Thursday, 25th August 2016

Drug Testing

The Significance of Blood & Urine Test Results

 

In the light of the serious consequences for the individual, and liabilities which can be incurred in the event of a positive or incorrect test result, Simpson et al[i] discussed the need for established procedures covering storage, chain of custody, confirmation of results and appropriate legal standards for ‘library’ matching of spectra from unknown substances (e.g. designer drugs) requiring identification.

Most blood and urine tests for the presence of cannabinoids differ from alcohol test results, as they measure inactive metabolites of cannabis, and not the active drug itself.  Alcohol produces clear dose-related impairment as measured by breath, blood or urine tests.  The presence of cannabinoids in urine merely signifies that the person had used or been exposed to cannabis at some point prior to the test[ii].  The Department for Transport recognizes that “tests should take into account that the effect of cannabis on driving is probably limited to a few hours at most after it is taken and therefore set aside inactive metabolites of cannabis, which remain well after it is taken by regular users[iii]

 

Cannabis Pharmacokinetics

 

The distribution of THC in body tissues is shown in fig 4 below.  Plasma levels drop dramatically following cessation of use, with increased absorption in the brain and high perfusion tissues.  Levels in body fat increase over a period of hours or days, and slowly release metabolites into the bloodstream thereafter.  The slow clearance rate from body fat is the main reason why cannabinoids can be detected in blood or urine for many days or weeks following cessation of use.

 

The major problem with measurement of metabolites is the very long detection times, owing to the rapid deposition of cannabinoids in inert fatty tissue following administration.  Johannson et al[iv] reported that total amount of THC metabolites and the levels of delta THC-acid could be followed up to 25 days after abstinence using EMIT cannabinoid assay and HPLC. Toennes & Kauert[v] noted that the nature of sample containers and preserving agents can affect test results for a variety of drugs.

 

The residual level of THC in the bloodstream occurs when THC is released from the adipose (fatty) tissues, where it is deposited shortly after smoking.  THC is also converted to its inert acid form within minutes of ingestion[vi].  The half-life of THC in fatty tissue is approximately 8 days[vii][viii].  There is no evidence that clearance rates for THC differ between naive and experienced cannabis users.  Chesher[ix] reported unmetabolised THC may be stored, and gradually released, from body fat for up to 28 days in chronic users.

 

Fig 4 - Distribution of THC in the Body

(Kreutz & Axelrod (1973)[x]

 

THC

 

Harder & Rietbrock[xi] noted the effects on plasma levels and intoxication produced by smoking different strengths of ‘joint’ at different intervals, finding that the effect of a strong (9mg) reefer  would last around 45min, or if smoked continuously a recovery within 100 minutes, with a continuous high if smoked hourly with a recovery after 150 minutes.  Weak (3mg) and hemp (1mg) reefers produced lower levels of intoxication and more rapid recovery times.

 

Chesher[xii] summarised that the inactive metabolite THC acid, formed in the liver from metabolism of THC, appears after THC in blood, and if present when the a subsequent dose is smoked, higher concentrations would ensue. He commented: “analytical data that provides a value only for the metabolite can only be validly interpreted as indicating recent consumption of cannabis ... a matter of hours or days.  For this reason quantitative determination of only the metabolite is of no value to determine possible impairment.”

 

McBurney et al [xiii]describe a study of plasma concentrations of THC in users where one subject was rejected as having a concentration of 37ng/ml prior to the test.  It is not stated when the subject had last smoked marijuana.  Perez-Reyes et al[xiv] tested concentrations in experienced marijuana smokers who had refrained for 6 days prior to the experiment.  Two cigarettes, with an average of 882mg cannabis at 1% THC (8.82mg THC), were smoked two hours apart, blood samples being taken every 5 minutes for the first 20 minutes after smoking, and at 10 minute intervals thereafter.  The first cigarette produced a level of 70ng/ml at 10 minutes roughly 17ng/ml at 20 min, and roughly 3ng/ml at 2 hours.  The second produced respective levels of 90, 17 and 5ng/ml at similar intervals after smoking.  There is a rapid rise in THC concentration during smoking, and then an equally rapid fall which levels off at roughly 30 min post-smoking and falls gradually thereafter.

 

Giroud et al[xv] studied levels of THC, 11-hydroxy THC and THC-acid in whole blood, serum and plasma samples, finding a 2.4:1 ratio between serum and whole-blood concentrations, and 1.6:1 between plasma and whole blood.

 

Agurell et al[xvi] studied THC levels in one “heavy marijuana user”.  His plasma THC was measured each day for four days before and one hour after smoking one cigarette laced with 10mg radioactively labelled THC, and for 8 days after ceasing all use.   Prior to the experiment his plasma THC was roughly 20ng/ml.  The levels of labelled and unlabelled THC both rose after smoking each cigarette, indicating that existing THC may be displaced from the fatty tissues as fresh THC is absorbed.  The pre-smoking unlabelled (i.e. residual) THC level fell steadily over the period of the experiment (20ng to 9ng to 8ng to 2ng/ml on successive days), still exceeding ten-fold the labelled (i.e. fresh) THC concentration.  After 8 days abstinence the levels were 1ng/ml unlabelled, and 0.1ng/ml labelled.  The decline during the first period of the experiment, when the subject was smoking 10mg THC per day, indicates that his normal consumption may have exceeded this level, possibly by ten-fold or more, i.e. 100mg THC per day.

 

Cone & Huestis[xvii] postulated a model for predicting the time of marijuana exposure from relative plasma concentrations of THC and THC-carboxy acid metabolite (THCCOOH).  These models were based on data from a controlled clinical study of marijuana smoking.  Such models allow prediction of the elapsed time since marijuana use based on analysis for cannabinoids from a single plasma sample and provide accompanying 95% confidence intervals around the prediction.  They noted that concentration estimates in the range of 7-29 ng/ml for amount of THC in blood is necessary for production of 50% of the maximal subjective high effect.  Their models were based on either THC concentration, or on the ratio of 11-nor-9-carboxy-delta 9-tetrahydro­cannabinol (THCCOOH) to THC in plasma[xviii], noting that their predicted times of exposure were generally accurate but tended to overestimate time immediately after smoking and tended to underestimate later times..

 

Sticht & Kaferstein[xix] estimated that the blood THC concentrations produced in a 70kg person smoking 15mg THC would peak at 7-8 minutes, after 30 minutes between 14-42ng/ml, and at 60 minutes between 7.5-14ng/ml.  Rosencranz[xx] reported that  blood levels of THC peak at 5 minutes, with a distribution half-life of 30 minutes, and elimination half-life of 18-36 hours.  For THC-acid, levels peaked at 20 minutes, with distribution and elimination half-lives of 15-30 minutes and 24-72 hours respectively.

 

Cami et al[xxi] studied the effects of expectancy on intoxication, noting a tendency toward more marked subjective effects in subjects who expected and received the drug, and that positive expectancy induced powerful subjective effects in the absence of active THC.

 

Augsberger et al[xxii] studied quantitative results of drug tests in Switzerland, finding “One or more psychoactive drugs were found in 89% of blood samples. Half of cases (223 of 440, 50.7%) involved consumption of mixtures (from 2 to 6) of psychoactive drugs. The most commonly detected drugs in whole blood were cannabinoids (59%), ethanol (46%), benzodiazepines (13%), cocaine (13%), amphetamines (9%), opiates (9%) and methadone (7%). Among these 440 cases, 11-carboxy-THC (THCCOOH) was found in 59% (median 25 ng/ml (1-215 ng/ml)), Delta(9)-tetrahydrocannabinol (THC) in 53% (median 3 ng/ml (1-35 ng/ml)), ethanol in 46% (median 1.19 g/kg (0.14-2.95 g/kg)), benzoylecgonine in 13% (median 250 ng/ml (29-2430 ng/ml)), free morphine in 7% (median 10 ng/ml (1-111 ng/ml)), methadone in 7% (median 110 ng/ml (27-850 ng/ml)), 3,4-methylenedioxymethamphetamine (MDMA) in 6% (median 218 ng/ml (10-2480 ng/ml)), nordiazepam in 5% (median 305 ng/ml (30-1560 ng/ml)), free codeine in 5% (median 5 ng/ml (1-13 ng/ml)), midazolam in 5% (median 44 ng/ml (20-250 ng/ml)), cocaine in 5% (median 50 ng/ml (15-560 ng/ml)), amphetamine in 4% (median 54 ng/ml (10-183 ng/ml)), diazepam in 2% (median 200 ng/ml (80-630 ng/ml)) and oxazepam in 2% (median 230 ng/ml (165-3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone, were found in less than 1% of the cases.”

 

Jones et al[xxiii] reported that in Australia “Over a 10-year period (1995-2004), between 18% and 30% of all DUID suspects had measurable amounts of THC in their blood (> 0.3 ng/ml) either alone or together with other drugs… The frequency distribution of THC concentrations (n = 8794) was skewed markedly to the right with mean, median and highest values of 2.1 ng/ml, 1.0 ng/ml and 67 ng/ml, respectively. The THC concentration was less than 1.0 ng/ml in 43% of cases and below 2.0 ng/ml in 61% of cases… THC concentrations in blood were higher when this was the only psychoactive substance present (n = 1276); mean 3.6 ng/ml, median 2.0 ng/ml compared with multi-drug users; mean 1.8 ng/ml, median 1.0 ng/ml (P < 0.001). In cases with THC as the only drug present the concentration was less than 1.0 ng/ml in 26% and below 2.0 ng/ml in 41% of cases… The concentration of THC in blood at the time of driving is probably a great deal higher than at the time of sampling (30-90 minutes later).

 

Metabolite or active drug?

 

It has been postulated, on the basis of experimental studies, that levels of 11-hydroxy THC (a psychoactive metabolite) in excess of 20ng/ml may be indicative of recent use[xxiv], however this study used single doses, or a short series of doses, of THC (150µg/kg) on volunteers, and would not measure residual cannabinoid levels in longer-term users.  There was a substantial variation in clearance rates, with several subjects showing total cannabinoids in urine samples (measured by EMIT) to be higher 18-22 hours after ingestion than 0-6 hours after consumption.

 

The vast majority of workplace urine tests measure not THC but the inactive metabolite - 11-Nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-acid, or THC-COOH). Manno et al[xxv] criticised reliance on THC-acid levels in urine as evidence of recent usage as “No accurate prediction of time of use is possible because THC-COOH has a half-life of 6 days” and concluded that only free THC could establish recent use - “Urinary concentrations of THC greater than 1.5 ng/mL suggests marijuana use during the previous 8-h time period.”  In 1984 Manno et al[xxvi] examined the effect of doses of 37.5µg/kg and 75µg/kg THC, finding urine cannabinoid levels to exceed the 50ng/ml cut-off for 3-4 days after the lower dose (fig 5)

 

Fig 5 – Urine Cannabinoid Profiles (Manno et al 1984)

urine cannabinoid profile 1 urine cannabinoid profile 2

 

Johansson et al[xxvii] found “An average elimination half-life (+/- SD) of 3.0 +/- 2.3 days was obtained for delta 1-THC-7-oic acid.”  Fraser & Worth[xxviii] studied urinary cannabinoids in chronic cannabis users, finding “The mean (range) of urinary Delta(9)-THC-COOH concentration was 1153ng/mL (78.7-2634) with a cut-off of 15ng/mL”. Studying oral doses of THC, Gustafson et al[xxix] found “the terminal urinary elimination t(1/2) of THCCOOH following oral administration was approximately two to three days for doses ranging from 0.39 to 14.8 mg/d.”

 

Skopp et al[xxx] studied serum cannabinoid levels of heavy (n = 12, > 1 joint/day), moderate (n = 11, < or = 1 joint/day) and light (n = 6, < 1 joint/week) smokers of cannabis for up to 48 hours after smoking cannabis, and found “For heavy users of cannabis, THC was detectable in 8 samples, and in 5 cases both biologically active compounds, THC and 11-hydroxy-THC, were present (1.3-6.4 ng THC/mL serum, 0.5-2.4 ng 11-hydroxy-THC/mL serum). Among moderate users, in 1 sample 1.8 ng THC/mL serum and 1.3 ng 11-hydroxy-THC/mL serum were determined, and another sample was tested positive with low concentrations close to the limit of detection. In serum samples of light users both analytes could not be detected, indicating that in those persons a positive finding of THC and 11-hydroxy-THC may rather result from recent consumption than from cannabis use 1 or 2 days prior to blood sampling. The concentrations of THC-COOH and its glucuronide covered a wide range in all groups of cannabis users. However, there was a trend to higher concentrations in heavy users compared to moderate users, and the mean concentration was smaller in light smokers than in moderate smokers.”

 

In a study of prison inmates following most recent reported use, Smith-Kielland et al[xxxi] reported “The plotting of THCCOOH/creatinine ratios (THCCOOH/C) versus time gave smoother excretion curves than THCCOOH concentrations alone. Based on THCCOOH/C the first 5 days after the last reported intake, the mean urinary excretion half-life was 1.3 days in infrequent users, and a median of 1.4 days was found in frequent users. In the latter group, apparent terminal urinary excretion half-lives up to 10.3 days were observed. The last positive specimens were found after 4 days for THCCOOH with cutoff 15.0 ng/mL (NIDA/SAMSHA), 5 days for THCCOOH with cutoff 10.3 ng/mL, and 12 days for cannabinoids (EMIT20) in infrequent users and after 17, 22, and 27 days, respectively, in frequent users.”

 

Lafolie et al[xxxii] established the importance of creatinine in normalising drug samples in urine of different dilutions, finding a near linear relationship between drug concentrations and urinary creatinine levels, with false negatives attributable to over-diluted urine samples. Following the effects of abstinence in a formerly chronic cannabis user, the cannabinoid-creatinine ratio showed a much steadier decline than the raw cannabinoid (THC-acid) data.

 

Fig 6 – Cannabinoid  Elimination following chronic use

(a) Cannabinoids (ng/ml)            (b) Cannabinoid/creatinine Ratio

cannabinoids creatine

Source – Lafolie et al [1991]

 

Leading researchers in this field include Cone and Huestis, who have undertaken a wide range of studies into cannabinoid excretion profiles and detection times in body fluids.  In 1998[xxxiii] they conducted a controlled clinical study “Subjects smoked a single marijuana cigarette (placebo, 1.75% or 3.55% THC) each week. Urine specimens (N=953) were analyzed under blind conditions for THCCOOH by gas chromatography-mass spectrometry. Mean+/-SEM half-lives calculated by the amount remaining to be excreted method after the low and high doses were 31.5+/-1.0 hours (range, 28.4 to 35.3 hours) and 28.6+/-1.5 hours (range, 24.9 to 34.5 hours), respectively, when a 7-day monitoring period was used. The amounts of THCCOOH excreted over a 7-day period were 93.9 +/-24.5 microg (range, 34.6 to 171.6 microg) and 197.4+/-33.6 microg after the low- and high-dose sessions. Longer half-lives, 44.3 to 59.9 hours, were obtained with a 14-day sample collection”.

 

Cannabinoid Levels after smoking single reefer cigarette of 3.55% potency (15.8mg THC)

(a)  Plasma -  Huestis et al [1992][xxxiv] (b)  Urine – Huestis & Cone [1998] [xxxv]

cannabinoid levels 1 cannabinoid levels 2

 

In a further study[xxxvi] Huestis & Cone monitored urine cannabinoids for up to 8 days following similar exposure.  In 1996[xxxvii] they reported “Mean peak urine THCCOOH concentrations averaged 89.8 +/- 31.9 ng/mL and 153.4 +/- 49.2 ng/mL after smoking of approximately 15.8 mg and 33.8 mg THC, respectively. The mean times of peak urine concentration were 7.7 +/- 0.8 h after the 1.75% THC and 13.9 +/- 3.5 h after the 3.55% THC dose. Mean GC-MS THCCOOH detection times for the last positive urine sample after the smoking of a single 1.75 or 3.55% THC cigarette were 33.7 +/- 9.2 h and 88.6 +/- 9.5 h, respectively, when a 15-ng/mL cutoff concentration was used”, and in 1995[xxxviii] studying effects of cut-off levels, found “Mean detection times increased from a maximum of 0.5 days after the low dose to 1.5 days after the high dose using the 100-ng/mL cutoff. Mean detection times were less than 1 day following the low dose and less than 2 days following high-dose exposure using the 50-ng/mL cutoff. Mean detection times ranged from 1 to 5 days after the low dose and from 3 to 6 days after the high dose using the 20-ng/mL cutoff immunoassay.

 

Ellis et al[xxxix] monitored urine cannabinoids in abstinent chronic users, and reported “under very strictly supervised abstinence, chronic users can have positive results for cannabinoids in urine at 20 ng/ml or above on the EMIT-d.a.u. assay for as many as 46 consecutive days from admission, and can take as many as 77 days to drop below the cutoff calibrator for 10 consecutive days. For all subjects, the mean excretion time was 27 days.”  Law et al[xl] noted “delta 9-THC metabolites were detected in blood for up to 5 days and in urine for up to 12 days following a single oral dose of delta 9-THC (20 mg).”

 

McBay[xli] compared THC and THC-COOH levels in a study involving smoked marijuana cigarettes.  THC-acid levels increased steadily following smoking, but were still detectable long after intoxication would have ceased.  Plasma THC levels declined rapidly following cessation of smoking, but were almost all still over 10ng/ml one hour later, and in the range of 1ng to 10ng/ml 2-4 hours after cessation of smoking.

 

Reeve et al[xlii] compared plasma THC levels with performance on the roadside sobriety test, finding that failures were associated with levels over 25-30ng/ml.  Sticht & Kaferstein[xliii] estimated that the blood THC concentrations produced in a 70kg person smoking 15mg THC would peak at 7-8 minutes, after 30 minutes between 14-42ng/ml, and at 60 minutes between 7.5-14ng/ml.

 

Although there are many papers reporting plasma THC levels, there are no papers which unequivocally relate plasma THC levels with overall consumption.  Most have been experimental studies matching short-term THC levels with perceived psychotropic effects.

 

Menetrey et al[xliv] proposed a ‘cannabis influence factor’ (CIF) value, which relies on the molar ratio of main active to inactive cannabinoids, finding a CIF “greater than 10 was found to correlate with a strong feeling of intoxication. It also matched with a significant decrease in the willingness to drive, and it matched also with a significant impairment in tracking performances.” Giroud et al[xlv] concluded “The cannabis influence factor (CIF) was demonstrated as a better tool to interpret the concentrations of THC and its metabolites in blood in forensic cases and therefore it was proposed to assume absolute driving inability because of cannabis intoxication from a CIF > or = 10. Additionally, a higher CIF is indicative of a recent cannabis abuse.”

 

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