Journal of Automatic Chemistry, Vol. 19, No. 5 (September-October 1997) pp. 169-173
Development and analytical performance
of an automated screening method for
cannabinoids on the Dimension(R)
clinical
chemistry system
D. M. Obzansky, E. G. Gorman, S. P. Kramer,
I. S. Masulli, E. A. Nuzzaci and W. F. Skogen
Dade Chemistry Systems Inc., PO Box 6101, Newark, DE 19714, USA
Afully automated, random access methodfor the determination of
cannabinoids (UTHC) was developed for the Dimension(R)
AR
and XL clinical chemistry systems. The method utilizes Abuscreen
ONLINE(R)
reagents and a multianalyte liquid calibrator con-
taining l-nor-Ag-THC-9-carboxylic acid. Within-run and
total reproducibility, determined using NCCLS protocol EP5-
T2, was less than 0.6% and 1.6% CV, respectively, at all
concentrations. Calibration stability was retainedfor at least 30
days. An extensive evaluation ofnon-structurally related drugs and
various physiological substances indicated lack of interference in
the method. No sample carry-over was observed following a
specimen containing 1886 ng/ml 11-nor-A
-THC-9-carboxylic
acid. A 99.1% agreement (N 445 samples) was found
between an EMIT< based method on the aca(R)
discrete clinical
analyser and the Dimension(R)
UTHC method.
Dimension@ clinical chemistry system and aca@ discrete clinical
analyser are registered trademarks ofDade International.
Introduction
Laboratory support has been recognized for many years
to be an integral part of the hospital service for patients
with drug abuse problems. There continues to be labora-
tory demand for high-quality screening immunoassays
for all of the abused drugs including cannabinoids. To
provide workstation consolidation and rapid turn-
around-times, assays for the detection of cannabinoids
need to be available on a high throughput analyser as
part of an overall drug of abuse panel. This paper
describes the development and performance of a canna-
binoid method (UTHC) for the Dimension(R)
AR and XL
clinical chemistry systems. The method provides a high
quality, rapid screening approach for measuring canna-
binoids on these general chemistry analysers. Stable
liquid reagents, calibrators and controls are utilized to
avoid reagent preparation steps. Since the Dimension@
clinical chemistry system is used for the measurement of
many other analytes [1], the benefits of workstation
consolidation are realized.
Materials and methods
Reagents
The Dimension@ UTHC method utilizes Abuscreen
ONLINE reagents (ONLINE is a registered trademark
of Roche Diagnostic Systems, Inc., Branchburg, NJ
08876-1760) packaged in FlexTM
cartridges (Cat. No.
DF95, Dade Chemistry Systems Inc., PO Box 6101,
Newark, DE 19714). Each UTHC cartridge contains
20 tests of matched reagents.
The UTHC method is calibrated with a multianalyte
liquid calibrator (Cat. No. DC40, Dade Chemistry
Systems Inc., PO Box 6101, Newark, DE 19714) that
contains eight drugs of abuse analytes in a human urine
matrix. The cut-off for the UTHC method is 50ng/ml
(11-nor-Ag-THC-9-carboxylic acid).
Positive and negative multianalyte liquid controls (Cat.
No. DC43 and DC44, Dade Chemistry Systems Inc., PO
Box 6101, Newark, DE 19714) have THC concentrations
at approximately 25 and 75 ng/ml 11-nor-Ag-THC-9
carboxylic acid, respectively.
Other materials
The UTHC method on the aca(R)
discrete clinical analy-
ser was used in accordance with the manufacturers
instructions (Dade Chemistry Systems Inc., PO Box
6101, Newark, DE 19714). Lyphochek(R)
urine toxicology
controls were obtained from Bio-Rad (ECS Division,
3726 E. Miraloma Ave., Anaheim, Ca., 92806). The
ONLINE@ THC calibrator was obtained from Roche
Diagnostic Systems, Inc. (Branchburg, NJ 08876-1760).
Instrumentation
The UTHC method is designed to run on Dimension(R)
AR and XL clinical chemistry systems (Dade Chemistry
Systems Inc., PO Box 6101, Newark, DE 19714).
Assay principle
Each test is initiated by the addition of 90 gl of diluent
and l15gl of monoclonal antibody to the reaction
cuvette. After a 60s incubation period, 26 gl of sample
and 59 gl of water are added to the reaction. After 78 s,
60 gl of microparticle reagent, with cannabinoid deriva-
tive bound to the particle surface, are added. Cannabi-
noids, if present in the sample, compete with the
microparticle reagent for the antibody, thereby decreas-
ing the rate of aggregation. Thus, the rate of aggregation
is inversely proportional to the concentration of canna-
binoids present in the sample. The rate of particle
aggregation, as measured by absorbance changes at 510
and 700 nm, is determined over the next 143 s.
The absorbance change at the cut-off (50ng/ml) cali-
brator is normalized during calibration to provide a
reference numerical value arbitrarily set to 1000. These
0142-0453/97 $12.00 (C) 1997 Taylor & Francis Ltd
169
D. M. Obzansky et al. Development and analytical performance of an automated screening method for cannabinoids
units are referred to as 'normalized qual units'. Negative
results are less than 1000 normalized qual units and
positive results are greater than or equal to 1000 normal-
ized qual units. The instrument automatically calculates
and prints the results of the UTHC analysis in normal-
ized qual units, as well as the interpretation of the result
as positive or negative.
Results
Assay optimization
Antibody, particle reagent, diluent and sample volumes
were determined using simultaneous variable co-optimi-
zation (RS/Discover by Bolt, Beranek and Newman,
Inc.) and linear optimization techniques. Diluent volume
had the most pronounced effect on the rate of particle
aggregation, especially with drug-free samples. Sample
size was the most significant factor to influence the shape
of the calibration curve (figure 1). A 26 gl sample size was
selected to provide optimal precision around the 50 ng/ml
cut-off, to provide a reasonable calibration curve up
to 100ng/ml and to minimize false positive and false
negative results with clinical samples.
Reproducibility
For within-run and total reproducibility experiments, the
method was calibrated on the first day of the study.
Calibrator, quality controls and urine pools were then
analysed in duplicate for 20 non-consecutive days with-
out recalibration. The within-run and total coefficients of
variation (% CV), expressed as normalized qual units,
were calculated using analysis of variance (NCCLS
protocol EP5-T2). The results, shown in table 1, indicate
that within-run reproducibility is less than 0.6% and
total reproducibility is less than 1-6% at all levels tested.
Expressed in concentration units (ng/ml), the within-run
and total reproducibility at the 50 ng/ml cut-off are 2.1%
and 6.5%, respectively.
In addition, the within-run reproducibility of the method
was further tested using drug-free urine samples spiked
with 40 and 60 ng/ml 11-nor-Ag-THC-9-carboxylic acid.
These samples were tested with 20 replicates per level on
two days across five analysers. All of the 400 results were
130
Sample Size
2 5
52 gl
2 0
40 gl
26 gL
17 gL
(aE 115
c 11 gL
eo 110
Em, 105
100
95:
9 0
0 2 0 4 0 6 0 8 0 100 120 140 160
ng/mL THC
Figure 1. Effect ofsample size on shape ofcalibration curve.
Table 1. Reproducibility of the UTHC method using NCCLS
protocol EP5- T2.
%cv
Mean (Normalized
(Normalized
qual units)
Material ng/ml qual units) Within-run Total
Urine pool 20 877 0.42 0.60
ONLINE(R)
25 897 0.37 1.0
THC calibrator
ONLINE(R)
50 996 0.46 1.51
THC calibrator
Lyphocheck 60 1091 0.52 0.90
Screen control
Urine pool 86 1123 0.57 0-75
ONLINE(R)
100 1131 0-50 0.92
THC calibrator
Lyphocheck 164 1133 0.41 0.46
Screen control
(Lyphocheck is a registered trademark of BIO-RAD, ECS
Division, 3726 E. Miraloma Avenue, Anaheim, CA 92806.)
classified correctly as positive or negative compared to
the 50 ng/ml cut-off calibrator.
Sample carry-over was evaluated by interleaving five
replicate groups of drug-free urine samples and a sample
containing 1886 ng/ml 11-nor-Ag-THC-9-carboxylic
acid. No detectable sample carry-over (<2 ng/ml) was
observed in any of the drug-free samples.
Stability
To determine the calibration interval for the UTHC
method, the method was calibrated on day with the
50 ng/ml cut-off calibrator and samples containing 25, 60
and 100 ng/ml 11-nor-A-THC-9-carboxylic acid were
tested (figure 2). The normalized qual units at each ofthe
levels were converted to analyte concentration (ng/ml)
using a logit equation and a linear regression analysis
performed (y =ng/ml THC, x day). The slopes of
these regression equations were -0.007, 0.155, and
1200
1100
lOOO
900
800
50 ng/mL Cutoff
00--6 o0OO
60 nglmL
25 nglmL
100 nglmL
0 5 10 15 20 25
Day
3O
Figure 2. Calibration stability" urine controls containing 25, 60
and 100 ng/ml 11-nor-A
-THC-9-carboxylic acid were processed
using four replicates per level per day. Results are expressed as
normalized qual units calculated using the day 1 calibration.
170
D. M. Obzansky et al. Development and analytical performance of an automated screening method for cannabinoids
1200
11oo
lOOO
900
8 0 0 25 nglmL 1
50 ng/mL
l
7 0 0 75 ng/mL
60 ng/mL
600
0 5 0 O0 150 200 250 300 350 400
Day
Table 2. The concentration ofcannabinoids required to produce a
response equivalent to that ofthe 50 ng/ml cut-offcalibrator
Concentration equivalent
to 50 ng/ml THC cut-
Compound offng/ml
8-c-Hydroxy-Ag-THC 157
11-Hydroxy-Ag-THC 386
8-/3-11-Dihydroxy-Ag-THC 344
A THC 242
11-Hydroxy-cannabinol 2714
Cannabinol 1360
11-Nor-Ag-THC-9-carboxylic acid 50
Figure 3. Long term stability" the mean results of10 replicate sets
per level were obtainedfrom reagents calibrated on day 1 of the
study.
0.147 ng/ml per day at the 25, 60 and 100 ng/ml 11-nor-
Ag-THC-9-carboxylic acid concentrations, respectively.
From these regression slopes, the analyte concentrations
had changed less than 10% after 30 days. Based on these
results, and results from multiple reagent lots, calibration
of the UTHC method was determined to be stable for at
least 30 days.
The long-term reagent stability of the UTHC method
was determined by testing urine controls containing 25,
50, 60 and 75 ng/ml 11-nor-Ag-THC-9-carboxylic acid
for 394 days. The means of the normalized qual units
(N 10 on each test date) at each of these levels is shown
in figure 3.
Specificity
The specificity of the UTHC method to various canna-
binoids and cannabinoid metabolites was determined by
spiking each compound into drug-free human urine and
determining the approximate quantity needed to give a
response equivalent to that of the 50ng/ml 11-nor-A
-
THC-9-carboxylic acid cut-off calibrator (table 2).
To additionally characterize the method's specificity, 79
non-structurally related drugs were added to aliquots of
pooled drug-free normal human urine at a concentration
of 100 000 ng/ml and found to be negative in the UTHC
method (table 3).
Potential interferences for the UTHC method were tested
by adding various physiological substances and other
compounds to a human urine pool containing 50ng/ml
l-nor-A-THC-9-carboxylic acid. All responses from
these samples corresponded to concentrations between
45 and 55 ng/ml ll-nor-A-THC-9-carboxylic acid
(table 4).
Method comparison
Four hundred and forty five urine specimens were tested
for cannabinoids using the Dimension and aca analysers.
The cannabinoid method on the aca analyser is based on
EMIT reagents (EMIT is a registered trademark of
Behring Diagnostics, Inc., San Jose, CA 95161-9103).
For both systems, results greater than or equal to the
50 ng/ml calibrator were considered positive. All samples
that tested positive by either or both methods were
confirmed by GC/MS. Results are shown in table 5.
One sample was negative on the aca analyser and
positive by both Dimension(R)
and GC/MS (86ng/ml).
Three samples were positive on the aca(R)
analyser and
negative on Dimension(R). These samples contained THC
concentrations of 12, 15 and 17 ng/ml by GC/MS.
Discussion
A qualitative screening method for the detection of
cannabinoids (UTHC), along with a multianalyte liquid
calibrator, and a positive and negative quality control,
have been developed for the Dimension(R)
AR and XL
clinical chemistry systems. The UTHC method running
in a random access mode delivers a result in 9min.
Studies (data not shown) where the UTHC method
was interleaved with other general chemistry methods,
demonstrated no interference.
The Dimension(R)
cannabinoid method uses ONLINE(R)
reagents and instrument specific method parameters that
were optimized for method reproducibility and clinical
performance using a single point 50ng/ml calibrator.
Previous reports have demonstrated that lowering the
cannabinoid cut-off from 100 to 50ng/ml can increase
efficiency and sensitivity with only a minor decrease in
specificity [2]. In addition, the US Department ofHealth
and Human Service Guidelines for forensic urine drug
testing now specify a 50 ng/ml cut-off.
Within-run and total reproducibility, determined using a
20 day NCCLS protocol (EP5-T2) were excellent for the
UTHC method. While authors often compare reprodu-
cibility for drug of abuse methods in terms of absorbance
units, it is more appropriate to transform absorbances to
concentrations and to compare reproducibility in con-
centration units [3]. The normalized qual units as shown
in table were converted to concentration units. At the
50 ng/ml cut-off the within-run and total reproducibility
were 2-1 and 6.5% respectively.
The calibration stability results presented in figure 2
clearly indicate that the method retains acceptable cali-
bration for at least one month. Results from the long-
171
D. M. Obzansky et al. Development and analytical performance of an automated screening method for cannabinoids
Table 3. lO0000ng/ml of these compounds were added to drug-free human urine and
determined to give a negative result in the UTHC method.
Acetaminophen Ecgonine Morphine
Acetylsalicylic acid Ecgonine methyl ester Naloxone
Aminopyrine Ephedrine Naltrexone
Amitriptyline Epinephrine Naproxen
Amobarbital Erythromycin Niacinamide
Amphetamine Estriol Norethindrone
Ampicillin Fenoprofen Oxazepam
Ascorbic acid Furosemide Penicillin G
Aspartame Gentisic acid Pentobarbital
Atropine Glutethimide Phencyclidine
Benzocaine Guaiacol glycerol ether Phenobarbital
Benzoylecgonine Hydrochlorothiazide Phenothiazine
Benzphetamine 5-Hydroxyindole- Phenylbutazone
Butabarbital 3-acetic acid Phenylpropanolamine
Caffeine 5-Hydroxyindole- Procaine
Calcium hypochlorite 2-carboxylic acid Promethazine
Chlordiazepoxide Ibuprofen Quinidine
Chloroquine Imipramine Quinine
Chlorpheniramine Isoproterenol Secobarbital
Chlorpromazine Ketamine Sulindac
Cocaine Lidocaine Tetracycline
Codeine LSD Tetrahydrozoline
Dextromethorphan Melanin Trifluoperazine
Dextropropoxyphene Meperidine Verapamil
Diazepam Methadone Zomepirac
Diphenhydramine d-Methamphetamine
Diphenylhydantoin Methaqualone
Dopamine Methyprylon
Table 4. These compounds were added at the concentrations shown to a human urine pool contain-
ing 50ng/ml ll-nor-A9- THC-9-carboxylic acid and tested in the UTHC method.
Ammonia 1.8 mg/1 Magnesium 1.0 g/1
Ascorbic acid 1.0 g/1 Methsuximide 0.08 g/1
Amikacin 0.2 g/1 NaOH 0.1%
Bilirubin 20 mg/1 Nicotine 0.1 g/1
Boric acid 0.5% Osmolality (as NaC1) 50mOsmol/kg
Calcium 1.5 g/1 1400 mOsmol/kg
Carbamazepine 0-2 g/1 Oxalic acid 1.0 g/1
Chloramphenicol 0.3 g/1 Penicillin V 0.8 g/1
Cimetidine 0.01 g/1 Phenytoin 0.1 g/1
Citric acid 2.0 g/1 Phosphorus 5-0 g/1
Creatinine 8.0 g/1 Primidone 0-1 g/1
Digoxin 3.0 gg/1 Protein 120 g/1
Ethanol 3.5 g/1 Salicylic acid 0.1 g/1
Ethosuximide 0.3 g/1 Sodium azide 0-5%
Gentamicin 0.2 g/1 Sodium fluoride 0.01 g/1
Glucose 25 g/1 Theophylline 0.1 g/1
HC1 0.1 N Thimerosal 0.1%
Haemoglobin 5.0 g/1 Thymol 2.0 g/1
Heparin (porcine) 8000 U/1 Urea nitrogen 17 g/1
Ketones (acetone) 0-4 g/1 Uric acid 0.15 g/1
Lithium 0-04 g/1 Valproic acid 0.5 g/1
term reagent stability study, shown in figure 3, demon-
strate that the flex reagents are stable for at least one year
after being manufactured.
The specificity of the UTHC method to various canna-
binoids and cannabinoid metabolites is shown in table 2.
The single most abundant marijuana metabolite found
in urine after smoking or ingestion is 11-nor-Ag-THC-9
carboxylic acid [4]. Therefore, a monoclonal antibody
having good reactivity to this metabolite was utilized
in the method. As 11-nor-Ag-THC-9-carboxylic acid is
used to calibrate this method, samples containing greater
than 50ng/ml l-nor--THC-9-carboxylic acid, or
other metabolites having reactivity equivalent to a
50ng/ml calibrator, will produce a positive result in the
method.
172
D. M. Obzansky et al. Development and analytical performance of an automated screening method for cannabinoids
Table 5. Cannabinoid method comparison between the Dimen-
sion(R)
and aca(R)
analysers.
aca(R)
aca(R)
Positive Negative
Dimension(R)
positive 85
Dimension(R)
negative 3 356
and a subset of 188 GC/MS confirmed positive samples
from 18800 total screened samples, RIA (Immunalysis
kits), EMIT(R)
II, CEDIA(R), ONLINE(R)
and TDx(R)
detected 100, 86, 89, 89 and 87%, respectively, of the
positives samples [7]. Variation of detection rates be-
tween immunoassay systems is probably due to differ-
ences among individual drug users' metabolism along
with differences in antibody specificity [8,9].
The clinical performance of the Dimension(R)
UTHC
method was demonstrated by comparison to an EMIT
based method on the aca(R)
analyser using 445 samples
(table 3). A 99-1% agreement between these methods
was observed. Three samples, positive on the aca(R)
and
negative on Dimension(R), with immunochemical reactiv-
ity near the cut-off of 50 ng/ml, contained concentrations
of 12-17 ng/ml by GC/MS. For the population ofsamples
evaluated in this study, aca(R)
(EMIT(R)
reagents) and
Dimension(R)
(ONLINE(R)
reagents) detected 99% and
97%, respectively, of samples confirmed positive by GC/
MS. All positive cannabinoid results were confirmed as
positive by GC/MS utilizing a cut-off of 10 ng/ml. These
differences in cannabinoid detection rates are typical.
RIA (Roche Abuscreen) and ONLINE(R)
reagents (on a
Hitachi 717 analyser) detected 99% of a population of
353 GC/MS confirmed positive samples, whereas
EMIT(R)
II reagents (on a Hitachi 717 analyser) detected
88% and TDx(R)
(TDx is a registered trademark of
Abbott Laboratories) detected 95% of these same sam-
ples [5]. In another study of 100 EMIT(R)
positive THC
samples, the Triage(R)
Panel for Drugs ofAbuse (Triage is
a registered trademark of Biosite Diagnostics, Inc.) de-
tected 93% [6]. Using the Hitachi 717 analyser with
EMIT(R)
II, CEDIA(R)
(CEDIA is a registered trademark
of Microgenics Corporation) and ONLINE(R)
reagents,
In conclusion, the cannabinoid method developed for use
on the Dimension(R)
AR and XL chemistry systems
provides a precise, accurate and rapid screening ap-
proach for measuring cannabinoids on these general
chemistry analysers. Since the Dimension(R)
is used for
the measurement of many other analytes in a true
random access mode, the benefits of workstation conso-
lidation may be realized.
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