BIINFO02 v 3Page 1 of 10
TESTING FOR SUBSTANCE ABUSE
TOXICOLOGY LABORATORY
CLINICAL BIOCHEMISTRY
LEEDS TEACHING HOSPITALS
BRITANNIA HOUSE
MORLEY
LEEDS
LS27 0DQ
October 2008
Contacts:
K R Allen FRCPathTel. 0113 3927850
Consultant Clinical Scientistemail
Shirley Twigger MSc, FIBMSTel. 0113 3927883
Advanced Biomedical Scientistemail
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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.
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 metabolismwhich 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.
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 SAMHSAcut-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.
Table 1. Drugs/Drug metabolites measured in Oral Fluid
Drug/Metabolite / Detection Time(Approx) / Cut-Off
(µg/L) / SAMHSA Cut-Off
(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
Amphetamine Group / 12-24 hrs
10-20 hrs / 0.4
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. Ifdiazepam 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.
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, acharacteristic 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 toEDDP 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
Cocaineis metabolised to benzoylecgonineand 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.
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 tothe 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.
Table 2. Drugs measured by Immunoassay
Drug Class / Detection Time (approx) / Cut-offOpiates / 2 days / 300 µg/L
Methadone / 2 days / 300 µg/L
Methadone
(maintenance dosing) / 7-9days / 300 µg/L
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)
triazolam / 1 day / 200 µg/L
Benzodiazepines (intermediate)
temazepam / 40-80 hours / 200 µg/L
Benzodiazepines (long acting)
Diazepam, Nitrazepam
Amphetamines / 7 days or more
2-3 days / 200 µg/L
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 Pholcodinedo 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):
Table 3. Cut-off values for specific opiates following urine extraction
Drug / Cut-off µg/LMorphine / 10
6MAM / 5
Codeine / 10
DHC / 10
3.3 Interpretationof 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 negativescreening 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.
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.
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: