudoephdrine and phenylpropanolamine - were,commonly found in drivers killed in heavy truck accidents. Similarresults have also been obtained based on data from emergencyrooms gathered over a five -year period [9] as well as data from

admissions in a trauma unit following motor vehicle accidents [10] ,[11]

In the National Transportation Safety Board study [8] , almost allthe amphetamine use was in California rather than in the otherseven states in the study, suggesting that drug use varies not onlyfrom country to country, but also within a given country.

Thus, the selection of a drug to be tested and monitored that isappropriate for one country or place may not necessarily be

appropriate for another.
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f06#f06http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f07#f07http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f08#f08http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f09#f09http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f10#f10http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f11#f11http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f08#f08http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f08#f08http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f11#f11http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f10#f10http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f09#f09http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f08#f08http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f07#f07http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f06#f06


6/35

B. Clinical effects

Drugs that manifest abuse potential and impair behavior to such anextent that job performance may -be affected are prime candidatesfor testing or monitoring in the workplace. Alcohol and cocaine are

examples of such drugs.

C. Analytical methods

A false -positive finding can have a serious impact on the livelihoodof the person being tested. Therefore-, special attention needs to bepaid to the testing methods used. Ideally,, the analytical methodshould be specific for the drug being tested (i.e. no false positive)and should be easy and inexpensive to use. Confirmation methodsshould also be readily available. The availability of technical and

scientific expertise to perform the tests is also essential.

The interpretation of the analytical results needs to be carefullyconsidered as even a normal diet can sometimes result in a positivedrug identification. Poppy seed ingestion [12] can result in a true-positive analytical report but it is a false positive for drug use. Someethnic diets may also lead to such confounding problems; forexample, food containing poppy seed is common during theRamadan period in India.

What should be analysed? Ideally, the parent drug, rather than itsmetabolite should be looked for in the analysis, although this maynot always be possible as some drugs are rapidly metabolized (e.g.heroin metabolism to morphine). The sensitivity of the analyticalprocedure should be dictated by the psychoactive pharmacologicalproperties of the drugs. If the drug is shown to be devoid of abusepotential, then its detection beyond the time of pharmacologicalactivity, although important in the clinical management of thepatient, does not necessarily serve a useful purpose in a workplacedrug-screening programme.

The guidelines developed by the National Institute on Drug Abuse(NIDA) [13] in the United States in April 1988 deal with five "illegal"drugs (marijuana, phencyclidine (PCP), amphetamine, cocaine andheroin). Rapid screening methods that allowed for 'mass screening'were available at that time, as were the confirmation methods forthose five drugs. Mood-altering substances, such asbenzodiazepines, barbiturates and stimulants, such asantihistamines, are at present excluded from the regulations in theUnited States. This is probably because of the much wideravailability of those drugs as medication and the technological

requirements for screening and monitoring them.
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f12#f12http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f12#f12


7/35

III. Types of testing. blood, urine and hair specimens

Blood and urine are the most commonly used fluids in the analysisfor drugs. Blood, obtained by an invasive procedure, is availableonly in small quantities and drug concentration levels in blood arelow. Urine is the preferred sample as it is available in larger volume,contains the metabolite and requires less invasive procedures in itscollection. Both sampling procedures, however, have limitations asthey only determine the absolute amount of drug present in thefluid being examined. That amount is dependent upon the amountof the drug used, when it was used last, and the half-life of thedrug.

Recently, hair samples have been used to detect drug use [14] ,[15] , [16] . There are a number of technical problems that must be

overcome, however, before hair can be used as definitive proof ofdrug use. An advisory committee of the Society of ForensicToxicology [17] has recently concluded that, 'because of thesedeficiencies, results of hair analysis alone do not constitutesufficient evidence of drug use for application in the workplace'.

Breath and various body fluids, such as sweat, saliva, blood andurine, have been used for alcohol analysis. Breath is commonlyused by law enforcement authorities for such analysis. Although anumber of variables [18] can affect the breath-blood ratio, the

2,100:1 alveolar breath-blood conversion ratio has been used andaccepted for the Breathalyser [19] . Breath-testing equipmentcalibrated with a blood- breath conversion factor of 2,100consistently underestimate actual blood alcohol concentrations [20]. The results of breath analysis may vary depending on theinstruments used and on biological factors [21] , [22] . Potentialerrors in breath analysis can also be caused by the presence ofresidual alcohol in the mouth. Immediately after drinking, there isenough alcohol vapour in the mouth to give artificially highconcentrations in breath analysis. Generally, this effect disappears

after 20minutes but high values for as long as 45 minutes havebeen reported [23] .

All existing technologies are limited in terms of their capacity todetermine how much of a drug was consumed or when it wasconsumed.

Blood and saliva concentrations reflect the current blood alcoholconcentration, but generally a blood sample is used in hospitals forpatients entering casualty wards. In programmes requiring

monitoring of alcohol use, urine is probably the sample of choice[24] . Urine alcohol concentration, which represents the average
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f14#f14http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f15#f15http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f16#f16http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f17#f17http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f18#f18http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f19#f19http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f20#f20http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f21#f21http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f22#f22http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f23#f23http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f24#f24http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f24#f24http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f23#f23http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f22#f22http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f21#f21http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f20#f20http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f19#f19http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f18#f18http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f17#f17http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f16#f16http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f15#f15http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f14#f14


8/35

blood alcohol concentration between voiding, has the potential ofbeing "positive" while the blood may be "negative".

IV. Measuring impairment

Except in alcohol analysis, the degree to which a person isinfluenced or impaired by a drug at the time of the testing cannotbe determined from test results alone. Correlations betweenpositive blood levels and degree of impairment are usually strongerthan correlations between urine levels and degree of impairment;however, neither blood nor urine tests are sufficiently accurate toindicate impairment even at high levels of concentration [25] , [26], [27] . Human studies using marijuana and cocaine have shownthat a "perceived high" is reached after the drug concentration haspeaked in the blood [2] , [28] . Generally, blood can only show

positive results for a short time after drug consumption, whereasurine can be positive for a few days or weeks after last use. Forexample, metabolites of THC that are lipid-soluble can be detectedin the urine for a few days or for many weeks, depending on thedrug habit of the user [3] . Excretion of the drug in urine and itsconcentrations are also effected by several factors, such as dilutionand acidity (pH) of the urine. The author has seen many caseswhere a urine sample was strong positive for cannabinoids in themorning, borderline positive in the afternoon and strong positive thenext morning. The author has made similar observations for

phenobarbital.

From a positive urine test, the form in which the drug was originallytaken or when and how much was taken cannot be determined. Forexample, crack, impure cocaine powder or cocaine paste (which canbe smoked, inhaled, injected or chewed) all give the same result ina urine test. The consumption of poppy seeds has been reported-togive positive results for opiate use because some seeds containtraces of opiates and some have been known to be contaminatedwith opium derivatives [12] . Similarly, consumption of herbal cocoa

tea has resulted in positive results for cocaine use. These incidentsclearly illustrate the difficulties involved in measuring impairmentusing urine test results.

The problem of interpreting urine test results is one of the mainarguments for restricting their use in the employment setting. In arecent study [29] , the effectiveness of pre-employment drug-screening tests has been questioned because of difficulties ininterpretation. Based on their analysis of 2,229 pre-employmentdrug-screening tests and follow- up, Ryan, Zwerling and Jones [291

concluded: 'Our findings raise the possibility that a pre-employmentdrug screening may be decreasingly effective in predicting adverse
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f25#f25http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f26#f26http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f27#f27http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f02#f02http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f28#f28http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f03#f03http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f12#f12http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f29#f29http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f29#f29http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f12#f12http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f03#f03http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f28#f28http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f02#f02http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f27#f27http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f26#f26http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f25#f25


9/35

outcomes associated with marijuana use after the first year ofemployment.' They made a similar comment about cocaine.

There is no threshold for alcohol effects on performance or motorvehicle accident risk. Although the effects of alcohol on impairment

and crash risk appear more dramatically above 80 mg/100 ml (0.08per cent), a review of the literature [30] , [31] would suggest thatimpairment may be observed at levels as low as 15 mg/100 ml(0.015 per cent). It is not possible to specify a blood alcoholconcentration (BAC) level above which all drivers are dangerous andbelow which they are safe or at "normal" risk [31] .

"Legal" BAC levels differ in different countries. Some even havemore than one legal limit over which the driver of a vehicle isconsidered "impaired". Some European countries have 50 mg/ 100,

ml (0.05 per cent) as their legal limit and others have 80 mg/ 100ml (0.08 per cent). In the United States, the legal limit varies from80 mg/ 100 ml (0.08 per cent) to 100 mg/100 ml (0.10 per cent) indifferent states, but employees who are regulated by the UnitedStates Department of Transportation have a BAC legal limit of 40mg/ 100 ml (0.04 per cent). In Canada, there are also two limits,50 mg/100 ml (0.05 per cent) and 80 mg/100 ml (0.08 per cent);BAC levels between 50 mg/100 ml and 80 mg/100 ml (0.05-0.08per cent) may result in suspension of driving privileges and levelsabove 80 mg/100 ml (0.08 per cent) may result in criminal charges.

V. Urine-testing methods

Urine is the most commonly used fluid for drug screening [32] . Themethods most commonly used in toxicology laboratories are asfollows:

1. Immunoassay:2. Enzyme immunoassay (EIA);3. Enzyme-multiplied immunoassay technique .(EMIT);4. Fluorescence polarization5. Radioimmunoassy (RlA);6. Chromatography.7. Thin-layer chromatography (TLC)8. High-performance liquid chromatography (HPLC);9. Gas chromatrography (GC.);.10. Chromatography coupled with mass spectrometry:11. Gas chromatography coupled with mass spectrometry

(GC/MS);12. High-performance liquid chromatography coupled with

mass spectrometry (HPLC/MS).
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f30#f30http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f31#f31http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f31#f31http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f32#f32http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f32#f32http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f31#f31http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f31#f31http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f30#f30


10/35

The methods vary considerably with respect to -their sensitivity andreliability. TLC is the least expensive and is reliable, GC/MS isconsidered nearly perfect, or a "gold standard", [33] ; it requireshighly trained technologists and the most expensive equipment.

A. Immunoassay

Immunoassay methods are used for preliminary screening (i.e.initial screening). Since the methods are based on an antibody-antigen reaction, small amounts of the drug or 'Metabolite(s) can bedetected. Antibodies specific to a particular drug are produced byinjecting laboratory animals with the drug. These antibodies arethen tagged with markers such as an enzyme (EIA), radio isotope(RIA) -or a fluorescence ((fluorescence polarization immunoassay(FPIA)) label. Reagents containing the labelled antibodies can then

be introduced into urine samples and, if the specific drug againstWhich the antibody was made is present,, a reaction will occur. RIAis the oldest immunoassay method used to detect drugs. The majordrawback of this method is that it requires a separation step andgenerates radioactive waste. RIA also requires special equipmentfor measuring radioactivity.

Immunoassays typically are designed for a class of drugs. Thus,their specificity (the ability to detect the presence of a specific drug)is not very good as 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 detectthe carboxylic acid metabolite of THC. Rollins, Jennison and Jones[34] , however, showed that non-steroidal anti-inflammatory drugs,ibuprofen (a non-prescription drug in Canada) and, naproxyn cangive random or sporadic false positives for cannabinoids. Codeinewill also give a positive reaction for the morphine (a metabolicproduct of heroin use) immunoassay and many antihistamines thatare available over the counter may yield positive reactions foramphetamines. While some reagent manufacturers claim to have

overcome many of these cross- reactivity problems, confirmation bya non-immunoassay is important. Immunoassays are consideredgood screening tests but not sufficient by themselves for aconclusion to be drawn. A non-immunoassay method is required todraw a conclusion.

Urine drug assay kits have been available in North America for thepast few years. More recently, single- and multiple-testimmunoassay kits designed for home and on-site testing have alsobeen introduced. Such kits generally carry a cautionary disclaimerthat positive test results must be confirmed by GC/MS, thereference method. When used in a non-laboratory environment,they are prone to procedural inaccuracies, poor quality control,


11/35

abuse and misinterpretations. Therefore, they are notrecommended for testing in the workplace. The risk of labelling aperson with a false positive is high without confirmatory analysis. Inaddition, confirmation analysis is generally expensive when anindividual sample is being tested. The advantages and

disadvantages of immunoassay testing may be summarized asfollows.

1. Advantages:2. Screening tests can be done quickly because automation and

batch processing are possible;3. Technologists doing routine clinical chemistry testing can be

easily trained;4. Detection limits are low and can be tailored to meet the

programmes screening requirements. For example, lower

detection thresholds can be raised to eliminate positivesresulting from passive inhalation of marijuana smoke;

5. Immunoassays are relatively inexpensive, although thesingle-test immunoassay kits can be expensive when quality-assurance and quality-control samples are included;

6. Immunoassays do not require a specialized laboratory. Mostclinical laboratories have automated instruments to do theprocedures;

7. Disadvantages:8. Although the tests are useful for detecting classes of drugs,

specificity for individual drugs is weak;9. Since the antibody is generated from laboratory animals,

there can be a lot-to-lot or batch-to-batch variation in theantibody reagents;

10. Results must be confirmed by another non.-immunoassay method;-,,

11. A radioactive isotope is used in RIA that requirescompliance with special licensing procedures, use of gammacounters to measure radioactivity and disposal of theradioactive waste;

12.

Only a single drug can be tested for at one time.

B. Chromatography

Separation of a mixture is the main outcome of thechromatographic method. If a drop of ink is put on a blotting paperand the tip of the paper is held in water, the water will rise in thepaper. After a period of time and under the right conditions, thesingle ink spot will separate into many different compounds (spots)of different colours (blue ink is a mixture of many dyes). Thisprocess, whereby a mixture of substances is separated in astationary medium (filter paper), is called chromatography. The


12/35

types of chromatographic processes used in the analysis of drugs.include thin-layer, gas, and liquid chromatography as well as acombination of gas or liquid chromatography with massspectrometry.

1. Thin-layer chromatography

TLC is most similar to the ink separation example mentioned above.This method requires extensive sample preparation and technicalexpertise on the part of the analyst, but it is inexpensive andpowerful if used properly. With the exception of cannabis, whichrequires separate sample preparation, a large number of drugs (e.g.cocaine, amphetamine, codeine and morphine) can be screened atthe 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 thesubjectivity involved in interpreting the results. To identify positiveTLC "spots", the technologist looks for the drugs and/or theirmetabolite patterns. A trained technologist can identify more than40 different drugs.

2. Gas chromatography

GC, which is similar to TLC, requires extensive sample preparation.In GC, the sample to be analysed is introduced into a narrow bore(capillary) column with a syringe. The. column, which sits inside anoven, is flushed with a carrier gas such as helium or nitrogen. In aGC system that has been properly set up, a mixture of substancesintroduced into the carrier gas is volatilized and the individualcomponents of the mixture migrate through the column at differentspeeds. Detection takes place at the end of the heated column. andis generally a destructive process. Often, the substance to beanalysed is "derivatized" to make it volatile or to change itschromatographic characteristics.

3. High-performance liquid chromatography

In contrast to GC, high-performance liquid chromatography (HPLC)requires a liquid under high. pressure, rather than a gas, to be usedto flush the column. Thus, this technology is sometimes referred toas high - pressure liquid chromatography. Typically, the columnoperates at room temperature or slightly above room temperature.This method is generally used for substances that are difficult tovolatilize (e.g. steroids) or are heat-labile (e.g. benzodiazepines).The two major differences between GC and HPLC are as follows:


13/35

1. GC is a "destructive" method (it destroys or burns thechemical in its detector to generate the signal), whereas HPLCdetection takes advantage of the electronic or chemicalstructure of the compound;

2. The mobile phase in GC is gas; in HPLC it is liquid.Consequently, less sample preparation is needed for HPLC.HPLC also has high specificity, but it is slower and generallyless sensitive than GC.

C. Gas chromatography coupled with mass spectrometry

GC/MS is a combination of the two sophisticated technologies. GCphysically separates (chromatographs or purifies) the compound,and MS fragments it so that a fingerprint of the chemical (or drug)can be obtained. Although sample preparation is extensive, when

the methods are used together the combination is regarded by mostauthorities as the "gold standard'. This combination is sensitive (itcan detect low levels), specific and able to identify all types of drugsin any body fluid. Furthermore, assay sensitivity can be enhancedby treating the test substance with reagents. When coupled withMS, HPLC/MS is the method of choice for substances that aredifficult to volatilize (e.g. steroids).

Given the higher costs associated with GC/MS, urine samples areusually tested in batches for broad classes of drugs by

immunoassay, and positive screens are later subjected toconfirmation by this more expensive technique. This is the mostcommon approach used in employment drug- screeningprogrammes and is the combination recommended by NIDA [13] inthe United States.

The advantages and disadvantages of the various methods ofchromatographic drug testing are as follows:

1. Advantages:2. All chromatographic methods: All the chromatographic

methods are specific and sensitive and can screen a largenumber of drugs at the same time;

3. TLC: Negligible capital outlay is needed;4. GC: The procedure can be automated;5. HPLC:

a. Of the chromatographic procedures, it has the easiestsample preparation requirements; b. The procedure can beautomated;

6. GC/MS:


14/35

a. It is regarded as the "gold standard" test; b. Computerizedidentification of fingerprint patterns makes identification easy;c. The procedure can be automated; d. It is currently thepreferred method for defence in the legal system;

7. Disadvantages:8. All chromatographic methods: All the chromatographic

methods are labour-intensive and require highly trained staff.Although all the chromatographic methods are specific,confirmation is still desirable;

9. TLC: Interpretation is subjective, hence training andexperience in interpretation capabilities of the technologist arecrucial;

10. GC or HPLC: Equipment costs are high, ranging betweenUS$ 25,000 and US$ 60,000, depending on the type of

detector and automation selected;11. GC/MS:

a. Equipment costs are the highest, ranging from US$120,000 to US$ 200,000, depending on the degree ofsophistication required; b. Due to the complexity of theinstrument, highly trained operators and technologists arerequired.

Table 2 presents a comparison of all the methods of testing.

Table 2. Comparison of all drug testing methods

AspectEMITandFPIA

RIA TLCGCandHPLC

GC/MS

Ease of samplepreparation

x x x

Less highlytrained

technologists

x x

required

Limitedequipmentrequired

x x x

Low detectionlimits

x x x x x

Adjustable lowerthreshold

x x

Highly specificand sensitive

x x x


15/35

Computerizedidentification

x

possible

Screen for

several drugs at x x x

a time

Procedure canbe automated

x x x x

Special atomicenergy licence

x

required

Confirmation ofresults

x x x x

required

Interpretation issubjective

x

Notes: EMIT =enzyme-multipliedimmunoassaytechnique

FPIA =fluorescencepolarizationimmunoassay

GC = gas chromatography

GCIMS =gas chromatographycoupled with massspectrometry

HPLC =high-performanceliquid chromatography

RIA = radioimmunoassay

TLC =thin-layer

chromatography

D. Procedures for alcohol testing

Since the introduction by Widmark [35] in 1922 of the "micro-method" for alcohol analysis in blood, many new methods andmodifications have been introduced. The distillation/oxidationmethods are generally non-specific for ethanol [36] , [37] , whereasbiochemical (spectrophotometric) methods using alcoholdehydrogenase (ADH) obtained from yeast [38] and the gas

chromatographic [39] method that are currently in use are specificfor ethanol. The radiative energy attenuation technique [40] and
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f35#f35http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f36#f36http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f37#f37http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f38#f38http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f39#f39http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f40#f40http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f40#f40http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f39#f39http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f38#f38http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f37#f37http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f36#f36http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f35#f35


16/35

those using the alcohol oxidase method are non-specific and willdetect not only ethanol but also other alcohols. The recentlyintroduced alcohol dipstick [41] , based on the ADH enzymesystem, is not only specific for ethanol, but also sensitive and doesnot require instrumentation. It can be used for the detection of

ethanol in all body fluids and can provide semi-quantitative resultsin ranges of pharmacological-toxicological interest. The alcoholdipsticks are being used in many alcohol treatment programmes aswell as in a number of laboratories as a screening device.

Breath can be analysed using a variety of instruments. Most of, theinstruments used today detect ethanol by using, for example,thermal conductivity, colorimetry, infrared spectroscopy or gaschromatography. In most countries, local statutes define theinstrument and method that can be used for evidentiary purposes.

A variety of Breathalyser instruments, the prices of which rangefrom US$ 100 to US$ 1,000, are available. The instruments arecompact and portable. The Canadian law enforcement authoritiesuse as a roadside alcohol-screening device a Breathalyserinstrument that gives a "pass" or "fail" result. A person who failsthat test is generally subjected to a Breathalyser test to measurethe BAC level before any charges are made. Many devices areavailable to preserve the breath sample for later analysis if aBreathalyser is not available immediately. In forensic laboratories inNorth America, gas chromatographic procedures are used to

analyse biological samples; in many European countries,biochemical procedures are used.

Blood samples that cannot be analysed soon after collection, shouldhave sodium fluoride (NaF) added as a preservative [42] . ADH, theenzyme responsible for the oxidation of alcohol, is also present inred blood cells and will slowly metabolize the alcohol, causing itsconcentration to drop if the preservative is not added. Largeamounts of alcohol can be produced in vitro in the urine samples ofdiabetic patients if samples are not processed immediately.

VI. Interpretation of test results

A. False negatives

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

Large amounts of fluids consumed prior to obtaining a sample foranalysis can affect detection of drugs in urine samples. Under


17/35

conditions of dilution, although the absolute amount of drug ormetabolite excreted may be the same over a period of time, thefinal concentration per millilitre will be reduced and may give afalse-negative result. Acidity levels in the urine may also affect theexcretion of the drug into the urine. In some cases, elimination is

enhanced; in others, the drug is reabsorbed.

Several measures can be used to decrease the likelihood ofobtaining a false-negative result. First, sensitivity of the method canbe enhanced by analysing for the metabolites of the drug inquestion. Heroin use, for example, is determined by the presence ofthe heroin metabolite, morphine. Increasing the specimen volumeused for analysis or treating it with chemicals can also makelaboratory methods more sensitive. Studies in the author'slaboratory have shown that one 5 mg dose of valium is usually

detected for 3-4 days. When these improved methods are utilized,however, sensitivity can be increased so that the same dose can bedetected for up to 20 days. One significant drawback of such highsensitivity is that estimates of when the drug was taken are far lessaccurate.

B. False positives

A false positive occurs if results show that the drug is present whenin 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. As discussed in the previous section onimmunoassays, an initially positive test based on immunoassaytechnique should always be confirmed with a non-immunoassaymethod. A confirmed positive finding only implies that the urinesample contains the detected drug and nothing more.

Sometimes false positives are attributable to ingested substancessuch as asthma or allergy medication [43] . Some authors havesuggested that employees subject to drug screening should refrain

from using certain brands of over-the-counter medication becausethey have caused false positives [44] . Some natural substancessuch as herbal teas and poppy seeds can also give positiveresponses to screens., These may be analytically true positives butthey need to be distinguished from those due to illegal drug use. Insome instances, false positives have been the result of mistakes orsabotage in the chain of custody for urine samples.

VII. Common adulteration methods

Substituting "clean" or drug-negative urine for drug-positive urine isthe most common way to fool the drug-screening system. A number


18/35

of entrepreneurs have attempted to bypass urine sample inspectionin this manner. A company in Florida sells lyophilized (freeze-dried)"clean" urine samples through newspaper and magazineadvertisements. Hiding condoms containing "clean" urine on thebody or inside the vagina is another common trick. Recently, a

patient at an addiction research clinic was, caught substituting"clean" urine when a glass bottle that had fallen into the toilet bowlwas discovered by the supervising nurse. It was later discoveredthat the bottle had been sealed with a thin aluminium wrap and hadbeen inserted into the patient's vagina.

Others have attempted to substitute apple juice or tea for urinesamples. Persons have been known to add to urine samples varioushouse - hold products, ranging from bleach to liquid soap to eye-drops, hoping that their drug use would be masked. Others have

hidden a masking substance under their fingernails and released itinto the urine specimen. Another method is to poke a small holeinto the urine sample container with a pin so that the sample leaksout by the time it reaches the laboratory [45] .

Since adding table salt (NaCl) or bleach to urine samples is acommon practice, many laboratories routinely test for sodium andchlorine in urine samples. Liquid soap and crystalline drain cleaners,strong alkaline products containing sodium hydroxide (NaOH), arealso used to adulterate urine samples. These contaminators can be

detected by checking for high pH levels in urine samples. In vivoalkalizing or acidifying the urine pH can also change the excretionpattern of some drugs, including amphetamines, barbiturates andPCP.

Water-loading (drinking large amounts of water prior to voiding)poses an interesting challenge to testing laboratories. Specificgravity has been used to detect dilution; however, themeasurement range is limited. Creatinine levels in random urinesamples have also been studied as a possible water-loading

detection method, but without much success.

Drug-using patients are resourceful and their ingenuity should notbe underestimated. In order to reduce the opportunities forspecimen contamination, some workplaces require that employeesprovide urine samples under direct supervision. Another way todetect any sample adulteration is to take the temperature of thesample. When the temperature of samples are taken within oneminute of voiding, the temperature range falls to between36.5deg; and 34deg; C, reflecting the body core temperature. Itis difficult to achieve this narrow temperature range by hiding acondom filled with urine in the armpit or adding water from a tap ortoilet bowl to the urine sample. The temperature of the sample


19/35

must be measured immediately after it is taken, since thetemperature drops rapidly.

VIII. Laboratory procedural and security standards

It is important that the laboratory drug testing facility has qualifiedindividuals who follow a specific set of laboratory procedures andmeet certain security standards. Laboratory management personnelmust have documented qualifications in analytical forensictoxicology in order to analyse urine samples for the presence ofdrugs [46] . A flow chart showing the various steps in the drug-testing process, from sample collection to the final disposition of theresults, is provided in figure I. Figure II shows the space, staffingand equipment requirements.

The laboratory should be secure at all times and access should belimited to authorized individuals only. The laboratory shouldestablish security measures to guarantee that specimens areproperly received, documented, processed and stored.Documentation of chain-of-custody procedures should includespecimen receipt, results during storage and final disposition ofspecimens. The laboratory must comply with any governmentallicence requirements, must be inspected routinely, must keepappropriate documentation and procedural manuals and must useproperly certified equipment.

Urine specimens should be inspected immediately upon arrival atthe laboratory in order to ensure that they have -not beentampered with during delivery. Specimens should be stored in asecure refrigeration unit if they are not tested within seven days ofarrival at the laboratory. The storage temperature should notexceed 6deg; C. Long-term storage must be at -20deg; C toensure that positive urine specimens will be available for anyretesting during administrative or disciplinary proceedings. Thelaboratory will be required to maintain any specimen under legal

challenge for an indefinite period.

Figure I. Schematic representation of the drug-testingprocess

Figure II. Space and staffing requirements of the drug-testing process
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f46#f46http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f46#f46http://www.unodc.org/images/odccp/bulletin/bulletin_1993-01-01_2_page005_img001_large.gif


20/35

IX. Initial and confirmatory testing requirements

The initial test (i.e. screening test) consists of an immunoassay

technique that meets the requirement for commercial distributionand eliminates "negative" urine specimens from furtherconsideration. Minimal (cut-off) levels should be used whenscreening a specimen to determine whether it is negative. Somenegative cut-off levels (in nanograms per millilitre) for initial tests

are as follows [13] :

The initial test (i.e. screening test) consists of animmunoassay technique that meets the requirement forcommercial distribution and eliminates "negative" urinespecimens from further consideration. Minimal (cut-off)levels should be used when screening a specimen to deter-mine whether it is negative. Some negative cut-off levels (innanograms per millilitre) for initial tests are as follows [13]:

Tetrahydrocannabinol metabolite 100

Cocaine metabolite 300

Opiate metabolites300a/

PCP 25

Amphetamines 1,000

Alcohol (mg/100ml) 10

a/ If immunoassay is specific for free morphine, 25nanograms per millilitre.

In the event of an identified positive, on the initial test, aconfirmatory test should be performed, whereby a second anddifferent analytical procedure is used to identify the presence of aspecific drug or metabolite. At present, GC/MS is the recommendedconfirmation method. Some minimal (cut-off) concentrations (innanograms per mililitre) for confirmatory tests are as follows [13] :

In the event of an identified positive, on the initial test, aconfirmatory test should be performed, whereby a second
http://www.unodc.org/images/odccp/bulletin/bulletin_1993-01-01_2_page005_img002_large.gifhttp://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/images/odccp/bulletin/bulletin_1993-01-01_2_page005_img002_large.gif


21/35

and different analytical procedure is used to identify thepresence of a specific drug or metabolite. At present, GC/MSis the recommended confirmation method. Some minimal(cut-off) concentrations (in nanograms per mililitre) forconfirmatory tests are as follows [13]:

Tetrahydrocannabinol metabolites a/ 15

Cocaine metabolites b/ 150

Opiates

Morphine 300

Codeine 300

PCP 25

AmphetaminesAmphetamine 500

Methamphetamine 500

Alcohol (mg/100 ml) 10

a/ For exampl e, 11- nor - ?- 9t er ahydr ocannabi nol i c aci d.

b/ Benzoyl ecgoni ne.

X. Regulations for drug testing *

The purpose of the regulations is to detail the standards andprocedures of laboratories that plan to provide drug testing for thework-place. There are two main parts to the present section. Thefirst part describes the requirements for the site collection of urinesamples and the second documents the essentials of laboratory

drug testing [47] .

Extreme caution must be exercised in the testing procedures. Usingspecimens for other types of analysis such as pregnancy or otherdisease criteria is expressly prohibited. In addition, the possibleimpact of a positive result on an individual's livelihood or rights,together with the possibility of a legal challenge of the results, setsthis type of testing apart from most clinical laboratory analysis.Urine drug testing should be considered a special application ofanalytical forensic toxicology. That is, in addition to the application

of appropriate analytical techniques, the specimens must be treated
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#n06#n06http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#n07#n07http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#nu05#nu05http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f47#f47http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f47#f47http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#nu05#nu05http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#n07#n07http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#n06#n06


22/35

as evidence and all aspects of the testing procedure should bedocumented and available for examination.

A. Specimen collection procedures

The purpose of the present subsection is to outline the proceduresused to ensure integrity of the specimens during urine collectionand during their transportation to a laboratory. A proper urinespecimen collection is the key to developing a successful testingprogramme.

The first concern of specimen collection is designating a specimencollection site. The collection site must have all the personnel,material, equipment and supervision necessary to allow the sampleto be collected, temporarily stored and transported to the drug-

testing facility. Collection site personnel must have successfullycompleted training to carry out these functions.

A second concern is ensuring the security of the collection site. Ifthe collection site facility is used solely for urine collection, it shouldbe secured at all times and accessible only to authorized persons.When urine sample specimens are being collected or stored, nounauthorized individual should be permitted in any part of thedesignated collection site unless he or she is accompanied by anauthorized person.

*The pr otocol s and pr ocedur es i n t he pr esent sect i on are based on t heaccumul at ed knowl edge of t he aut hor i n hi s capaci t y as I nspect or f ort he Col l ege of Amer i can Pat hol ogi st s ( CAP), a cer t i f yi ng agency f ordr ug- t est i ng l abor at or i es i n t he Uni t ed St at es. I n addi t i on,gui del i nes suggest ed by Kwong and other s [ 46] , CAP [47] , NI DA [ 13]and the Canadi an l aborat or y st andards f or dr ug scr eeni ng i n thewor kpl ace were extensi vel y consul t ed.

Procedures for collecting urine samples should allow for individualprivacy unless there is reason to believe that a particular individualmay alter or substitute a sample to be provided.

1. Minimal procedures to ensure integrity and identity ofspecimens

The following minimal procedures should be followed to ensure theintegrity and identity of the specimen at the collection site, to helpto safeguard against specimen adulteration or dilution during thecollection procedure and to allow for proper identification of theindividual being tested:

1. Immediately after the specimen is collected, the collection siteperson should also inspect the specimen to determine its
http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f46#f46http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f47#f47http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f13#f13http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f47#f47http://www.unodc.org/unodc/bulletin/bulletin_1993-01-01_2_page005.html#f46#f46


23/35

colour and should took for any signs of contaminants. Anyunusual findings should be noted in a permanent record orlogbook;

2. All specimens suspected of being adulterated should beforwarded to the laboratory for testing;

3. Both the individual being tested and the collection site personshould keep the specimen in view at all times prior to it beingsealed and labelled. If the specimen is transferred to a secondbottle, the collection site person should request the individualbeing tested to observe the transfer of the specimen and theplacement of a tamper-proof seal over the bottle cap anddown the side of the bottle;

4. The collection site person should securely place anidentification label on the bottle that contains the date, theindividual specimen number and any other identifying

information provided or required- by the laboratory;5. The individual being tested should identify the label on the

specimen bottle to certify that the specimen was. actuallycollected from him or her;

6. The collection site, person should, enter into the permanentrecord or logbook all information identifying the specimen.The collection site person should then sign the permanentrecord or logbook next to the identifying information. Anindividual being tested should be asked to read and sign astatement in the permanent record or logbook certifying that

the specimen identified was collected from him or her;7. The collection site person should complete the chain-of-

custody form;8. If the specimen is not immediately prepared for shipment, it

should be appropriately safeguarded during temporarystorage in a refrigerator at a temperature of about 4deg; C.The specimen should normally be shipped within 48 hours anddefinitely no longer than one week after it has been placed instorage. If stored longer, the temperature should be -20deg;C;

9.In the event that a collection site is not accessible and there isimmediate requirement for specimen collection (e.g. anaccident investigation), a public rest room may be usedaccording to the following procedures:

10. A collection site person of the same gender as theindividual being tested may accompany the individual into thepublic rest room, which shall be made secure during thecollection procedure;

11. If possible, a toilet bluing agent should be placed in thetoilet bowl and any accessible toilet tank to deter specimendilution. If no bluing agent is available, the collection siteperson should instruct the individual; being tested not to flush


24/35

the toilet until the specimen is, delivered to the collection siteperson;

12. It is essential that the urine specimen and the custodydocument be under the control of the collection site person. Ifthe collection site person leaves the workstation for a short

period, the specimen and the chain-of-custody form should betaken with him or her or should be secured. After thecollection site person returns to the workstation the custodyprocess should continue. If the collection site person leavesthe site for an extended period of time, the specimen shouldbe packaged for transportation beforehand;

13. Collection control site personnel should keep thespecimen bottle within sight both before and after theindividual has urinated. After the specimen is collected, itshould be properly sealed and labelled and an approved

chain-of-custody form should be used to control and accountfor each specimen from the point of collection to finaldisposition of the specimen;

14. The date and purpose should be documented on anapproved chain-of-custody form each time a specimen ishandled or transferred and every individual in the chain ofcustody should be identified. Every effort should be made toreduce to a minimum the number of persons handling thespecimen;

15. Collection site personnel should arrange to ship thecollected specimen to the drug -testing laboratory (see thesubsection below entitled "Guidelines for transportingspecimens"). Specimens should be placed in a containerdesigned to keep to a minimum the possibility of damageduring shipment. Specimen boxes or padded mailers, forexample, should be securely Scaled to eliminate thepossibility of undetected tampering. On the tape used to sealthe container, the collection site supervisor should place his orher signature and enter the date on which the specimen isbeing scaled for transportation. Collection site personnel

should ensure that the chain-of-custody document is-attachedto each container and is being sealed for shipment to thedrug-testing laboratory.

2. Procedures to maintain the integrity of specimens

The following procedures are recommended to ensure thatunadulterated specimens are obtained:

1. An individual being tested should be instructed to wash anddry 'his or her hands prior to urination. After washing his or

her hands, the individual being tested should remain in thepresence of the collection site person and should not have


25/35

access to any water fountains, faucets, soap dispensers,cleaning agents or any other material that could be used toadulterates the specimen;

2. The collection site person should note any unusual behaviouror appearance of the individual being tested in the permanent

record or logbook;3. To deter: dilution of the specimen at the collection site, toilet

bluing agents should be placed in toilet tanks wheneverpossible so that the reservoir of water in the toilet bowlalways remains blue. Ideally, there should lie no source ofrunning water (i.e. no sink in the room where the urine iscollected);

4. The individual giving the urine sample should be requested toremove any unnecessary outer garments, such as a coat or

jacket or hand- bag, that might conceal items or substances

that could be used to tamper with or adulterate the urinesample;

5. Upon receiving the sample from the individual, the personsupervising the collection should determine whether itcontains a minimum of 60 ml of urine. If there is less than 60ml of urine in the container, then an additional urine sampleshould be collected in a separate container. The individualbeing tested may be given a reasonable amount of liquid todrink for this purpose;

6. After the specimen has been submitted to the personsupervising the collection, the individual being tested shouldbe allowed to wash his or her hands;

7. Immediately after the specimen is collected, the personsupervising the collection shall measure and record thetemperature of the specimen. The time from urination totemperature measurement is critical and in no case shouldexceed four minutes. Normal temperature ranges from32.5deg; to 37.7deg; C (90.5deg;-99.8deg; F). (In theauthor's laboratory, temperatures of 100 consecutive urinesamples ranged between 34.5deg; and 36.5deg; C when

measured within two minutes of voiding.);8. If the temperature of the specimen is outside the range of32.5deg;-37.7deg; C (90.5deg;-99.8deg; F) or the pH isnot between 4.8 and 8, there is reason to believe that theindividual being tested may have altered or substituted thespecimen and another specimen should be collected underdirect observation by the collection site person. Bothspecimens should be identified and forwarded, withappropriate comments, to the laboratory for testing. Theindividual being tested may volunteer to have his or her bodytemperature taken to refute the suggestion that the specimenwas altered or substituted. Diluted urine can also be detected


26/35

by its pale colour or by its smell if adulterants such asammonia or furniture polish have been added;

9. Immediately after the specimen is collected, the collection siteperson should also inspect the specimen to determine itscolour and to look for any signs of contaminants. Any unusual

findings should be recorded in the permanent record orlogbook;

10. All specimens suspected of adulteration should beforwarded, with appropriate comments, to the laboratory fortesting;

11. It is important that the sample container is properlylabelled before it is given to the individual being tested.Ideally, the scaling of the specimen container should bewitnessed by the individual being tested.

3. Guidelines for transporting specimens

To maintain effective control while transporting the urine sample tothe testing laboratory, the following guidelines for transportingspecimens should be kept in mind:

1. All containers (i.e. plastic urine bottles) must be checked forcracks before filling, and only intact containers should beused;

2. Chain-of-custody forms and containers must be clearlylabelled;3. Appropriate protective equipment must be worn to maintainbody substance precaution techniques during retrieval of allspecimens;

4. All specimen caps or lids should be checked beforetransporting to see if they are securely closed;

5. Specimen containers should be placed in the designatedtransportation container;

6. Specimens should always be handled carefully to avoidbreakage.

B. Laboratory procedures

Laboratory personnel requirements

Laboratory management personnel must have documentedqualifications in analytical forensic toxicology in order to carry outthe analysis of urine samples for drug testing. They must be able toreview data and quality-control results, to supervise the routineoperations of drug testing, to train personnel and to keep files onpersonnel.


27/35

The laboratory should have a qualified director to assume theprofessional, organizational, educational and administrativeresponsibilities for its urine drug-testing facility. That individualshould have documented scientific qualifications in analyticalforensic toxicology. The minimum qualifications are as follows:

1. A Doctor of Philosophy (Ph.D.) in one of the natural sciencesand an adequate undergraduate and graduate education inbiology, chemistry and pharmacology or toxicology; ortraining and experience comparable to a Ph.D. in one of thenatural sciences such as a medical or scientific degree withadditional training and laboratory research experience inbiology, chemistry and pharmacology or toxicology;

2. Appropriate experience in analytical forensic toxicology,including experience in the analysis of biological materials for

drugs of abuse;3. Appropriate training and or experience in forensic applications

of analytical toxicology (e.g. publications, court testimony,research concerning analytical toxicology of drugs of abuse orother factors that qualify an individual as an expert witness inforensic toxicology).

In jurisdictions where certification as a laboratory director isrequired, the director should be certified or be eligible to apply forthat certificate. The director will be responsible for certifying the

final test results being reported.

The laboratory urine drug-testing facility should have qualifiedindividuals who review all pertinent data and quality- control resultsin order to attest to the validity and certification of the laboratorytest reports. The laboratory may designate more than one person toperform this function. Such individuals may be any employees whoare qualified to be responsible for the day-to-day management oroperation of the drug- testing laboratory. Individuals responsible forthe day-to-day operations and supervision of the technical analysis

should at least have Bachelor's degrees in chemical or biologicalsciences or medical technology or the equivalent. They should havetraining and experience in the theory and practice of proceduresused in the laboratory and should thoroughly understand quality-control practices and procedures. They will be responsible forreviewing, interpreting and reporting test results and formaintenance of the chain of custody. Additionally, these individualsshould ensure that proper remedial actions are taken in response totest systems that are out of control limits or when erroneous resultsare detected.

Other technical and non-technical staff should have the necessarytraining and skills for the tasks that they will be assigned. The


28/35

laboratory programme should make continuing educationprogrammes available to meet the needs of the laboratory,professionals., The personnel file of such an individual shouldinclude the following:

1. A rsum or documentation of training and experience;2. Certification and licence, if any;3. References;4. Job descriptions;5. A record of performance evaluation and advancement;6. Incident reports and results of tests that establish employee

competence and advancement.

2. Laboratory security and chain of custody

The laboratory should be secure at all times. Sufficient securitymeasures should be in place to control access to the premises andto ensure that no unauthorized personnel handle specimens or gainaccess to the laboratory processes or the record or log storageareas. Access to these secured areas should be limited tospecifically authorized and documented individuals. With theexception of personnel authorized to conduct inspections, all visitorsand maintenance service personnel should be escorted at all times.Records of individuals entering these areas and the date, time andpurpose of entry must also be maintained. Documentation of chain -

of -custody procedures should include specimen receipt, resultsduring storage and final disposition of specimen. The date andpurpose should also be documented on an appropriate chain-of-custody form each time the specimen is handled or transferred, andevery individual in the chain should be identified. Accordingly,authorized technologists should be responsible for each urinespecimen in their possession and must sign a complete chain-of-custody form when such specimens are received.

The drug-testing laboratory must have security measures toguarantee that specimens are properly received, documented,processed and stored. The laboratory must also comply with anygovernmental licensing requirements,. must be inspected routinely,must keep appropriate documentation and procedure manuals andmust use properly certified equipment.

3. Receiving specimens

When a shipment or specimen is received, the laboratory personnelshould inspect each package for evidence of tampering and shouldcompare information on specimen bottles with each package of

information accompanying the chain-of-custody forms. Any directevidence of tampering or discrepancy between the information on


29/35

the specimen bottle and the accompanying chain-of-custody formshould be recorded on the chain-of-custody form and accompanythe specimen while it is in the laboratory. Specimen: bottles shouldnormally be retained within the laboratory area until all analyseshave been completed.

4. Storing specimens

Specimens that do not receive an initial test within seven days ofarrival at the laboratory must be placed in a secure refrigerationunit. The temperature must not exceed 6deg; C. Emergencyelectrical power equipment should. be available in case of prolongedpower failure.

Long-term storage must be at a temperature of 20deg; C-to

ensure that positive urine specimens are available for any retestingduring administrative or disciplinary proceedings. Unless otherwiseauthorized in writing, the laboratory must retain and place inproperly secured long- term storage all specimens confirmedpositive for a minimum of one year. Within this one-year period, theclient may request the laboratory to retain the specimen for anadditional period of time. If no such request is received thelaboratory should discard the specimen after one year. Thelaboratory will be required to maintain any specimen under legalchallenge for an indefinite period.

5. Initial and confirmatory testing

Specimens can be processed in batches for either initial orconfirmatory toasts. When conducting either initial or confirmatorytests, every batch should contain an appropriate number ofstandards for calibrating the instrument and a minimum of twospecimen controls or 10 per cent specimen controls, whichever ishigher. Both quality-control and blind performance test samples willappear as ordinary samples to the laboratory analyst.

The initial test (screening test) involves an immunoassay techniquethat meets the requirement for commercial distribution andeliminates a "negative" urine specimen from further consideration.The initial cut-off levels (see the section above entitled "Initial andconfirmatory testing requirements') should be used when screeninga specimen to determine whether it is negative for the drugs orclasses of drugs being tested. For alcohol, the initial test may be anenzymatic test based on the ADH system.

In the event of an identified positive on the initial test, a

confirmatory test in which a second analytical procedure is used toidentify the presence of a specific drug or metabolite must be


30/35

performed. The confirmatory test method must use a differenttechnique and chemical principle from that of the initial test. Atpresent, GC/MS is the recommended confirmation method forreasonable analytical accuracy of cocaine, marijuana, opiates,amphetamines and PCP. In the case of alcohol, confirmation must

be done by using a gas chromatographic procedure. The valuesobtained by the initial test and the confirmatory test for alcoholmust be within 10 per cent of each other. All confirmations mustinclude quantitative analysis. Concentrations that exceed the linearregion of the standard curve should be documented in thelaboratory records as greater than the highest standard curvevalue.

The results of retesting of specimens (being challenged) mustconfirm the presence of the drug or metabolite in question but are

not required to correspond to any specific cut-off level since someanalyte may deteriorate during the freezing, thawing or storageprocedures.

6. Operational guidelines

(a) Laboratory facilities

The laboratory must comply with provisions of the localgovernmental licensing requirements. In order to attaincertification, the laboratory must be capable of performing both the

initial and the confirmatory tests for each drug or metabolite forwhich the service is being offered. Urine specimens collected for thepurpose of drug testing should only be used to test for those drugsincluded in their clients' approved drug- free workplace plan andmay not be used to conduct any other analysis or tests.

(b) Inspection

A certification body or any client utilizing the laboratory mayreserve the right to inspect the laboratory at any time. Anyemployer who has contracted the laboratory for drug testing orcollection site services is permitted to conduct unannouncedinspections. In addition, prior to awarding a contract, a client maycarry out pre-award inspections and evaluations of the proceduralaspects of the laboratory drug-testing operation.

(c) Subcontracting

Unless authorized to do so, the drug-testing laboratory may notsubcontract and should perform all, work with its own personneland equipment unless otherwise authorized by the client. If

subcontracting is authorized, then the subcontracted laboratorymust be capable of performing tests for the agreed upon classes of


31/35

drugs. Same-site screening and confirmation is ideal for themaintenance of strict quality control in the chain of custody and intransporting and processing samples, as well as in reporting results.The subcontracting laboratory must adhere to the guidelines statedin the present paper.

(d) Information collection requirement

The drug-testing laboratory must maintain and make available forat least two years documentation of all aspects of the testingprocess. The required documentation must include the following:

1. Personnel files on all individuals authorized to have access tothe specimen;

2. Chain-of -custody documents;3. Quality-assurance and quality-control records;4. Operating procedure manuals according to which the tests

were performed;5. All test results including calibration curves and any

calculations used in determining the test results;6. Reports;7. Performance records on performance testing;8. Performance on certification inspections;9. Hard copies of any computer-generated results.

(e) Procedure manuals

The laboratory procedure manual must include the following:

1. The principle of each test;2. Preparation of reagents;3. Standards and controls;4. Equipment set-up and calibration procedures;5. Schedule for critical operational checks on equipment;6. Derivation of results;7. Linearity of methods;8. Sensitivity of the methods;9. Cut-off values;10. Mechanism of reporting results;11. Controls;12. Criteria for unacceptable specimens and results;13. Remedial actions to be taken if test systems are outside

acceptable limits;14. Reagents and expiration dates;15. References.

Copies of all procedures and dates on which they are in effectshould be maintained as part of the laboratory procedure manual


32/35

under the signatory approval of the director of the drug-testinglaboratory.

(f) Standards and controls

Laboratory standards should be prepared with pure drug standardsthat are properly labelled, indicating their content and purity. Thestandards are to be labelled with the following dates:

1. Date received;2. Date opened or prepared;3. Expiration date.

When reagents are prepared, the date and the initials of the personpreparing them should be recorded. Control samples should be

labelled with a code allowing the analysts to test them in a blindmanner. The temperature of the refrigerator used for storing thereagents should be checked and recorded daily. The results forcontrols and standards must be recorded along with the date, timeand name of the analyst conducting the test procedure.

(g) Instruments and equipment

The laboratory, supervisor should oversee the day-to-daymaintenance of the laboratory equipment. Volumetric pipettes andother measuring devices must be certified for accuracy or checked

by gravimetric, colorimetric or other verification procedures.Automatic pipettes and diluters should be checked for accuracy andreproducibility before being placed in service and should be checkedmonthly thereafter. The procedure manual should include writtenprocedures for instrument setup, calibration, critical operatingcharacteristics, tolerance limits for acceptable function checks andinstructions for major trouble-shooting and repair. Records mustalso be available on preventative maintenance for the instruments.

(h) Reporting of results.

The laboratory should report results to the medical review officerwithin an average of 10 working days after receipt of the specimen.Before a test result is reported, it should be reviewed and test-certified as an accurate report by the individual responsible for thisactivity. The report should identify the drug metabolites tested for(whether positive or negative), cut-off levels, the specimen numberassigned by the test- requesting facility and the drug-testinglaboratory specimen identification number. The results, whetherpositive or negative, should be reported back to the medical review

officer, at the same time. The laboratory should report as negativeall specimens that are negative on the initial test or negative on the


33/35

confirmatory test. Only specimens confirmed positive should bereported positive for a specific drug. The laboratory must providequantitative. test results at the request of the medical reviewofficer.

(i) Quality,-assurance and quality-control procedures

The drug-testing laboratory have a quality-assurance and quality-control programme that encompasses all aspects of the testingprocess, from specimen collection to reporting of the results and thefinal disposition of the specimen. Quality-assurance proceduresshould be designed, implemented and reviewed to monitor eachstep of the process.

The quality-control requirements for initial tests should be

structured so that each analytical run of specimen to be screenedincludes:

1. A urine specimen certified to contain no drug;2. A urine specimen fortified ("spiked") with known standards;3. Positive controls with a drug or metabolite at or near the

threshold or cut-off level.

In addition, with each batch of samples, a sufficient number ofstandards should be included to document the linearity of the assaymethod over time in the concentration range of the cut-off. Aminimum of 10 per cent of test samples should be used as quality-control samples.

The quality-control requirements for confirmatory tests should bestructured so that each analytical run of specimen to be confirmedincludes:

1. A urine specimen containing no drugs;2. A urine specimen fortified ("spiked") with known standards;3. A positive and positive controls with a drug or metabolite at or

near the threshold or cut-off level.

Linearity and precision of the method must be periodicallydocumented. Implementation of procedures to ensure that carry-over does not contaminate the testing of the individual specimenshould also be documented.

7. Review of results

(a) Medical review officer

The medical review officer should be a licensed physician with aknowledge of substance abuse disorders. A positive result does not


34/35

auto- matically identify an individual as an illegal drug user. Therole of the medical review officer is to review, verify and interpretpositive test results obtained through the client's drug- testingprogramme. In carrying out his or her responsibilities, the medicalreview officer should examine alternate medical explanations for a

positive test result by, for example, conducting a medical interviewwith the individual, reviewing the individual's medical history anddetermining the clinical evidence of illegal use of any opiate oropium derivatives. The medical review officer should review allmedical records available on the tested individual to ascertainwhether or not a confirmed positive could be explained by the useof a legally prescribed medication. Prior to making a final decision toverify a positive result, the medical review officer should give theindividual an opportunity to discuss the test results with him or her.If the medical review officer determines that there is a legitimate

medical explanation for the positive test, the result is consistentwith the legal drug use and no further action should be taken.

Should any question arise as to the accuracy or validity of a positivetest result, the medical review officer is authorized to order areanalysis of the original sample. Additionally, based on the reviewand inspection of reports, quality-control data, multiple samples andother pertinent results, the medical review officer may determinethat the result is scientifically insufficient for further action anddeclare the test specimen negative. The director of the laboratory

should be available to consult with the medical review officer asrequired. The employee being tested has a right to identify aprescribed medication when the urine sample is being provided.

(b) Protection of employee records

All laboratory contracts should require that the contractor complywith the privacy act. In addition, the laboratory contract shouldrequire compliance with the access and confidentiality provisionsestablished within the jurisdiction where the laboratory practice ismaintained. The contract and the privacy act should specificallyrequire that employee records be maintained and used with thehighest regard for employee privacy.

(c) Individual access to test results and laboratory certification results

Any employee who is subject to drug testing should, upon writtenrequest, have access to any records relating to his or her drug testand any records relating to the results of any relevant certificationreview proceedings.

8. Certification of laboratory


35/35

The laboratory must meet all pertinent provisions and guidelinesstated in the present paper. In determining whether to certify alaboratory or to accept the certification, the following criteria shouldbe considered:

1. The adequacy of the laboratory facilities;2. The expertise and experience of the laboratory personnel;3. The level of the laboratory's quality-assurance and quality-

control programme;4. The performance of the laboratory on annual performance

tests (once in place);5. The laboratories compliance with the standards as reflected in

any laboratory inspection;'6. Any other factors affecting there liability and accuracy of drug

tests and reporting done by the laboratory.

In order to remain certified the laboratory must participate in blinddrug-testing challenges initiated by the certifying body. During theprocess, 90 per cent of the number of tests performed must becorrectly identified (i.e. identification and confirmation of 90 percent of the total drug challenges) or suspension or revocation of thecertificate may take place

Documents

Drug testing methods.pdf