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Drug-testing methods and clinical interpretations of test results

Article  in  Bulletin on Narcotics · February 1993

DOI: 10.1007/978-1-4615-2399-4_5 · Source: PubMed

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Drug-testing methods and clinical interpretations of
test 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 are
discussed. For example, drug-testing techniques measure the
presence of a drug but are not sophisticated enough to measure
impairment from drug use. Moreover, it is difficult to determine the
route of drug administration, quantity or frequency, as well as when
the drug was taken, on the basis of the laboratory results.

Selection of the drug to be tested should depend on the local

availability of the drug, its abuse potential and clinical effects, as
well as on the availability of analytical technology and expertise in
testing and in interpreting laboratory results. The most
sophisticated drug-testing approach is gas chromatography coupled
with mass spectrometry (GC/MS), which is regarded as a "gold
standard"; it is used in confiramtory testing. Typically, GC/MS is
preceded by a rapid immunoassay method to eliminate the majority
of the "negative" samples.

Despite the existence of sophisticated drug-testing methods, it is

still possible to obtaining incorrect test results and to detect
adulterated urine samples.

A "positive" drug finding can have a serious impact on the livelihood

of an individual, therefore, persons conducting such tests should
adhere to the strictest standards of laboratory performance. Only
qualified and experienced individuals with proper laboratory
equipment should perform these analyses. The standards of
laboratory performance ,must meet local legal and forensic
requirements. Access to patient samples and laboratory records
must be restricted in order to prevent the tampering of samples and
results. In order to maintain confidentiality, the results must be
communicated only to the medical review officer. Chain-of-custody
documents and all file so that they can be examined in case of a
legal challenge. The laboratory must have a complete record on
quality control. Finally, specific initial and confirmatory testing
requirements should be met.

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

I. Drug properties: absorption, distribution and elimination

phases
Detection of a drug depends largely on its absorption, distribution
and elimination properties. There are various routes of drug
administration; oral drinking, e.g. alcohol), intravenous (injecting
into a vein, e.g. heroin) and inhalation (smoking, e.g. marijuana;
snorting, e.g. cocaine; and sniffing, e.g. glue). Drugs taken orally
are usually the slowest to be absorbed (i.e. by the brain and other
body organs), whereas the intravenous and inhalation routes result
in the fastest absorption. Once the drug enters the bloodstream it is
rapidly distributed to the various tissues in the body. The amount of
drug stored depends on the nature of the drug, the quantity, the
duration of ingestion, the tissue holding the drug and the frequency
of use.

Some drugs are fat-soluble and are deposited in fat tissues. For
example, 9-tetrahydrocannabinol (THC), the active ingredient in
marijuana, is highly fat-soluble, resulting in a rapid reduction in
levels of THC in the blood as the drug is distributed to the various
tissues [1] . Some studies have shown that THC levels peak and
start to decline in half the time it takes to smoke a marijuana
"joint". Concentrations are known to fall by almost 90 per cent in
the first hour [1] , [2] suggesting that a higher degree of
sophistication in laboratory analysis is needed to detect fat-soluble
drugs. 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 after
last use [3] . Ethanol or ethyl alcohol is the beverage alcohol that is
consumed 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
drugs like THC. The absorption and distribution phases are followed
by an elimination phase. The liver is the major detoxification centre
in the body; drugs are metabolized as blood circulates through the
organ. The metabolites are then excreted into the urine through the
kidneys. At the same time, drugs deposited in fat tissues are also
slowly released into the bloodstream and metabolized.

Drugs vary by their elimination half -lives, which is the time

required 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 clinical
status. A compromised liver and the concurrent presence of another
disease or drug have the potential of enhancing the toxic effects of
the drug by slowing down the elimination process. Under different
clinical conditions, however, the process may be speeded up.
Therefore, great variation may be found in the half -lives of the
same drug.

Approximately six half -lives are required to eliminate 99 per cent of

any drug. Because the half-life of cocaine is relatively short,
averaging one hour [5] , only six hours are needed for the
elimination of 99 per cent of the drug. Cocaine metabolites have a
longer half -life and can be detected for a considerably longer period
of 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 cent
of the drug. Since there is much variation in the half -lives of
different drugs and the absolute amount of drug present can be
very small, it is crucial that the appropriate body fluid for analysis is
selected for testing.

Elimination of ethanol follows a different pattern. Its levels decline

almost linearly over time. The average elimination rate is between
15 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 alcoholic
patient, the elimination rate is generally higher. In forensic
calculations, a rate of 15 mg/100 ml (0.015 per cent) per hour is
usually used.

Table 1.

Drug half-lives and approximate urine detection periods

Half-life Detection
Drug
a/ period
12-34
Methamphetamine 2-3 days
hours
Amphetamine (metabolite of
7-34
methamphetamine)
hours
60-90
Heroin Minutes
minutes
Morphine (metabolite of
1.3-6.7
heroin)
hours
7-16
Phencyclidine. (PCP) 2-3 days
hours
0.5-1.5
Cocaine A few hours
hours
Benzoyltegonine (metabolite
of cocaine) 5-7 hours 3-5 days
90 per cent
14-38
?-9-Tetrahydrocannabinol fall in 1 hour
hours
(blood)
Depending on
?-9-Tetrahydrocannabinolic
use, any-
where
acid (marijuana metabolite in between a
few days
and many
urine)
weeks
Blood
1.5-12 hours,
Alcohol (ethanol) levels fall
depending on
by
an the peak
average blood level;
of 15- urine
18 mg/ is typically
100 positive for
ml/hour an
additional 1-2
hours

Source-R. C. Baselt, Disposition of Toxic

Drugs and Chemicals in Man, 2nd ed.
(Davis, California, Biomedical Publications,
1982)..
a/ The detection period is dose-
dependent. The larger the dose, the
longer the period that the drug or,
metabolite can be detected in the urine.

II. Selection of drugs to be tested

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

A. Availability

Prescription patterns of psychoactive and other drugs vary from

place to place and from Country to Country [6] , [7] . The abuse of
the benzodiazepine nitrazepam is common in Europe but almost
unknown in Canada and the United States of America, as it is not
available on markets in North America. The psychoactive chemical
cathine is the active ingredient in the leaves of the khat (Catha
edulis) plant, which are chewed in north-eastern Africa; the practice
does not appear to have crossed over to the European or the North
American continent. In Canada, the narcotic drug codeine is
available as over-the-counter, non- prescription medication,
whereas in the United States it is available only with a physician's
prescription.

The wide availability of "legal" stimulants poses an interesting

problem since they are commonly found in accident victims. A study
carried out in the United States by. the National Transportation
Safety Board [8] from October 1987 to September 1988 showed
that over-the- counter stimulants - antihistamines.such as
ephedrine, pseudoephdrine and phenylpropanolamine - were,
commonly found in drivers killed in heavy truck accidents. Similar
results have also been obtained based on data from emergency
rooms 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 all

the amphetamine use was in California rather than in the other
seven states in the study, suggesting that drug use varies not only
from country to country, but also within a given country.

Thus, the selection of a drug to be tested and monitored that is

appropriate for one country or place may not necessarily be
appropriate for another.
B. Clinical effects

Drugs that manifest abuse potential and impair behavior to such an

extent that job performance may -be affected are prime candidates
for 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 livelihood

of the person being tested. Therefore-, special attention needs to be
paid to the testing methods used. Ideally,, the analytical method
should be specific for the drug being tested (i.e. no false positive)
and should be easy and inexpensive to use. Confirmation methods
should 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 carefully

considered as even a normal diet can sometimes result in a positive
drug identification. Poppy seed ingestion [12] can result in a true-
positive analytical report but it is a false positive for drug use. Some
ethnic diets may also lead to such confounding problems; for
example, food containing poppy seed is common during the
Ramadan period in India.

What should be analysed? Ideally, the parent drug, rather than its
metabolite should be looked for in the analysis, although this may
not always be possible as some drugs are rapidly metabolized (e.g.
heroin metabolism to morphine). The sensitivity of the analytical
procedure should be dictated by the psychoactive pharmacological
properties of the drugs. If the drug is shown to be devoid of abuse
potential, then its detection beyond the time of pharmacological
activity, although important in the clinical management of the
patient, does not necessarily serve a useful purpose in a workplace
drug-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 and
heroin). Rapid screening methods that allowed for 'mass screening'
were available at that time, as were the confirmation methods for
those five drugs. Mood-altering substances, such as
benzodiazepines, barbiturates and stimulants, such as
antihistamines, are at present excluded from the regulations in the
United States. This is probably because of the much wider
availability of those drugs as medication and the technological
requirements for screening and monitoring them.
III. Types of testing. blood, urine and hair specimens
Blood and urine are the most commonly used fluids in the analysis
for drugs. Blood, obtained by an invasive procedure, is available
only in small quantities and drug concentration levels in blood are
low. Urine is the preferred sample as it is available in larger volume,
contains the metabolite and requires less invasive procedures in its
collection. Both sampling procedures, however, have limitations as
they only determine the absolute amount of drug present in the
fluid being examined. That amount is dependent upon the amount
of the drug used, when it was used last, and the half-life of the
drug.

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 of
drug use. An advisory committee of the Society of Forensic
Toxicology [17] has recently concluded that, 'because of these
deficiencies, results of hair analysis alone do not constitute
sufficient evidence of drug use for application in the workplace'.

Breath and various body fluids, such as sweat, saliva, blood and
urine, have been used for alcohol analysis. Breath is commonly
used by law enforcement authorities for such analysis. Although a
number of variables [18] can affect the breath-blood ratio, the
2,100:1 alveolar breath-blood conversion ratio has been used and
accepted for the Breathalyser [19] . Breath-testing equipment
calibrated with a blood- breath conversion factor of 2,100
consistently underestimate actual blood alcohol concentrations [20]
. The results of breath analysis may vary depending on the
instruments used and on biological factors [21] , [22] . Potential
errors in breath analysis can also be caused by the presence of
residual alcohol in the mouth. Immediately after drinking, there is
enough alcohol vapour in the mouth to give artificially high
concentrations in breath analysis. Generally, this effect disappears
after 20minutes but high values for as long as 45 minutes have
been reported [23] .

All existing technologies are limited in terms of their capacity to

determine how much of a drug was consumed or when it was
consumed.

Blood and saliva concentrations reflect the current blood alcohol

concentration, but generally a blood sample is used in hospitals for
patients 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
blood alcohol concentration between voiding, has the potential of
being "positive" while the blood may be "negative".

IV. Measuring impairment

Except in alcohol analysis, the degree to which a person is
influenced or impaired by a drug at the time of the testing cannot
be determined from test results alone. Correlations between
positive blood levels and degree of impairment are usually stronger
than correlations between urine levels and degree of impairment;
however, neither blood nor urine tests are sufficiently accurate to
indicate impairment even at high levels of concentration [25] , [26]
, [27] . Human studies using marijuana and cocaine have shown
that a "perceived high" is reached after the drug concentration has
peaked in the blood [2] , [28] . Generally, blood can only show
positive results for a short time after drug consumption, whereas
urine can be positive for a few days or weeks after last use. For
example, metabolites of THC that are lipid-soluble can be detected
in the urine for a few days or for many weeks, depending on the
drug habit of the user [3] . Excretion of the drug in urine and its
concentrations are also effected by several factors, such as dilution
and acidity (pH) of the urine. The author has seen many cases
where a urine sample was strong positive for cannabinoids in the
morning, borderline positive in the afternoon and strong positive the
next morning. The author has made similar observations for
phenobarbital.

From a positive urine test, the form in which the drug was originally
taken or when and how much was taken cannot be determined. For
example, crack, impure cocaine powder or cocaine paste (which can
be smoked, inhaled, injected or chewed) all give the same result in
a urine test. The consumption of poppy seeds has been reported-to
give positive results for opiate use because some seeds contain
traces of opiates and some have been known to be contaminated
with opium derivatives [12] . Similarly, consumption of herbal cocoa
tea has resulted in positive results for cocaine use. These incidents
clearly illustrate the difficulties involved in measuring impairment
using urine test results.

The problem of interpreting urine test results is one of the main

arguments for restricting their use in the employment setting. In a
recent study [29] , the effectiveness of pre-employment drug-
screening tests has been questioned because of difficulties in
interpretation. Based on their analysis of 2,229 pre-employment
drug-screening tests and follow- up, Ryan, Zwerling and Jones [291
concluded: 'Our findings raise the possibility that a pre-employment
drug screening may be decreasingly effective in predicting adverse
outcomes associated with marijuana use after the first year of
employment.' They made a similar comment about cocaine.

There is no threshold for alcohol effects on performance or motor

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

"Legal" BAC levels differ in different countries. Some even have

more than one legal limit over which the driver of a vehicle is
considered "impaired". Some European countries have 50 mg/ 100,
ml (0.05 per cent) as their legal limit and others have 80 mg/ 100
ml (0.08 per cent). In the United States, the legal limit varies from
80 mg/ 100 ml (0.08 per cent) to 100 mg/100 ml (0.10 per cent) in
different states, but employees who are regulated by the United
States Department of Transportation have a BAC legal limit of 40
mg/ 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 (0.05-0.08
per cent) may result in suspension of driving privileges and levels
above 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] . The
methods most commonly used in toxicology laboratories are as
follows:

1. Immunoassay:
2. Enzyme immunoassay (EIA);
3. Enzyme-multiplied immunoassay technique .(EMIT);
4. Fluorescence polarization
5. 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).
The methods vary considerably with respect to -their sensitivity and
reliability. TLC is the least expensive and is reliable, GC/MS is
considered nearly perfect, or a "gold standard", [33] ; it requires
highly 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 be
detected. Antibodies specific to a particular drug are produced by
injecting laboratory animals with the drug. These antibodies are
then tagged with markers such as an enzyme (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 against
Which the antibody was made is present,, a reaction will occur. RIA
is the oldest immunoassay method used to detect drugs. The major
drawback of this method is that it requires a separation step and
generates radioactive waste. RIA also requires special equipment
for 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 detect
the 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 can
give random or sporadic false positives for cannabinoids. Codeine
will also give a positive reaction for the morphine (a metabolic
product of heroin use) immunoassay and many antihistamines that
are available over the counter may yield positive reactions for
amphetamines. While some reagent manufacturers claim to have
overcome many of these cross- reactivity problems, confirmation by
a non-immunoassay is important. Immunoassays are considered
good screening tests but not sufficient by themselves for a
conclusion to be drawn. A non-immunoassay method is required to
draw a conclusion.

Urine drug assay kits have been available in North America for the
past few years. More recently, single- and multiple-test
immunoassay kits designed for home and on-site testing have also
been introduced. Such kits generally carry a cautionary disclaimer
that positive test results must be confirmed by GC/MS, the
reference method. When used in a non-laboratory environment,
they are prone to procedural inaccuracies, poor quality control,
abuse and misinterpretations. Therefore, they are not
recommended for testing in the workplace. The risk of labelling a
person with a false positive is high without confirmatory analysis. In
addition, confirmation analysis is generally expensive when an
individual sample is being tested. The advantages and
disadvantages of immunoassay testing may be summarized as
follows.

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 positives
resulting from passive inhalation of marijuana smoke;
5. Immunoassays are relatively inexpensive, although the
single-test immunoassay kits can be expensive when quality-
assurance and quality-control samples are included;
6. Immunoassays do not require a specialized laboratory. Most
clinical laboratories have automated instruments to do the
procedures;
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 the
antibody reagents;
10. Results must be confirmed by another non.-
immunoassay method;-,,
11. A radioactive isotope is used in RIA that requires
compliance with special licensing procedures, use of gamma
counters to measure radioactivity and disposal of the
radioactive waste;
12. Only a single drug can be tested for at one time.

B. Chromatography

Separation of a mixture is the main outcome of the

chromatographic method. If a drop of ink is put on a blotting paper
and the tip of the paper is held in water, the water will rise in the
paper. After a period of time and under the right conditions, the
single ink spot will separate into many different compounds (spots)
of different colours (blue ink is a mixture of many dyes). This
process, whereby a mixture of substances is separated in a
stationary medium (filter paper), is called chromatography. The
types of chromatographic processes used in the analysis of drugs
.include thin-layer, gas, and liquid chromatography as well as a
combination of gas or liquid chromatography with mass
spectrometry.

1. Thin-layer chromatography

TLC is most similar to the ink separation example mentioned above.

This method requires extensive sample preparation and technical
expertise on the part of the analyst, but it is inexpensive and
powerful if used properly. With the exception of cannabis, which
requires separate sample preparation, a large number of drugs (e.g.
cocaine, amphetamine, codeine and morphine) can be screened at
the same time. By combining different

TLC, systems, a high degree of :specificity. can be obtained,

although the training of the analyst is crucial because of the
subjectivity involved in interpreting the results. To identify positive
TLC "spots", the technologist looks for the drugs and/or their
metabolite patterns. A trained technologist can identify more than
40 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 an
oven, is flushed with a carrier gas such as helium or nitrogen. In a
GC system that has been properly set up, a mixture of substances
introduced into the carrier gas is volatilized and the individual
components of the mixture migrate through the column at different
speeds. Detection takes place at the end of the heated column. and
is generally a destructive process. Often, the substance to be
analysed is "derivatized" to make it volatile or to change its
chromatographic 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 used
to flush the column. Thus, this technology is sometimes referred to
as high - pressure liquid chromatography. Typically, the column
operates at room temperature or slightly above room temperature.
This method is generally used for substances that are difficult to
volatilize (e.g. steroids) or are heat-labile (e.g. benzodiazepines).
The two major differences between GC and HPLC are as follows:
 1. GC is a "destructive" method (it destroys or burns the
chemical in its detector to generate the signal), whereas HPLC
detection takes advantage of the electronic or chemical
structure 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 generally
less sensitive than GC.

C. Gas chromatography coupled with mass spectrometry

GC/MS is a combination of the two sophisticated technologies. GC

physically 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 most
authorities as the "gold standard'. This combination is sensitive (it
can detect low levels), specific and able to identify all types of drugs
in any body fluid. Furthermore, assay sensitivity can be enhanced
by treating the test sub- stance with reagents. When coupled with
MS, HPLC/MS is the method of choice for substances that are
difficult to volatilize (e.g. steroids).

Given the higher costs associated with GC/MS, urine samples are
usually tested in batches for broad classes of drugs by
immunoassay, and positive screens are later subjected to
confirmation by this more expensive technique. This is the most
common approach used in employment drug- screening
programmes and is the combination recommended by NIDA [13] in
the United States.

The advantages and disadvantages of the various methods of

chromatographic drug testing are as follows:

1. Advantages:
2. All chromatographic methods: All the chromatographic
methods are specific and sensitive and can screen a large
number 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 easiest

sample preparation requirements; b. The procedure can be
automated;

6. GC/MS:
 a. It is regarded as the "gold standard" test; b. Computerized
identification of fingerprint patterns makes identification easy;
c. The procedure can be automated; d. It is currently the
preferred 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 and
experience in interpretation capabilities of the technologist are
crucial;
10. GC or HPLC: Equipment costs are high, ranging between
US$ 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 of
sophistication required; b. Due to the complexity of the
instrument, highly trained operators and technologists are
required.

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

Table 2. Comparison of all drug testing methods

EMIT GC
GC/M
Aspect and RIA TLC and
S
FPIA HPLC
Ease of sample
x x x
preparation
Less highly
trained x x
technologists
required
Limited
equipment x x x
required
Low detection
x x x x x
limits
Adjustable lower
x x
threshold
Highly specific
x x x
and sensitive
Computerized
x
identification
possible
Screen for
x x x
several drugs at
a time
Procedure can
x x x x
be automated
Special atomic
x
energy licence
required
Confirmation of
x x x x
results
required
Interpretation is
x
subjective
enzyme-multiplied
Notes: EMIT = immunoassay
technique
fluorescence
FPIA = polarization
immunoassay
GC = gas chromatography
gas chromatography
GCIMS = coupled with mass
spectrometry
high-performance
HPLC =
liquid chromatography
RIA = radioimmunoassay
thin-layer
TLC =
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 and
modifications have been introduced. The distillation/oxidation
methods are generally non-specific for ethanol [36] , [37] , whereas
biochemical (spectrophotometric) methods using alcohol
dehydrogenase (ADH) obtained from yeast [38] and the gas
chromatographic [39] method that are currently in use are specific
for ethanol. The radiative energy attenuation technique [40] and
those using the alcohol oxidase method are non-specific and will
detect not only ethanol but also other alcohols. The recently
introduced alcohol dipstick [41] , based on the ADH enzyme
system, is not only specific for ethanol, but also sensitive and does
not require instrumentation. It can be used for the detection of
ethanol in all body fluids and can provide semi-quantitative results
in ranges of pharmacological-toxicological interest. The alcohol
dipsticks are being used in many alcohol treatment programmes as
well as in a number of laboratories as a screening device.

Breath can be analysed using a variety of instruments. Most of, the

instruments used today detect ethanol by using, for example,
thermal conductivity, colorimetry, infrared spectroscopy or gas
chromatography. In most countries, local statutes define the
instrument and method that can be used for evidentiary purposes.
A variety of Breathalyser instruments, the prices of which range
from US$ 100 to US$ 1,000, are available. The instruments are
compact and portable. The Canadian law enforcement authorities
use as a roadside alcohol-screening device a Breathalyser
instrument that gives a "pass" or "fail" result. A person who fails
that test is generally subjected to a Breathalyser test to measure
the BAC level before any charges are made. Many devices are
available to preserve the breath sample for later analysis if a
Breathalyser is not available immediately. In forensic laboratories in
North 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, should

have sodium fluoride (NaF) added as a preservative [42] . ADH, the
enzyme responsible for the oxidation of alcohol, is also present in
red blood cells and will slowly metabolize the alcohol, causing its
concentration to drop if the preservative is not added. Large
amounts of alcohol can be produced in vitro in the urine samples of
diabetic patients if samples are not processed immediately.

VI. Interpretation of test results

A. False negatives

A positive or negative result is highly dependent on the sensitivity

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

Large amounts of fluids consumed prior to obtaining a sample for

analysis can affect detection of drugs in urine samples. Under
conditions of dilution, although the absolute amount of drug or
metabolite excreted may be the same over a period of time, the
final concentration per millilitre will be reduced and may give a
false-negative result. Acidity levels in the urine may also affect the
excretion of the drug into the urine. In some cases, elimination is
enhanced; in others, the drug is reabsorbed.

Several measures can be used to decrease the likelihood of

obtaining a false-negative result. First, sensitivity of the method can
be enhanced by analysing for the metabolites of the drug in
question. Heroin use, for example, is determined by the presence of
the heroin metabolite, morphine. Increasing the specimen volume
used for analysis or treating it with chemicals can also make
laboratory methods more sensitive. Studies in the author's
laboratory 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 be
detected for up to 20 days. One significant drawback of such high
sensitivity is that estimates of when the drug was taken are far less
accurate.

B. False positives

A false positive occurs if results show that the drug is present when
in fact it is not. False-positive tests are obtained if an interfering
drug -or substance is present in the biological fluid and it cross -
reacts with the reagents. As discussed in the previous section on
immunoassays, an initially positive test based on immunoassay
technique should always be confirmed with a non-immunoassay
method. A confirmed positive finding only implies that the urine
sample contains the detected drug and nothing more.

Sometimes false positives are attributable to ingested substances

such as asthma or allergy medication [43] . Some authors have
suggested that employees subject to drug screening should refrain
from using certain brands of over-the-counter medication because
they have caused false positives [44] . Some natural substances
such as herbal teas and poppy seeds can also give positive
responses to screens., These may be analytically true positives but
they need to be distinguished from those due to illegal drug use. In
some instances, false positives have been the result of mistakes or
sabotage in the chain of custody for urine samples.

VII. Common adulteration methods

Substituting "clean" or drug-negative urine for drug-positive urine is
the most common way to fool the drug-screening system. A number
of entrepreneurs have attempted to bypass urine sample inspection
in this manner. A company in Florida sells lyophilized (freeze-dried)
"clean" urine samples through newspaper and magazine
advertisements. Hiding condoms containing "clean" urine on the
body or inside the vagina is another common trick. Recently, a
patient at an addiction research clinic was, caught substituting
"clean" urine when a glass bottle that had fallen into the toilet bowl
was discovered by the supervising nurse. It was later discovered
that the bottle had been sealed with a thin aluminium wrap and had
been inserted into the patient's vagina.

Others have attempted to substitute apple juice or tea for urine

samples. Persons have been known to add to urine samples various
house - 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 it
into the urine specimen. Another method is to poke a small hole
into the urine sample container with a pin so that the sample leaks
out by the time it reaches the laboratory [45] .

Since adding table salt (NaCl) or bleach to urine samples is a

common practice, many laboratories routinely test for sodium and
chlorine in urine samples. Liquid soap and crystalline drain cleaners,
strong alkaline products containing sodium hydroxide (NaOH), are
also used to adulterate urine samples. These contaminators can be
detected by checking for high pH levels in urine samples. In vivo
alkalizing or acidifying the urine pH can also change the excretion
pattern of some drugs, including amphetamines, barbiturates and
PCP.

Water-loading (drinking large amounts of water prior to voiding)

poses an interesting challenge to testing laboratories. Specific
gravity has been used to detect dilution; however, the
measurement range is limited. Creatinine levels in random urine
samples have also been studied as a possible water-loading
detection method, but without much success.

Drug-using patients are resourceful and their ingenuity should not

be underestimated. In order to reduce the opportunities for
specimen contamination, some workplaces require that employees
provide urine samples under direct supervision. Another way to
detect any sample adulteration is to take the temperature of the
sample. When the temperature of samples are taken within one
minute of voiding, the temperature range falls to between
36.5¦deg; and 34¦deg; C, reflecting the body core temperature. It
is difficult to achieve this narrow temperature range by hiding a
condom filled with urine in the armpit or adding water from a tap or
toilet bowl to the urine sample. The temperature of the sample
must be measured immediately after it is taken, since the
temperature drops rapidly.

VIII. Laboratory procedural and security standards

It is important that the laboratory drug testing facility has qualified
individuals who follow a specific set of laboratory procedures and
meet certain security standards. Laboratory management personnel
must have documented qualifications in analytical forensic
toxicology in order to analyse urine samples for the presence of
drugs [46] . A flow chart showing the various steps in the drug-
testing process, from sample collection to the final disposition of the
results, is provided in figure I. Figure II shows the space, staffing
and equipment requirements.

The laboratory should be secure at all times and access should be

limited to authorized individuals only. The laboratory should
establish security measures to guarantee that specimens are
properly received, documented, processed and stored.
Documentation of chain-of-custody procedures should include
specimen receipt, results during storage and final disposition of
specimens. The laboratory must comply with any governmental
licence requirements, must be inspected routinely, must keep
appropriate documentation and procedural manuals and must use
properly certified equipment.

Urine specimens should be inspected immediately upon arrival at

the laboratory in order to ensure that they have -not been
tampered with during delivery. Specimens should be stored in a
secure refrigeration unit if they are not tested within seven days of
arrival at the laboratory. The storage temperature should not
exceed 6¦deg; C. Long-term storage must be at -20¦deg; C to
ensure that positive urine specimens will be available for any
retesting during administrative or disciplinary proceedings. The
laboratory will be required to maintain any specimen under legal
challenge for an indefinite period.

Figure I. Schematic representation of the drug-testing

process

Figure II. Space and staffing requirements of the drug-

testing process
IX. Initial and confirmatory testing requirements
The initial test (i.e. screening test) consists of an immunoassay
technique that meets the requirement for commercial distribution
and eliminates "negative" urine specimens from further
consideration. Minimal (cut-off) levels should be used when
screening a specimen to determine whether it is negative. Some
negative cut-off levels (in nanograms per millilitre) for initial tests
are as follows [13] :

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

immunoassay technique that meets the requirement for
commercial distribution and eliminates "negative" urine
specimens 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 (in
nanograms per millilitre) for initial tests are as follows [13]:

Tetrahydrocannabinol metabolite 100

Cocaine metabolite 300
300
Opiate metabolites
a/
PCP 25
Amphetamines 1,000
Alcohol (mg/100ml) 10

a/ If immunoassay is specific for free morphine, 25

nanograms per millilitre.

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

confirmatory test should be performed, whereby a second and
different analytical procedure is used to identify the presence of a
specific drug or metabolite. At present, GC/MS is the recommended
confirmation method. Some minimal (cut-off) concentrations (in
nanograms per mililitre) for confirmatory tests are as follows [13] :

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

confirmatory test should be performed, whereby a second
and different analytical procedure is used to identify the
presence of a specific drug or metabolite. At present, GC/MS
is the recommended confirmation method. Some minimal
(cut-off) concentrations (in nanograms per mililitre) for
confirmatory tests are as follows [13]:

Tetrahydrocannabinol metabolites a/ 15
Cocaine metabolites b/ 150
Opiates
Morphine 300
Codeine 300
PCP 25
Amphetamines
Amphetamine 500
Methamphetamine 500
Alcohol (mg/100 ml) 10

a/ For example, 11-nor-?-9terahydrocannabinolic acid.

b/ Benzoylecgonine.

X. Regulations for drug testing *

The purpose of the regulations is to detail the standards and
procedures of laboratories that plan to provide drug testing for the
work-place. There are two main parts to the present section. The
first part describes the requirements for the site collection of urine
samples and the second documents the essentials of laboratory
drug testing [47] .

Extreme caution must be exercised in the testing procedures. Using

specimens for other types of analysis such as pregnancy or other
disease criteria is expressly prohibited. In addition, the possible
impact of a positive result on an individual's livelihood or rights,
together with the possibility of a legal challenge of the results, sets
this type of testing apart from most clinical laboratory analysis.
Urine drug testing should be considered a special application of
analytical forensic toxicology. That is, in addition to the application
of appropriate analytical techniques, the specimens must be treated
as evidence and all aspects of the testing procedure should be
documented and available for examination.

A. Specimen collection procedures

The purpose of the present subsection is to outline the procedures

used to ensure integrity of the specimens during urine collection
and during their transportation to a laboratory. A proper urine
specimen collection is the key to developing a successful testing
programme.

The first concern of specimen collection is designating a specimen

collection site. The collection site must have all the personnel,
material, equipment and supervision necessary to allow the sample
to be collected, temporarily stored and transported to the drug-
testing facility. Collection site personnel must have successfully
completed training to carry out these functions.

A second concern is ensuring the security of the collection site. If

the collection site facility is used solely for urine collection, it should
be secured at all times and accessible only to authorized persons.
When urine sample specimens are being collected or stored, no
unauthorized individual should be permitted in any part of the
designated collection site unless he or she is accompanied by an
authorized person.

*The protocols and procedures in the present section are based on the
accumulated knowledge of the author in his capacity as Inspector for
the College of American Pathologists (CAP), a certifying agency for
drug-testing laboratories in the United States. In addition,
guidelines suggested by Kwong and others [46] , CAP [47] , NIDA [13]
and the Canadian laboratory standards for drug screening in the
workplace were extensively consulted.

Procedures for collecting urine samples should allow for individual

privacy unless there is reason to believe that a particular individual
may alter or substitute a sample to be provided.

1. Minimal procedures to ensure integrity and identity of

specimens

The following minimal procedures should be followed to ensure the

integrity and identity of the specimen at the collection site, to help
to safeguard against specimen adulteration or dilution during the
collection procedure and to allow for proper identification of the
individual being tested:

1. Immediately after the specimen is collected, the collection site

person should also inspect the specimen to determine its
 colour and should took for any signs of contaminants. Any
unusual findings should be noted in a permanent record or
logbook;
2. All specimens suspected of being adulterated should be
forwarded to the laboratory for testing;
3. Both the individual being tested and the collection site person
should keep the specimen in view at all times prior to it being
sealed and labelled. If the specimen is transferred to a second
bottle, the collection site person should request the individual
being tested to observe the transfer of the specimen and the
placement of a tamper-proof seal over the bottle cap and
down the side of the bottle;
4. The collection site person should securely place an
identification label on the bottle that contains the date, the
individual 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. actually
collected from him or her;
6. The collection site, person should, enter into the permanent
record or logbook all information identifying the specimen.
The collection site person should then sign the permanent
record or logbook next to the identifying information. An
individual being tested should be asked to read and sign a
statement 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 temporary
storage in a refrigerator at a temperature of about 4¦deg; C.
The specimen should normally be shipped within 48 hours and
definitely no longer than one week after it has been placed in
storage. If stored longer, the temperature should be -20¦deg;
C;
9. In the event that a collection site is not accessible and there is
immediate requirement for specimen collection (e.g. an
accident investigation), a public rest room may be used
according to the following procedures:
10. A collection site person of the same gender as the
individual being tested may accompany the individual into the
public rest room, which shall be made secure during the
collection procedure;
11. If possible, a toilet bluing agent should be placed in the
toilet bowl and any accessible toilet tank to deter specimen
dilution. If no bluing agent is available, the collection site
person should instruct the individual; being tested not to flush
 the toilet until the specimen is, delivered to the collection site
person;
12. It is essential that the urine specimen and the custody
document be under the control of the collection site person. If
the collection site person leaves the workstation for a short
period, the specimen and the chain-of-custody form should be
taken with him or her or should be secured. After the
collection site person returns to the workstation the custody
process should continue. If the collection site person leaves
the site for an extended period of time, the specimen should
be packaged for transportation beforehand;
13. Collection control site personnel should keep the
specimen bottle within sight both before and after the
individual has urinated. After the specimen is collected, it
should be properly sealed and labelled and an approved
chain-of-custody form should be used to control and account
for each specimen from the point of collection to final
disposition of the specimen;
14. The date and purpose should be documented on an
approved chain-of-custody form each time a specimen is
handled or transferred and every individual in the chain of
custody should be identified. Every effort should be made to
reduce to a minimum the number of persons handling the
specimen;
15. Collection site personnel should arrange to ship the
collected specimen to the drug -testing laboratory (see the
subsection below entitled "Guidelines for transporting
specimens"). Specimens should be placed in a container
designed to keep to a minimum the possibility of damage
during shipment. Specimen boxes or padded mailers, for
example, should be securely Scaled to eliminate the
possibility of undetected tampering. On the tape used to seal
the container, the collection site supervisor should place his or
her signature and enter the date on which the specimen is
being scaled for transportation. Collection site personnel
should ensure that the chain-of-custody document is-attached
to each container and is being sealed for shipment to the
drug-testing laboratory.

2. Procedures to maintain the integrity of specimens

The following procedures are recommended to ensure that

unadulterated specimens are obtained:

1. An individual being tested should be instructed to wash and

dry 'his or her hands prior to urination. After washing his or
her hands, the individual being tested should remain in the
presence of the collection site person and should not have
 access to any water fountains, faucets, soap dispensers,
cleaning agents or any other material that could be used to
adulterates the specimen;
2. The collection site person should note any unusual behaviour
or 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 whenever
possible so that the reservoir of water in the toilet bowl
always remains blue. Ideally, there should lie no source of
running water (i.e. no sink in the room where the urine is
collected);
4. The individual giving the urine sample should be requested to
remove 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 urine
sample;
5. Upon receiving the sample from the individual, the person
supervising the collection should determine whether it
contains a minimum of 60 ml of urine. If there is less than 60
ml of urine in the container, then an additional urine sample
should be collected in a separate container. The individual
being tested may be given a reasonable amount of liquid to
drink for this purpose;
6. After the specimen has been submitted to the person
supervising the collection, the individual being tested should
be allowed to wash his or her hands;
7. Immediately after the specimen is collected, the person
supervising the collection shall measure and record the
temperature of the specimen. The time from urination to
temperature measurement is critical and in no case should
exceed four minutes. Normal temperature ranges from
32.5¦deg; to 37.7¦deg; C (90.5¦deg;-99.8¦deg; F). (In the
author's laboratory, temperatures of 100 consecutive urine
samples ranged between 34.5¦deg; and 36.5¦deg; C when
measured within two minutes of voiding.);
8. If the temperature of the specimen is outside the range of
32.5¦deg;-37.7¦deg; C (90.5¦deg;-99.8¦deg; F) or the pH is
not between 4.8 and 8, there is reason to believe that the
individual being tested may have altered or substituted the
specimen and another specimen should be collected under
direct observation by the collection site person. Both
specimens should be identified and forwarded, with
appropriate comments, to the laboratory for testing. The
individual being tested may volunteer to have his or her body
temperature taken to refute the suggestion that the specimen
was altered or substituted. Diluted urine can also be detected
 by its pale colour or by its smell if adulterants such as
ammonia or furniture polish have been added;
9. Immediately after the specimen is collected, the collection site
person should also inspect the specimen to determine its
colour and to look for any signs of contaminants. Any unusual
findings should be recorded in the permanent record or
logbook;
10. All specimens suspected of adulteration should be
forwarded, with appropriate comments, to the laboratory for
testing;
11. It is important that the sample container is properly
labelled before it is given to the individual being tested.
Ideally, the scaling of the specimen container should be
witnessed by the individual being tested.

3. Guidelines for transporting specimens

To maintain effective control while transporting the urine sample to

the testing laboratory, the following guidelines for transporting
specimens should be kept in mind:

1. All containers (i.e. plastic urine bottles) must be checked for

cracks before filling, and only intact containers should be
used;
2. Chain-of-custody forms and containers must be clearly
labelled;
3. Appropriate protective equipment must be worn to maintain
body substance precaution techniques during retrieval of all
specimens;
4. All specimen caps or lids should be checked before
transporting to see if they are securely closed;
5. Specimen containers should be placed in the designated
transportation container;
6. Specimens should always be handled carefully to avoid
breakage.

B. Laboratory procedures
Laboratory personnel requirements

Laboratory management personnel must have documented

qualifications in analytical forensic toxicology in order to carry out
the analysis of urine samples for drug testing. They must be able to
review data and quality-control results, to supervise the routine
operations of drug testing, to train personnel and to keep files on
personnel.
The laboratory should have a qualified director to assume the
professional, organizational, educational and administrative
responsibilities for its urine drug-testing facility. That individual
should have documented scientific qualifications in analytical
forensic toxicology. The minimum qualifications are as follows:

1. A Doctor of Philosophy (Ph.D.) in one of the natural sciences

and an adequate undergraduate and graduate education in
biology, chemistry and pharmacology or toxicology; or
training and experience comparable to a Ph.D. in one of the
natural sciences such as a medical or scientific degree with
additional training and laboratory research experience in
biology, 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 or
other factors that qualify an individual as an expert witness in
forensic toxicology).

In jurisdictions where certification as a laboratory director is

required, the director should be certified or be eligible to apply for
that certificate. The director will be responsible for certifying the
final test results being reported.

The laboratory urine drug-testing facility should have qualified

individuals who review all pertinent data and quality- control results
in order to attest to the validity and certification of the laboratory
test reports. The laboratory may designate more than one person to
perform this function. Such individuals may be any employees who
are qualified to be responsible for the day-to-day management or
operation of the drug- testing laboratory. Individuals responsible for
the day-to-day operations and supervision of the technical analysis
should at least have Bachelor's degrees in chemical or biological
sciences or medical technology or the equivalent. They should have
training and experience in the theory and practice of procedures
used in the laboratory and should thoroughly understand quality-
control practices and procedures. They will be responsible for
reviewing, interpreting and reporting test results and for
maintenance of the chain of custody. Additionally, these individuals
should ensure that proper remedial actions are taken in response to
test systems that are out of control limits or when erroneous results
are detected.

Other technical and non-technical staff should have the necessary

training and skills for the tasks that they will be assigned. The
laboratory programme should make continuing education
programmes available to meet the needs of the laboratory,
professionals., The personnel file of such an individual should
include 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 security

measures should be in place to control access to the premises and
to ensure that no unauthorized personnel handle specimens or gain
access to the laboratory processes or the record or log storage
areas. Access to these secured areas should be limited to
specifically authorized and documented individuals. With the
exception of personnel authorized to conduct inspections, all visitors
and maintenance service personnel should be escorted at all times.
Records of individuals entering these areas and the date, time and
purpose of entry must also be maintained. Documentation of chain -
of -custody procedures should include specimen receipt, results
during storage and final disposition of specimen. The date and
purpose should also be documented on an appropriate chain-of-
custody form each time the specimen is handled or transferred, and
every individual in the chain should be identified. Accordingly,
authorized technologists should be responsible for each urine
specimen 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 to

guarantee that specimens are properly received, documented,
processed and stored. The laboratory must also comply with any
governmental licensing requirements,. must be inspected routinely,
must keep appropriate documentation and procedure manuals and
must use properly certified equipment.

3. Receiving specimens

When a shipment or specimen is received, the laboratory personnel

should inspect each package for evidence of tampering and should
compare information on specimen bottles with each package of
information accompanying the chain-of-custody forms. Any direct
evidence of tampering or discrepancy between the information on
the specimen bottle and the accompanying chain-of-custody form
should be recorded on the chain-of-custody form and accompany
the specimen while it is in the laboratory. Specimen: bottles should
normally be retained within the laboratory area until all analyses
have been completed.

4. Storing specimens

Specimens that do not receive an initial test within seven days of

arrival at the laboratory must be placed in a secure refrigeration
unit. The temperature must not exceed 6¦deg; C. Emergency
electrical power equipment should. be available in case of prolonged
power failure.

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

ensure that positive urine specimens are available for any retesting
during administrative or disciplinary proceedings. Unless otherwise
authorized in writing, the laboratory must retain and place in
properly secured long- term storage all specimens confirmed
positive for a minimum of one year. Within this one-year period, the
client may request the laboratory to retain the specimen for an
additional period of time. If no such request is received the
laboratory should discard the specimen after one year. The
laboratory will be required to maintain any specimen under legal
challenge for an indefinite period.

5. Initial and confirmatory testing

Specimens can be processed in batches for either initial or

confirmatory toasts. When conducting either initial or confirmatory
tests, every batch should contain an appropriate number of
standards for calibrating the instrument and a minimum of two
specimen controls or 10 per cent specimen controls, whichever is
higher. Both quality-control and blind performance test samples will
appear as ordinary samples to the laboratory analyst.

The initial test (screening test) involves an immunoassay technique

that meets the requirement for commercial distribution and
eliminates a "negative" urine specimen from further consideration.
The initial cut-off levels (see the section above entitled "Initial and
confirmatory testing requirements') should be used when screening
a specimen to determine whether it is negative for the drugs or
classes of drugs being tested. For alcohol, the initial test may be an
enzymatic 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 to
identify the presence of a specific drug or metabolite must be
performed. The confirmatory test method must use a different
technique and chemical principle from that of the initial test. At
present, GC/MS is the recommended confirmation method for
reasonable 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 values
obtained by the initial test and the confirmatory test for alcohol
must be within 10 per cent of each other. All confirmations must
include quantitative analysis. Concentrations that exceed the linear
region of the standard curve should be documented in the
laboratory records as greater than the highest standard curve
value.

The results of retesting of specimens (being challenged) must

confirm the presence of the drug or metabolite in question but are
not required to correspond to any specific cut-off level since some
analyte may deteriorate during the freezing, thawing or storage
procedures.

6. Operational guidelines
(a) Laboratory facilities

The laboratory must comply with provisions of the local

governmental licensing requirements. In order to attain
certification, the laboratory must be capable of performing both the
initial and the confirmatory tests for each drug or metabolite for
which the service is being offered. Urine specimens collected for the
purpose of drug testing should only be used to test for those drugs
included in their clients' approved drug- free workplace plan and
may not be used to conduct any other analysis or tests.

(b) Inspection

A certification body or any client utilizing the laboratory may

reserve the right to inspect the laboratory at any time. Any
employer who has contracted the laboratory for drug testing or
collection site services is permitted to conduct unannounced
inspections. In addition, prior to awarding a contract, a client may
carry out pre-award inspections and evaluations of the procedural
aspects of the laboratory drug-testing operation.

(c) Subcontracting

Unless authorized to do so, the drug-testing laboratory may not

subcontract and should perform all, work with its own personnel
and equipment unless otherwise authorized by the client. If
subcontracting is authorized, then the subcontracted laboratory
must be capable of performing tests for the agreed upon classes of
drugs. Same-site screening and confirmation is ideal for the
maintenance of strict quality control in the chain of custody and in
transporting and processing samples, as well as in reporting results.
The subcontracting laboratory must adhere to the guidelines stated
in the present paper.

(d) Information collection requirement

The drug-testing laboratory must maintain and make available for

at least two years documentation of all aspects of the testing
process. The required documentation must include the following:

1. Personnel files on all individuals authorized to have access to

the 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 effect

should be maintained as part of the laboratory procedure manual
under the signatory approval of the director of the drug-testing
laboratory.

(f) Standards and controls

Laboratory standards should be prepared with pure drug standards

that are properly labelled, indicating their content and purity. The
standards 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 person
preparing them should be recorded. Control samples should be
labelled with a code allowing the analysts to test them in a blind
manner. The temperature of the refrigerator used for storing the
reagents should be checked and recorded daily. The results for
controls and standards must be recorded along with the date, time
and name of the analyst conducting the test procedure.

(g) Instruments and equipment

The laboratory, supervisor should oversee the day-to-day

maintenance of the laboratory equipment. Volumetric pipettes and
other 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 and
reproducibility before being placed in service and should be checked
monthly thereafter. The procedure manual should include written
procedures for instrument setup, calibration, critical operating
characteristics, tolerance limits for acceptable function checks and
instructions for major trouble-shooting and repair. Records must
also be available on preventative maintenance for the instruments.

(h) Reporting of results.

The laboratory should report results to the medical review officer

within 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 this
activity. The report should identify the drug metabolites tested for
(whether positive or negative), cut-off levels, the specimen number
assigned by the test- requesting facility and the drug-testing
laboratory specimen identification number. The results, whether
positive or negative, should be reported back to the medical review
officer, at the same time. The laboratory should report as negative
all specimens that are negative on the initial test or negative on the
confirmatory test. Only specimens confirmed positive should be
reported positive for a specific drug. The laboratory must provide
quantitative. test results at the request of the medical review
officer.

(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 testing
process, from specimen collection to reporting of the results and the
final disposition of the specimen. Quality-assurance procedures
should be designed, implemented and reviewed to monitor each
step of the process.

The quality-control requirements for initial tests should be

structured so that each analytical run of specimen to be screened
includes:

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 of

standards should be included to document the linearity of the assay
method over time in the concentration range of the cut-off. A
minimum of 10 per cent of test samples should be used as quality-
control samples.

The quality-control requirements for confirmatory tests should be

structured so that each analytical run of specimen to be confirmed
includes:

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 periodically

documented. Implementation of procedures to ensure that carry-
over does not contaminate the testing of the individual specimen
should also be documented.

7. Review of results
(a) Medical review officer

The medical review officer should be a licensed physician with a

knowledge of substance abuse disorders. A positive result does not
auto- matically identify an individual as an illegal drug user. The
role of the medical review officer is to review, verify and interpret
positive test results obtained through the client's drug- testing
programme. In carrying out his or her responsibilities, the medical
review officer should examine alternate medical explanations for a
positive test result by, for example, conducting a medical interview
with the individual, reviewing the individual's medical history and
determining the clinical evidence of illegal use of any opiate or
opium derivatives. The medical review officer should review all
medical records available on the tested individual to ascertain
whether or not a confirmed positive could be explained by the use
of a legally prescribed medication. Prior to making a final decision to
verify a positive result, the medical review officer should give the
individual 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 consistent
with the legal drug use and no further action should be taken.

Should any question arise as to the accuracy or validity of a positive

test result, the medical review officer is authorized to order a
reanalysis of the original sample. Additionally, based on the review
and inspection of reports, quality-control data, multiple samples and
other pertinent results, the medical review officer may determine
that the result is scientifically insufficient for further action and
declare the test specimen negative. The director of the laboratory
should be available to consult with the medical review officer as
required. The employee being tested has a right to identify a
prescribed medication when the urine sample is being provided.

(b) Protection of employee records

All laboratory contracts should require that the contractor comply

with the privacy act. In addition, the laboratory contract should
require compliance with the access and confidentiality provisions
established within the jurisdiction where the laboratory practice is
maintained. The contract and the privacy act should specifically
require that employee records be maintained and used with the
highest regard for employee privacy.

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

Any employee who is subject to drug testing should, upon written

request, have access to any records relating to his or her drug test
and any records relating to the results of any relevant certification
review proceedings.

8. Certification of laboratory
 The laboratory must meet all pertinent provisions and guidelines
stated in the present paper. In determining whether to certify a
laboratory or to accept the certification, the following criteria should
be 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 blind

drug-testing challenges initiated by the certifying body. During the
process, 90 per cent of the number of tests performed must be
correctly identified (i.e. identification and confirmation of 90 per
cent of the total drug challenges) or suspension or revocation of the
certificate may take place

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