Journal of Analytical Toxicology, Vol. 22, September 1998
Letter to the Editor
I
Evaluation of DRI Drug-of-Abuse Reagentson the
Hitachi 911
To the Editor:
Diagnostic Reagents, Inc. (DRI, Sunnyvale, CA) recently introduced a series of ready-to-use liquid homogeneous drug-ofabuse immunoassay reagents for automated analyzers. Antibodies, either monoclonal or polyclonal (depending on the assay),
are used. The principle is based on competition of a drug-labeled enzyme, glucose-6-phosphate dehydrogenase, and the free
drug in the urine sample for a fixed amount of specific antibody binding sites. In the absence of free drug in the urine, the druglabeled enzyme is bound by antibody, inhibiting enzyme activity. Thus, there is a direct relationship between drug
concentration in the urine sample and enzyme activity. The activity is determined spectrophotometrically at 340 nm by
measuring the ability to convert NADto NADH.We report here an evaluation of these reagents on a Hitachi 911, along with
correlation of results with Syva EMIT II drug-of-abuse reagents
Table I. Hitachi 911 Instrument Parameters for a Typical
for amphetamine, barbiturates, benzodiazepines, cocaine
DRI Drug-of-Abuse Immunoassay
rnetabolite, opiates, methadone, THC, tricyclic antidepressants,
and ethanol.
Samplevolume
10 pL
The DRI reagents and calibrators are liquid, require no
Reagentvolumes(each)
125 pL
preparation, and are stable until the expiration date on the
Primary wavelength
340 nm
materials (usually several months). The manufacturer
Secondarywavelength
415 nm
recommends calibration with a new lot of reagents. Parameters
Rate reaction
RateA
initially provided to us by the vendor were developed for use on
Assaypoint
16-21
a Hitachi 717. Adjustments of sample volumes, reagent
volumes, and read times were done to optimize performance on
Table II. Number of Speciments Tested Positive by Either
the 911. The parameters for a sample drug-of-abuse assay are
EMIT or DRI Reagents for Select Analytes
listed in Table I.
Negative and positive urine samples were assayed by both
SyvaEMIT
DRI
methodologies. Twelvesamples negative by Syva EMIT yielded
negative results for all DRI analytes. Table II shows the results
Amphetamines
20
13
on positive specimens. The EMIT II monoclonal amphetamine
Barbiturates
9
I0
assay yielded significantly more positive results than the DRI
Benzodiazepines
20
14
monoclonal assay. All seven additional positives by EMIT
Cocaine metabolite
20
20
contained phenothiazines, and did not have amphetamine or
Opiates
20
17
methamphetamine present. Phenothiazine interference in EMIT Methadone
12
12
rnonoclonal amphetamine assays has been described (1). The
THC
20
20
I0
I0
EMIT polyclonal amphetamine assay has much less interference Tricyclic antidepressants
Ethanol
15
13
(2). For example, chlorprornazine is not detected at 12 pg/mL.
In the past, we used the polyclonal kit in combination with the
* There was complete agreement on all nesative samples.
monoclonal EMITassay to rule out such false-positive samples.
The DRI reagent exhibits much less interference; the package
insert notes phenothiazines do not interfere up to 10,000 ng/mL Table III. Recovery of THCCOOH from Calibrators, DRI
Assay
(3).
Barbiturate analysis showed one sample positive by DRI that
was negative by Syva EMIT.The DRI reagents have a lower
threshold for detection of phenobarbital (600 ng/rnL) compared
with Syva (1500 ng/mL). This sample contained 1000 ng/mL
phenobarbital, so the observed discrepancy was expected. The
DRI benzodiazepine assay was unable to detect the presence of
7-amino-clonazepam, as shown by the six fewer positive
benzodiazepine results. Gas chromatography-mass
Assignedvalue(ng/mL)
10
20
50
100
200
Measuredvalue(ng/mL)
% Recovery
13
23
53
97
121
130
115
106
97
60
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403
Journal of Analytical Toxicology,Vol. 22, September1998
spectrometry (GC-MS) verified that these six samples contained cionazepam metabolite. This is a significant deficiency of the
DRI reagents for our patient population, in which usage of clonazepam has been a problem. There were three fewer positive
opiate samples detected by the DRI reagents. GC-MS showed that these three samples all contained morphine at a
concentration near 100 ng/mL, which is below the cutoff of both assay systems. We have occasionally noted that the Syva
reagents were more sensitive than the manufacturer's cutoff (i.e., 300 ng/mL), yielding positive opiate results that are
subsequently shown by GC-MS to be at levels below this threshold.
There was complete agreement between the two assays with regard to cocaine, methadone, THC, and tricyclic antidepressant
results. The 10 samples positive for a tricyclic antidepressant were from patients prescribed either doxepin, nortriptyline,
arnitriptyline, imipramine or desiprarnine. There were two discrepancies noted for ethanol that were close to the assay's
sensitivities and not considered clinically important. Two samples read just above the ethanol cutoff of 10 mg/dL by Syva and
just below this cutoff by DRI.
Calibrations were shown to be stable over a 9-day period. SentryTM positive and negative controls (J&S Medical Associates,
Inc., Natick, MA) were assayed each day; these controls contained negative and positive values at • 25% of the assay's threshold.
Absorbance values were stable over a 9-day period, with no decreasing trend observed. We did not assay the quality control
sample beyond 9 days. In addition, the absorbance values of calibrators and controls obtained with DRI reagents spanned a
greater range than those obtained with the corresponding Syva EMIT reagents. This indicated a steeper calibration slope was
obtained with the DRI reagent system, which is an advantage in terms of accuracy and precision of an assay, even if just
qualitative.
The ability to semiquantitate THC using the DRI reagents was assessed. Calibrator levels of 20, 50, and 100 ng/mL were
provided by DRI along with a 200 ng/mL assayed control; these were run as unknowns. A 10-ng/mL concentration was made by
diluting the 20-ng/rnL calibrator. Recovery of these samples is shown in Table III. There was good recovery demonstrated from
10 to 100 ng/mL, but the curve flattened out beyond 100 ng/mL. Levels at 75 and 125 ng/mL were made by diluting the
200-ng/mL assayed control. The mean of six separate runs for each of these calibrators was 77.5 • 5.2 and 113 • 2.8,
respectively. There was good recovery (103%) of the 75-ng/mL standard, but much less recovery (90.4%) of the 125-ng/mL
standard. We agree with the manufacturer's claim for linearity of this assay up to 100 ng/mL. Five patient samples were
quantitated for THC by both Syva and DRI methods, and results were equivalent.
In summary, we found the performance of all the DRI assays, with the exception of the benzodiazepines, to be suitable for our
patient population drug-of-abuse testing needs. The DRI amphetamine assay offers advantages over the Syva EMIT monoclonal
amphetamine reagent and demonstrates much less interference from phenothiazines. The THC assay showed suitable accuracy
for semi-quantitation of this analyte. However, the Syva reagents detect clonazepam metabolite far better, so we will continue to
use these reagents for detection of benzodiazepines.
Acknowledgments
We thank Diagnostic Reagents, Inc. for providing reagents for the study and Linda Nishimoto for technical assistance.
V. Haver, D. Black, and J. Garbin
VAPuget Sound Health Care System
Pathology and Laboratory Medicine Service (113)
1660 South Columbian Way
Seattle, Washington 98108
References
1. A. Warner, D. Hohnadel, and A. Pesce. Professional Practice in Toxicology: A Review. AACC, Inc., 1993, p 351.
2. Syva Company EMIT II monoclonal amphetamine/methamphetamine assay package insert, 1992.
3. Diagnostic Reagents, Inc., amphetamines enzyme immunoassay package insert, 1997.
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