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TESTING FOR SUBSTANCE ABUSE

TOXICOLOGY LABORATORY
CLINICAL BIOCHEMISTRY
LEEDS TEACHING HOSPITALS
BRITANNIA HOUSE
MORLEY
LEEDS
LS27 0DQ

October 2008

Contacts:

K R Allen FRCPath Tel. 0113 3927850

Consultant Clinical Scientist email keith.allen@leedsth.nhs.uk
Email toxicology@leedsth.nhs.uk

Shirley Twigger MSc, FIBMS Tel. 0113 3927883

Advanced Biomedical Scientist email shirley.twigger@leedsth.nhs.uk

------------------------

Documentation subject to future revision. Not to be redistributed or

photocopied.
Information contained is only applicable to users of the Leeds Teaching Hospitals
Toxicology Service.
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1.0 SERVICE OVERVIEW

This laboratory provides a routine clinical service for the measurement of a specified
range of illicit and therapeutic drugs in patients attending substance abuse clinics. We
do not carry out medico-legal work which would require samples collected under
chain-of-custody procedures. We measure drugs in either urine or oral fluid samples
depending upon the preference for the particular clinic. Both these types of matrix
have advantages and disadvantages which will be highlighted in their respective
following sections. A section has been appended giving details relating to drug
metabolism which is important with regard to interpretation of results.

2.0 Oral Fluids

2.1 Sample Collection

This is achieved using the Intercept device manufactured by Orasure Technologies

Inc. and supplied via the UK agent Altrix. This device uses a cotton pad to collect a
mixture of saliva and oral mucosal transudate in accordance with the instructions
which are clearly printed on each package. The device is placed into a tube
containing a fluid which has been altered by this laboratory from that supplied by
Orasure. The clear fluid in the collecting device contains 4mM ammonium acetate in
15% methanol. The volume of the fluid in the tube is very small as is the
concentration of methanol. However, methanol is toxic if taken orally or by contact
with the skin and all precautions must be taken to avoid this risk. Any exposed skin
coming into contact with this solution should be washed with water immediately. If
any of the fluid is swallowed then the mouth should be washed with water and plenty
of water given to drink. Medical attention should then be sought. On no account
should the patient be allowed to have contact with the collection tube. The Intercept
collection stick should be handled by the drug worker following the collection of the
oral fluid.

As many of the illicit drugs are chemically basic they tend to be retained within the
oral cavity following salivary secretion. This is due to the mildly acidic nature of
unstimulated saliva. Consequently, concentrations of basic drugs in oral fluid may be
several fold higher than that of the blood plasma. Changes in salivary pH due to
stimulation by chewing or use of agents such as lemon juice can affect oral fluid drug
collection leading to possible false negative results. This is particularly the case for
amphetamines although other drugs such as methadone may also be affected. The
Intercept device collects unstimulated saliva and in addition collects a transudate from
the blood capillaries supplying the gums and cheek of the mouth thus avoiding these
problems.
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2.2 Advantages/Disadvantages of Oral Fluid

The 4 main disadvantages between oral fluid collection and urine collection are as
follows:

 Shortened detection window for oral fuid.

 Methadone cannot be confirmed using its metabolite EDDP*
 Buprenorphine results are difficult to interpret. High levels of parent drug
may be due to oral contamination. Concentrations of the metabolite
Norbuprenorphine are very low in oral fluid.
 THC (Cannabis) metabolite generally not detectable**

*EDDP (2-ethylidene- 1,5-dimethyl-3,3-diphenylpyrrolidine) only occurs in 70% of

oral fluid samples from subjects taking methadone due to its poor salivary:plasma
ratio.

** The major Cannabis metabolite (11-nor-delta-9-tetrahydrocannabinol-9-carboxylic

acid is not secreted in saliva. The parent compound delta 9 THC may be found
indicating very recent usage.

The main advantages of oral fluid collection are:

 Better sample integrity

 Better matrix for the analyst with less potential interferences
 Ease of collection
 Identification of specific drugs and metabolites with regards to the
benzodiazepines

2.3 Drug Measurements Available

Drugs are measured using liquid chromatography linked to tandem mass spectrometry
and measurements related to cut-off concentrations provided by the Substance Abuse
and Mental Health Services Administration (SAMHSA) of the USA. A 1 in 4 dilution
of the oral fluid in the collecting device solution is allowed for with regard to the cut-
off concentrations. Exceptions to the SAMHSA cut-offs include 6
Monoacetylmorphine (6MAM) which is set at 3 µg/L whereas SAMHSA advise
1µg/L (allowing for dilution). We feel that 1µg/L is too low and could lead to false
positive results due to carry over in the analytical method. Also, cocaine and its
metabolite benzoylecgonine have cut-offs equivalent to 5 µg/L rather than the
SAMHSA level of 2 µg/L. We experienced too many “false positive” cocaine
samples at the low cut-off value. The possibility of passive inhalation may be a
problem with low cut-offs for cocaine. All oral fluid drugs are reported quantitatively
and levels less than their respective cut-offs are considered to be not-detectable. We
do on occasions report some drugs e.g. morphine and diazepam (nordiazepam, its
metabolite) as detectable but below the cut-off. This is done when for example we see
a positive 6-MAM, we also like to see the presence of morphine even if it is less than
10ug/L to help with confirmation. We will also look for diazepam or its metabolite
nordiazepam if it is less than the cut-off of 0.4 ug/L if the subject is reported as taking
this medication.
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Table 1. Drugs/Drug metabolites measured in Oral Fluid

Drug/Metabolite Detection Time Cut-Off SAMHSA Cut-Off

(Approx) (µg/L) (µg/L) (Factored by
1/4)
6MAM 6 hrs 3 1
Morphine 12 hrs 10 10
Codeine 12 hrs 10 10
DHC 12 hrs 10 10
Methadone Up to 48 hrs 5 -

Cocaine 6 hrs (12-36 hrs) 5 2

Benzoylecgonine 6 hrs (12-26 hrs) 5 2

Diazepam 12-24 hrs 0.4 -

Nordiazepam 12-24 hrs 0.4 -
Temazepam ? 0.4 -
Nitrazepam 12-24 hrs 0.4 -
7-Aminonitrazepam 12-24 hrs 0.4

Amphetamine Group 10-20 hrs 12 ug/L 12 ug/L

Notes:

 Cocaine detection time is increased in subjects with chronic use of the drug
 Methadone detection time shows high degree of intra-individual variation
 Nordiazepam the main metabolite of diazepam may be seen in the absence of
diazepam. If diazepam is detected in high amounts without nordiazepam then
oral contamination due to recent drug exposure is suspected. Note,
Nordiazepam is also a metabolite of other benzodiazepines.
 7-aminonitrazepam is the metabolite of Nitrazepam

2.4 Interpretive Guidelines for Drugs in Oral Fluids

From February 2008, oral fluid results are reported quantitatively. The cut-offs are
shown in Table 1 above. When the drug is present in the sample, its concentration is
reported in µg/L. If the concentration is high, it will be reported as “greater then (>)”
the upper limit of our top standard i.e. 50 µg/L. When the drug is not detected it is
reported as “less than (<)” the cut-off. On occasions a drug/metabolite that is less
than the cut-off but still detectable will be reported as “present but below cut-off”.
This is used in situations where confirmation of a particular drug is required by
presence of it’s metabolite.
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Opiates
Heroin (diacetylmorphine) has an extremely short half life being metabolised
sequentially first to 6-monacetylmorphine (6MAM) and then to morphine. Illicit
heroin also contains acetylcodeine which is metabolised to codeine. Hence, a
characteristic metabolic profile for heroin abuse is the presence of 6MAM, morphine
and codeine. However, in oral fluid samples we do see just the presence of 6MAM
and morphine or morphine alone following heroin use. Only 6MAM is characteristic
for heroin use as morphine may arise from metabolism of codeine, from poppy seed
containing foods or from morphine itself.

DHC is not metabolised to morphine or codeine.

Codeine is metabolised to morphine in the majority of subjects. 10% of the

population do not metabolise codeine to morphine.

Methadone
Methadone is metabolised to EDDP which is used for confirmation of this drug in
urine samples i.e. its presence rules out the possibility of the sample being spiked with
methadone. However, EDDP has a low saliva:plasma ratio and is not secreted into the
oral cavity as well as the basic drugs. We can look for EDDP in oral fluid if
specifically asked for although its absence is not indicative of non-compliance with
methadone treatment. We do occasionally see extremely high levels of methadone in
oral fluid which suggests recent ingestion of the drug just prior to sampling and hence
oral contamination.

Cocaine
Cocaine is metabolised to benzoylecgonine and both compounds are often seen
together in oral fluid samples. In the case of samples collected relatively late
following cocaine use then only the metabolite benzoylecgonine may be detected.

Benzodiazepines
Compliance for specific benzodiazepines i.e. diazepam, nitrazepam and
temazepam is assessed. Confirmation of diazepam will be made by the presence of
its metabolite nordiazepam. Temazepam may also be seen as a metabolite of
diazepam or from administration of the drug itself. Nitrazepam is confirmed by the
presence of 7-aminonitrazepam. On occasions only the metabolites of these drugs
may be seen. We also see very high levels of diazepam with no metabolite detected
which suggests possible non-compliance and ingestion of the drug just prior to
sampling resulting in oral contamination.

Amphetamines
The amphetamine group that we test for presently consists of amphetamine,
methamphetamine, MDMA (its metabolite MDA). The amphetamines may result in
reduced salivary excretion and produce false negative results.
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3.0 URINE TESTING

Sample Requirements – 20ml of urine collected into a plain universal which must be
tightly secured. Leaking samples cannot be analysed for health & safety reasons.
Please ensure that all Universal Containers are properly sealed before
despatching to the laboratory. All request details must be placed on the
accompanying form and match those on the specimen container. At least 2 points of
identification are required e.g. patient full surname and forename name and date of
birth.

Testing Procedure.

Urine testing is a 2 phase process.

3.1 Phase 1: Immunoassay

All samples are initially screened using immunoassay for the following drug/drug
groups:

Opiates
Cocaine
Cannabis
Methadone
Amphetamines
Benzodiazepines
Alcohol

In addition creatinine is measured to assess sample integrity.

Samples with a creatinine < 2mmol/L will be flagged as “dilute” and may not be valid
for drug screening.

The cut-off values and detection times for drugs in urine by immunoassay are shown
in Table 2 below.
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Table 2. Drugs measured by Immunoassay

Drug Class Detection Time (approx) Cut-off

Opiates 2 days 300 µg/L
Methadone 2 days 300 µg/L
Methadone 7-9days 300 µg/L
(maintenance dosing)
Cocaine (metabolite) 2-3 days 300 µg/L
THC (cannabis) single use 3 days 50 µg/L
THC moderate use 4 days 50 µg/L
THC heavy use (daily) 10 days 50 µg/L
THC chronic heavy use Up to 36 days 50 µg/L
Benzodiazepines (short acting) 1 day 200 µg/L
triazolam
Benzodiazepines (intermediate) 40-80 hours 200 µg/L
temazepam
Benzodiazepines (long acting) 7 days or more 200 µg/L
Diazepam, Nitrazepam

Amphetamines 2-3 days 1000µg/L

Notes:

Opiate immunoassay measures a range of opiate drugs and metabolites including the
conjugated forms of these drugs. False positive results due to Pholcodine do occur.
Buprenorphine is not detected by this opiate immunoassay method.

3.2 Phase 2: Tandem Mass Spectrometry

All urine samples which are positive for the opiate and amphetamine group are
subjected to a screen by tandem mass spectrometry for the following reasons:

 Confirmation of the type of opiate present, i.e. 6-Monoacetylmorphine,

Morphine, Codeine, DHC, pholcodine, oxycodone
 Detection and typing of any amphetamine present i.e. Amphetamine, MDMA
(its metabolite (MDA), ephedrine (pseudoephidrine), methamphetamine
(metabolite hydroxy-methamphetamine).

The above screening method enables us to confirm the majority of opiate positive
samples but has a high limit of detection to avoid saturation of the instrument with
excessive drugs in patients’ samples. Therefore further mass spectrometry, after drug
extraction, is carried out on any samples that meet the following criteria:

 Samples that remain positive by immunoassay for opiates but not confirmed
by the initial mass spectrometry screening method.

A semi-quantitative method is used for the opiates meeting the above criteria based on
the following cut-off levels (Table 3):
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Table 3. Cut-off values for specific opiates following urine extraction

Drug Cut-off µg/L

Morphine 10
6MAM 5
Codeine 10
DHC 10

3.3 Interpretation of Drugs in Urine

Opiates

The criteria for detection of heroin is the same as that for oral fluids above. However,
the opiate immunoassay may also detect pholcodine and on occasions an opiate
positive urine cannot be identified and an appropriate comment made to this effect.
An opiate positive urine does not always indicate that heroin has been abused.
Only the presence of 6-MAM is indicative of heroin abuse. Morphine may be
detected either as a product of heroin metabolism, codeine metabolism, administration
of morphine or from poppy seed containing foods. If we see a codeine concentration
that is very much higher than that of morphine and no 6-MAM is present then it is
more likely that the morphine was derived from codeine metabolism.

Methadone

There is considerable intra-individual variation in the metabolism of methadone with

both slow and fast metabolisers of this drug. This may result in some individuals who
are being treated with methadone showing a negative screening result by
immunoassay. Any samples that show borderline methadone levels (just under the
urine cut-off range) are further analysed by tandem mass spectrometry for methadone
and its metabolite EDDP.

Amphetamines

At present we only look for methamphetamine, amphetamine, MDMA, MDA and

ephedrine (pseudoephidrine). We do not distinguish between street amphetamine and
prescribed amphetamine. Urine samples for the latter have to be despatched to another
laboratory. If any amphetamine other than those listed needs to be looked for then the
laboratory must be informed.
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Cannabis

The immunoassay detects the carboxylic acid metabolite of cannabis and generally
this assay is interference free. Cannabis can remain in the body for a considerable
period of time in heavy users.

Cocaine

The immunoassay uses an antibody directed against the cocaine metabolite i.e.
benzoylecgonine and is generally interference free.

Benzodiazepines

The immunoassay detects a wide range of benzodiazepines and their metabolites.

However, no specific benzodiazepine is identified in this laboratory in urine.

Interpretation of drugs is related to clinical cut-off values. These cut-offs are biased to
prevent the reporting of false positive results which may occur due to interferences or
assay carry over.

4.0 OTHER DRUGS

The following can be measured if specifically requested:

Ketamine

Naltrexone : Naltrexone and its metabolite naltrexol can be measured in urine and
oral fluid.

Buprenorphine: Buprenorphine and its metabolite norbuprenorphine will be

measured in urine when requested but only for compliance purposes. The assay
detects down to 5 µg/L of these drugs.

Tramadol

Any other drug requirements than please contact laboratory.

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5.0 TURNAROUND TIMES

5.1 Oral Fluid Samples

Measured daily in batches of 50 samples.

5.2 Urines

Measured daily followed by mass spectrometry confirmation the following day.

Therefore samples are completed in 2 days following receipt. If further confirmatory
work is required then this may take 3 days to complete the analysis.

6.0 CONTACTS

Any problems relating to interpretation of results of problems regarding the service

should be repeorted to the Consultant in charge of this service or the Senior
Biomedical Scientist by phone or by e-mail (see front cover). Please do inform us
immediately of any problems so that we can investigate and rectify the situation if
need be. Ideally, please use the following email address which is opened daily and
accessed by several members of staff.

Email: toxicology@leedsth.nhs.uk