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Journal of Analytical Toxicology, Vol. 35, January/February 2011
Technical Note
Evaluation of the Integrated E-Z Split Key® Cup II for
Rapid Detection of Twelve Drug Classes in Urine
Dina N. Greene, Christopher M. Lehman, and Gwendolyn A. McMillin*
University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, Utah
Abstract
The availability of point-of-care (POC) medical devices for drug
testing has surged. Reduction in turnaround time, and hence, rapid
results are attractive, particularly to acute care facilities,
rehabilitation facilities and specialized clinics such as pain
management clinics. Here we describe our validation results for
the Integrated E-Z Split Key Cup II, a low-cost, rapid urine test that
utilizes competitive immunoassay technology to detect 12 drugs or
drug classes of commonly abused drugs. Positivity is based on the
absence of a colored band at a labeled portion of the detection
strip; a negative result produces a distinct, colored band. Using
reagent-grade standards, the apparent cut-off for each of the drugs
was challenged. The stability of the results was monitored over
time. Five urine samples known to be negative for all drug
categories and 24 patient samples confirmed positive for a total of
95 drugs and/or drug metabolites claimed to be detected by the
device were tested. Adulterants and potential cross-reacting
compounds were also evaluated. One false-positive result for
benzodizepines was observed. One false-negative result for
barbiturates was observed, but was resolved. Overall, the cups
demonstrated excellent sensitivity, specificity, and diagnostic
efficiency for all drugs represented.
Introduction
Point-of-care (POC) testing provides a means for health practitioners to receive accurate and timely laboratory test results
at the bedside. By definition, a POC test is performed near the
patient, outside of a clinical laboratory, and therefore has the potential to make results available within minutes of specimen
collection. Many POC testing methods are subject to minimal
regulatory requirements, and can be performed with specimens such as urine, capillary blood, or oral fluid, that do not require phlebotomy. POC testing may improve patient counseling
* Author to whom correspondence should be addressed: Department of Pathology, University
of Utah School of Medicine, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108.
Email: [email protected].
46
and management because results are available during a patient
visit. Successful examples of POC testing used routinely include glucose and prothrombin time monitoring to guide
glycemic and anti-coagulation management. Advances in technology have increased the availability of POC devices, and the
potential clinical benefits are attractive; however, to be effective,
POC must be easy to use, cost effective, and meet accepted
quality standards. Recent work has shown that many POC devices fail to meet quality standards, emphasizing the need for
stringent validation criteria (1).
Inappropriate drug use and drug abuse remains a common
problem clinically. It is well recognized that self-reporting of
drug use is of limited accuracy and utility. For example, when
screening for prenatal exposure to methamphetamine, a notable
percent of women that denied abuse gave birth to infants who
tested positive (2). Further, a large review of national surveys
showed that although some people will self-report drug history
accurately, external factors such as pregnancy and need for approval increase the rate of inaccurate reporting (3). Because patient management decisions regarding drug use and abuse depend on accurate drug histories, detection of drugs using POC
testing may benefit outpatient drug rehabilitation facilities,
whether used to monitor abstinence, verify compliance with
prescribed therapy, or identify inappropriate drug use. Specifically, a counselor may better manage the patient session having
real-time drug screen results available rather than waiting for
results from a test that was sent to a central laboratory and may
require several days to generate results.
Here, we evaluate the performance of the Integrated E-Z
Split Key Cup II, a user-friendly and cost-effective POC device
that detects 12 commonly abused drugs (amphetamine, barbiturates, benzodiazepines, buprenorphine, cocaine, marijuana,
methadone, methamphetamine, MDMA, opiates, oxycodone,
and propoxyphene). Using a validation procedure consistent
with CLIA guidelines, we evaluated the ability of the drug screen
cup to detect purified reagent standards for each drug class at
concentrations above and below the cutoff; the stability of the
results over time; the ability of the cup to accurately identify
multiple drugs in patient urine; and the effect of potential
cross-reacting, interfering compounds, and common adulter-
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
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ants on the results. To the best of our knowledge, this is the only
published validation for a POC device that detects 12 drug
classes.
says. There are six test strips/cup; each strip detects specific
drugs. Each strip also has its own colored control band. Urine
is distributed into the cup. The “key” is removed from the cap
and pushed into the siding of the apparatus. This unlocks the
urine and allows it to wick the membrane, traveling to the top
of the membrane via capillary action. Embedded in the memMaterials and Methods
brane are drug-protein conjugates; each test strip has corresponding anti-drug mouse monoclonal antibodies that will
POC device
travel with the urine. The anti-drug antibodies are labeled
The Integrated E-Z Split Key Cup II (Lot DOA9100739) was
with a colored conjugate. Therefore, if there is drug in the
purchased from Rapid Detect (Poteau, OK). The manufacurine, the drug will sequester the antibody, and no band will
turer’s published cutoffs for the 12 drug classes detected by the
appear at the specific area of the membrane containing the
device are listed in Table I.
drug-protein conjugate. In contrast, if the patient is negative
The drugs are detected based on competitive immunoasfor a drug, the anti-drug antibody will migrate to and bind the
embedded drug, which is visualized by a
colored band. More specifically, if urine is
Table I. Limit of Detection Measured for Each Drug Class
positive for a drug, the band is absent; if
urine is negative for a drug, the band is
Published
Confirmed
present (Figure 1).
Drug
Reference
Cutoff Concentration
LOD
Class
Standard
(ng/mL)
(ng/mL)
The control band on each strip is used
to detect proper volume of specimen,
Amphetamine
d,l-Amphetamine
3000
1250
membrane wicking, and correct proceAmphetamine
d-Amphetamine
1000
420*
dural technique. The control band does
Barbiturates
Secobarbital
300
375
not specifically detect the presence of
Benzodiazepines
Oxazepam
300
450
urine. Embedded in the membrane at the
Buprenorphine
Buprenorphine
10
10
control position is rabbit IgG. Disbursed
Cocaine
Benzoylecognine
150
150
9
with the anti-drug mouse monoclonal anMarijuana
11-nor-∆ -THC-9-COOH
50
62.5
tibodies are color conjugated goat polyMethadone
Methadone
300
450
clonal anti-rabbit IgG. Hence, any liquid
Methamphetamine
d,l-Methamphetamine
4500
1250
Methamphetamine
d-Methamphetamine
1000
300*
that properly moistens the membrane
MDMA†
MDMA
500
375
will result in the appearance of a strong
Opiate
Morphine
300
375
band in the control region of the strip.
Oxycodone
Propoxyphene
Oxycodone
Propoxyphene
100
300
100
300
* These values were calculated using measured cut offs for the racemic mixture and the published cut offs for
d,l and d isoforms (amphetamines) or d and l isoforms (methamphetamine).
† 3,4-Methylenedioxymethamphetamine.
Limit of detection and
time course study
The cutoff for each drug was challenged
using Cerilliant purified reference standards (Round Rock, TX). The standard
specific to the drug class was the calibrator published in the product package insert. The standards
were run using 0%, 25%, 50%, 75%, 100%, 125%, and, if
needed, 150% of the apparent drug-specific cutoff. We used five
separate cups for each percent tested and defined our limit of
detection to be the concentration that gave a positive drug result in all five cups after 5 min, consistent with manufacturer’s
instructions. To evaluate the stability of test results, the cups
were re-read after 30, 60, 120, and 1440 min.
Patient urine samples
Figure 1. Images generated by photocopying the Integrated E-Z Split Key
Cup II after screening urine positive for methamphetamine, amphetamine,
and propoxyphene. Positive control is indicated by the “C”; drug tested indicated either by number or “T”.
Patient urine previously confirmed positive or negative by
validated gas chromatography–mass spectrometry (GC–MS)
or liquid chromatography (LC)–tandem MS assays for drugs of
abuse were pooled to create 24 samples containing various
amounts of the drugs of interest (2–5 drugs/urine pool, see
Table II for a detailed description of sample composition) and
5 negative samples. If necessary, the pools were diluted with
blank urine. The 24 drug-positive pools and 5 drug-negative
urines were blinded before analysis. Thirty-five milliliters of the
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Table II. Detailed Description of the 24 Patient Samples Screened Using
the EZ Cup
Drug(s)
Present
Concentration
(ng/mL)
Positive Using
E-Z Cup
1
THC
Morphine
Oxycodone
Amphetamine
Methamphetamine
Methadone
160
420
170
170
4700
460
THC
Opioids
Oxycodone
Amphetamine
Methamphetamine
Methadone
2
THC
Benzoylecognine
Methamphetamine
Amphetamine
95
1220
2000
360
THC
Cocaine
Methamphetamine
Amphetamine
THC
Lorazepam
Alprozolam
α-Hydroxyalprazolam
7-Aminoclonazepam
Amphetamine
Methamphetamine
65
150
30
130
150
3680
2600
THC
Benzodiazepines
THC
Nordiazepam
Oxazepam
Temazepam
Lorazepam
Morphine
Oxycodone
Norbuprenorphine
210
830
1660
1920
470
350
550
13
5
THC
MDMA
Oxycodone
Benzoylecognine
60
410
135
280
6
THC
MDMA
Nordiazepam
Oxazepam
Temazapam
Alprazolam
Methadone
Propoxyphene
55
600
450
640
340
50
1600
370
α-Hydroxyalprazolam
7-Aminoclonazepam
Alprazolam
Nordiazepam
Oxazepam
Temazepam
Amphetamine
Oxycodone
Morphine
Propoxyphene
180
40
50
280
1100
880
4690
2800
3000
310
Amphetamine
Oxycodone
Opioids
Propoxyphene
Alprazolam
Amphetamine
370
2000
Benzodiazepines
Amphetamine
Sample
3
4
7
8
48
Amphetamine
Methamphetamine
THC
Benzodiazepines
Opioids
Oxycodone
Buprenorphine
THC
MDMA
Oxycodone
Cocaine
THC
MDMA
Benzodiazepines
urine in question was allowed to react in
the drug screen cup for 5 min before results were read. If there was a false-positive or false-negative result, the urine was
poured into a fresh cup to assess reproducibility. If a specimen was positive for a
drug that was unexpected, the drug identity was confirmed and the concentration
was quantified using an appropriate, previously validated GC–MS or LC–MS–MS
assay. The Institutional Review Board of
the University of Utah in Salt Lake City,
Utah approved all studies using human
samples.
Adulterants and additional drug
specificity
The following adulterants were individually dissolved in 30 mL drug positive
urine: 5 mL dry eye drops, 5 g Comet®
Cleaner with Bleach, 3 mL Backdown®
antibacterial hand soap, 5 mL 5.0%
gluteraldehyde, 5 mL 1.0 M sodium nitrite, 6.5 g pyridinium chlorochromate
(PCC), and 5 mL 0.1 M acetic acid. The
urine used had previously screened positive for multiple drugs, including THC.
Further adulteration studies were accomplished by testing 30 mL of non-urine
liquid: Monster® energy drink, Kroger®
sterile eye drops, and water. For all
screens, results were read after incubating
for 5 min. Cross-reactivity was assessed
using either purified reference standards
(Cerilliant) or patient samples confirmed
positive for the drug. The standards or
the patient samples were diluted in blank
urine, and results were read after 5 min.
Results
Methadone
Propoxyphene
Benzodiazepines
Limit of detection and time course
The limit of detection was defined as
the drug concentration that gave a positive result for all 5 cups after 5 min.
This value was identical to the manufacturer’s published cutoff concentration
for the following drug classes: buprenorphine, cocaine, opiates, and propoxyphene (Table I). Three of the drug classes
showed a limit of detection at 125% of
the cutoff concentration: opiates, barbiturates, and marijuana. Two drug
classes, methadone and benzodiazepines,
required 150%; 3,4-methylenedioxymethamphetamine (MDMA) was the only
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Table II (continued). Detailed Description of the 24 Patient Samples Screened
Using the EZ Cup
Drug(s)
Present
Concentration
(ng.mL)
Positive Using
E-Z Cup
8
Buprenorphine
Methamphetamine
920
1230
Buprenorphine
Methamphetamine
9
Oxycodone
Benzoylecognine
Norbuprenorphine
750
640
25
Sample
Oxycodone
Cocaine
Buprenorphine
10
Methadone
Morphine
Buprenorphine
1110
840
150
Methadone
Opioids
Buprenorphine
Benzodiazepines*
11
Hydromorphone
Morphine
Oxycodone
Oxymorphone
40
1510
400
20
Opioids
Lorazepam
Butalbitol
Codeine
Morphine
Methadone
Oxycodone
Propoxyphene
Benzoylecognine
Norbuprenorphine
Buprenorphine-glucuronide
350
4830
340
11
780
1860
210
60
7
29
MDMA
2000
Propoxyphene
Butalbitol*
26
3400
Oxazepam
Lorazepam
Methamphetamine
Amphetamine
MDMA
Propxyphene
2930
380
800
1550
400
1370
Methadone
Morphine
Codeine
6-Acetylmorphine
Methamphetamine
MDMA
THC
475
830
90
170
2030
480
4
Propoxyphene
Amphetamine
Codeine
Buprenorphine
Norbuprenorphine
Norbuprenorphine-glucuronide
THC
90
3200
510
9
29
1100
18
12
13
14
15
16
* False-positive or false-negative result.
Oxycodone
Benzodiazepines
Barbiturates
Opioids
Methadone
Oxycodone
Propoxyphene
Cocaine
Buprenorphine
MDMA
Methamphetamine
Propoxyphene
Benzodiazepines
Methamphetamine
Amphetamine
MDMA
Propoxyphene
Methadone
Opioids
Methamphetamine
MDMA
THC
Propoxyphene
Amphetamine
Opioids
Buprenorphine
THC
drug to give consistent positive results
at 75% of the expected value. The observed cutoff levels for methamphetamine and amphetamine were measured using a racemic mixture standard.
The antibodies against these stimulants
are designed to more specifically detect
the illegal d-form of the drugs, and
hence the published cutoff for the
racemic mixture is greater. The observed cutoffs for the racemic mixtures
were much lower (< 45%) than the published cutoffs: 1250 ng/mL for both amphetamine and methamphetamine compared to the published 3000 and 4500
ng/mL, respectively. Using these values
and assuming that there would be a proportional increase in sensitivity with a
pure d-isomer, the cutoff concentrations
for d-amphetamine and d-methamphetamine were calculated. For example, the measured LOD was 58% less
than the published LOD for the racemic
mixture of amphetamines. Thus, we
assumed that the d-isomer LOD would
behave similarly, and the LOD was calculated to be 58% less than the manufacturer states. A comparison of the published cutoff concentrations and the
validated concentrations are listed in
Table I.
One advantage of most POC devices,
including the Integrated E-Z Split Key
Cup II, is the rapid production of results. According to the manufacurer’s
instructions, results should be visualized after 5 min. In order to determine
how incubation time influences test results, cups used for the LOD study were
resulted after 5, 30, 60, 120, and 1440
min. The results were similar for every
concentration tested. The results for the
cups incubated with 125% or similar
concentration of the published cutoff
are shown in Table III. In general, results were stable up to 60 min, but
began to give false-negative results after
2 h. Results interpreted after 1440 min
were markedly affected, producing a significant amount of false-negative results. Of note, the two drugs that were
determined to require 150% of standard
to produce consistently positive results
at 5 min (methadone and benzodiazepines) had improved sensitivity after
30 min. These two drugs also showed
consistently positive results after 120
min using concentrations at 100% of
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the published cutoffs; concentrations at 75% of the published
cutoff did not give positive results, even after overnight
incubation.
samples screened negative, the specificity and sensitivity were
calculated to be 96.3% and 100.0%, respectively. The diagnostic efficiency was 96.6%. The root cause of the false-positive
urine result was evaluated by testing for the presence of a rePatient urine samples
acting compound (22 benzodiazepines/metabolites were exTwenty-four urine samples confirmed positive for a total of
amined); however, none was detected. The sample also tested
95 drugs of abuse and 5 urine samples negative for all relevant
negative for sertraline, a commonly prescribed antidepressant
drugs were screened (29 samples total; Tables II, IV, and V). One
that the manufacturer suggests can cause positive benzodifalse-positive and one-false negative result were observed.
azepine results, leaving the false positive unresolved.
The false-positive result was for benzodiazepines. Given that
The false-negative result was for barbiturates. This sample
13 true-positive urines screened positive and 26 true-negative
was confirmed to be positive for butalbitol at concentrations
well above the cutoff. When the specimen
was decanted into a new screening cup,
Table II (continued). Detailed Description of the 24 Patient Samples Screened
the barbiturates gave a positive result,
Using the EZ Cup
suggesting that there may be variation
between cups. Thus, with 5 true-positive
Drug(s)
Concentration
Positive Using
screening results and 23 true-negative
Sample
Present
(ng/mL)
E-Z Cup
screening results, the sensitivity, specificity, and diagnostic efficiency are 83.3%,
17
Benzoylecognine
170
Cocaine
100.0%, and 96.6%, respectively.
Phenobarbitol
2370
Barbiturates
All other drug classes had a specificity,
Temazepam
270
Benzodiazepines
sensitivity, and diagnostic efficiency of
Oxazepam
325
Nordiazepam
65
100% for the samples tested. It is noteworthy that many of the drugs gave pos18
Methamphetamine
6100
Methamphetamine
itive results at concentrations well below
THC
6
THC
the published cutoff and the limit of dePhenobarbitol
1030
Barbiturates
tection quantified using purified stanAmphetamine
1600
Amphetamine
dards. This result is not unexpected, conLorazepam
60
Benzodiazepines
sidering
the large amounts of metabolites
7-Aminoclonazepam
30
structurally similar to the parent drug of
Buprenorphine-glucuronide
5
Buprenorphine
interest that are expected to be present in
Norbuprenorphine
4
the urine of drug users. In accordance,
Norbuprenorphine-glucuronide
13
the patient results for methadone were
19
THC
2
THC
positive at levels well below the reagent
Phenobarbitol
590
Barbiturates
standard cutoff because the assay recogα-Hydroxyalprazolam
900
Benzodiazepines
nizes both methadone and 2-ethylideneAlprazolam
320
1,5-dimethyl-3,3-diphenylpyrrolidine
Benzoylecognine
700
Cocaine
(EDDP) equally. Similarly, high levels of
MDMA (> 2000 ng/mL) can lead to false20
Methamphetamine
5300
Methamphetamine
positive results for methamphetamine.
Amphetamine
830
Amphetamine
Although MDMA is not a metabolite of
Propoxyphene
440
Propoxyphene
methamphetamine, the structure of
21
Tramadol
16030
MDMA and the structures of MDMA
metabolites are very similar to metham22
Hydrocodone
8300
Opioids
phetamine. This cross-reactivity was reOxazepam
130
Benzodiazepines
ported by the manufacturer and supported by our results (Table IV).
23
7-Aminoclonazepam
750
Benzodiazepines
24
50
Hydrocodone
THC
1050
16
Fentanyl
THC
Nordiazepam
Oxazepam
Temazapam
7-Aminoclonazepam
Phenobarbitol
Oxycodone
5000
25
440
1800
1400
580
404
400
Opioids
THC
THC
Benzodiazepines
Barbiturates
Oxycodone
Adulterants
The use of household or proprietary
chemicals to alter drug screen results is
recognized to occur. Thus, knowing the
effect that frequently used adulterants
might have on the Integrated E-Z Split
Key Cup II is important. Samples that
had previously screened positive for multiple drug classes were manipulated with
liquid soap, Kroger sterile eye drops, pow-
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dered bleach, acetic acid, gluteraldehyde, or sodium nitrite.
There were no instances of these substances giving a falsenegative result; gluteraldehyde gave a false-positive result for
methamphetamine. Both the liquid soap and the gluteraldehyde weakened the intensity of the control band. False positives
Table III. Time Course of Results Using Purified Reagent
Standards at 125% the Published Cutoff*
Drug
d,l-Methamphetamine
d,l-MDMA
11-nor-∆9-THC-9 COOH
Oxazepam (BZO)
Methadone
Secobarbitol (BAR)
d,l-Amphetamine
Morphine
Oxycodone
Propoxyphene
Benzoylecgonine (COC)
Buprenorphine
5
(min)
30
(min)
60
(min)
120
(min)
1440
(min)
5
5
5
3
2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
4
5
5
5
5
5
5
5
5
5
5
0
0
5
4
4
5
4
5
5
2
5
2
* The results represent the number of cups positive from a total of 5. Bold results
indicate that 100% of cups were positive for a drug.
or weakened intensity of negative bands is expected after the
addition of these substances, as all are known to interfere with
protein interactions, and hence, antibody antigen interactions.
Most interesting was the sample adulterated with PCC. The addition of PCC inhibited the binding of all antibodies to the
membrane giving a false-positive result for all drugs being
tested, while also blocking the formation of the control band.
A more troubling result was obtained after the addition of
non-urine liquid to the screening cup. Because the control
band is only used to detect proper volume of specimen, membrane wicking, and correct procedural technique, it will produce a colored line regardless of the matrix added to the cup,
not specifically in the presence of urine. The addition of water,
Monster energy drink, or Kroger sterile eye drops all gave results that mimicked blank urine.
Cross-reactivity of additional drugs
The manufacturer provided an extensive list of compounds
that do or do not cross-react with the various drug classes
screened by the Integrated E-Z Split Key Cup II. However,
there were still commonly prescribed or abused drugs that
were not present on their list. For these compounds, patient
urine or blank urine plus purified reference standard were
screened using the cup at concentrations similar to what would
be observed in urine. Patient urine was used to test cross-reactivity of fentanyl (5000 ng/mL) and tramadol (16,030 ng/mL);
purified reagent standards were used to test cross-reactivity of
Table IV. Overview of Patient Urine Screening Results: Compounds that Screened Positive for the Various Drug Classes, the
Range of Concentrations Present in the Specimens, the Number of Samples (n) for the Specific Compound, and the Mean
Concentration of All Positive Specimens
Drug
Class
Compound
n
Concentration
Range
(ng/mL)
Mean
(ng/mL)
%
CrossReactivity*
Amphetamines
Amphetamine
8
170–4700
1920
100%
Cocaine
Benzoylecognine
6
60–1220
510
100%
Methamphetamine
Methamphetamine
MDMA
8
1
1230–17340
2000
5410
2000
100%
50%
MDMA
MDMA
5
400–2000
780
100%
Barbiturates
Butalbitol
Phenobarbitol
2
4
3400–4830
400–2370
4120
1100
12%
300%
Methadone
Methadone
5
475–1600
890
100%
Opiates
Morphine
Codeine
Hydrocodone
6
2
2
420–3010
345–830
1050–8300
1160
590
4680
100%
100%
0.60%
Oxycodone
Oxycodone
9
170–4100
1270
100%
Propoxyphene
Propoxyphene
7
15–1370
400
100%
Buprenorphine
Buprenorphine
Norbuprenorphine
2
4
150–920
4–29
540
20
100%
70%
Marijuana
11-nor-∆9-THC-9 COOH
12
2–210
60
100%
* Estimated from the detection concentration listed in the package insert by dividing the LOD for the drug class standard by the LOD of the specific drug present.
51
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tapentadol (2500 ng/mL), phentermine (17,000 ng/mL), ritalinic acid (17,000 ng/mL), selegiline (10 ng/mL), desmethylselegiline (1000 ng/mL), zolpiclone (5000 ng/mL), and zolpidem
(5000 ng/mL). The results revealed no apparent cross-reactivity with any of the tested compounds.
Discussion
Rapid detection of drugs in patient
specimens may be beneficial to acute care
facilities, rehabilitation facilities, and specialty clinics such as those used for pain
management, and emergency departments. In this study we evaluated the performance of the Integrated E-Z Split Key
Cup II, a chromatographic immunoassay
that qualitatively detects multiple drugs
and drug metabolites in urine. Overall,
the device met quality standards, displaying high diagnostic sensitivity, specificity, and diagnostic efficiency. The device
is accurate, easy to use, and cost effective, making it an ideal screen for institutions where timely results can have a
profound effect on patient care. The tight
seal of the cup allows the results to easily
be photocopied (Figure 1), facilitating patient record maintenance and standardization of reporting procedures.
Although CLIA has identified the cup to
be a moderately complex test, the drug
screening cup is remarkably easy to use.
Many POC devices are waived, meaning
that the basic regulatory guideline is to
follow the manufacturer’s instructions.
However, because the Integrated E-Z Split
Key Cup II is a moderately complex test,
the institution employing the screen
must follow more stringent guidelines regarding the training of testing personnel
(including regular competency evaluations). Most importantly, when implementing a moderately complex test, the
institution must demonstrate proficiency
through participation in an accredited
program such as that offered through the
College of American Pathologists.
The results of the cup are generally
very clear, especially if the urine is negative for a drug/multiple drugs. However, if
the cup is closely scrutinized the reader
may be able to convince himself or herself
that a drug is or is not present. For example, the cup used to analyze benzodiazepine sample 9 (Table V) showed a very
faint band. Because the intensity was
52
markedly decreased compared to surrounding bands, the specimen was resulted as positive. On confirmation, the specimen
was found to contain quantities of benzodiazepines well below
the cutoff. Thus, interpretation of results may be subjective. To
Table V. Concentrations of Benzodiazepines and Metabolites Present in Patient
Urine Samples that Tested Positive for Benzodiazepines Using the E-Z Cup
Sample
Benzodiazepine
Concentration
(ng/mL)
% Cross-Reactivity
Relative to Standard*
150
30
130
150
20%
150%
25%
40%
1
Lorazepam
Alprazolam
α-Hydroxyalprazolam
7-Aminoclonazepam
2
Nordiazepam
Oxazepam
Temazepam
Lorazepam
830
1660
1920
470
80%
100%
300%
20%
3
Nordiazepam
Oxazepam
Temazepam
Alprazolam
450
640
340
50
80%
100%
300%
150%
180
40
50
280
1100
880
25%
40%
150%
80%
100%
300%
4
α-Hydroxyalprazolam
7-Aminoclonazepam
Alprazolam
Nordiazepam
Oxazepam
Temazepam
5
Alprazolam
370
150%
6
Lorazepam
350
20%
7
Oxazepam
Lorazepam
2930
380
100%
20%
8
Temazepam
Oxazepam
270
325
300%
100%
9
Lorazepam
Nordiazepam
7-Aminoclonazepam
60
70
30
20%
80%
40%
10
α-Hydroxyalprazolam
Alprazolam
900
320
25%
150%
11
7-Aminoclonazepam
750
40%
12
Nordiazepam
Oxazepam
Temazepam
7-Aminoclonazepam
440
1800
1400
580
80%
100%
300%
40%
13
Oxazepam
130
100%
* Estimated from the detection concentration listed in the package insert by dividing the LOD for the drug class
standard by the LOD of the specific drug present.
Greene.qxd:JATLynneTemplate
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12:42 PM
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Journal of Analytical Toxicology, Vol. 35, January/February 2011
minimize bias of results, it is helpful to employ the following
guidelines: 1. read the results at least an arm’s length distance; 2. blind the reader to the patient name; and 3. run a negative control or blank specimen in parallel with the patient
sample. Archiving a photocopy of the cup may assist with resolving any discrepancies or inconsistencies over time and
among analysts. Confirmation of any result inconsistent with
expectations will also improve interpretation of results. However, sensitivity for detection of drugs, and hence, interpretation of results, is also a function of available confirmation
testing. Thus, positive results in an immunoassay such as the
EZ-Cup may reflect drugs or metabolites that are not detected
by the confirmation testing available. It is likely that this latter
scenario explains the false-positive benzodiazepine result observed in this study.
These experiments clearly support the diagnostic capabilities
of the drug testing device. However, it is important to note that
the study is limited by the use of samples with a high prevalence population (24 positive/29 negative patient samples).
The study was designed in this manner mainly for overall efficiency, but also because we expected the device to be utilized
in facilities with high prevalence of drug abuse (such as rehabilitation and pain management centers) and not for the general population. A more broadly applicable study design would
have been to test individuals at random and thus measure the
predictive values of the device in a lower prevalence population.
Government agencies require random and regular drug
abuse testing. The initial screening cutoffs for the mandatory
drug classes are published by the Substance Abuse and Mental
Health Services Administration (SAMSHA) and were recently
modified in May 2010 to be 50 ng/mL marijuana; 150 ng/mL
cocaine; 2000 ng/mL opiates; 25 ng/mL phencyclidine; 500
ng/mL amphetamines; 500 ng/mL MDMA; and 10 ng/mL 6acetylmorphine. With these cutoffs, this device would be ap-
propriate for only MDMA, cocaine, and marijuana. Although
this cup is not ideal for a SAMSHA-certified agency, the excellent performance of the screening cup encourages the validation and use of similar devices in institutions where rapid initial testing may be practical and advantageous.
Acknowledgments
The authors would like to thank Chantry Clark and Heidi
Carlisle for their helpful laboratory assistance. We are grateful
for the funding to support this study provided by ARUP Laboratories and the ARUP Institute for Clinical and Experimental
Pathology.
References
1. E. Lenters-Westra and R.J. Slingerland. Six of eight hemoglobin
A1c point-of-care instruments do not meet the general accepted
analytical performance criteria. Clin. Chem. 56(1): 44–52 (2010).
2. T.R. Gray, L.L. LaGasse, L.M. Smith, C. Derauf, P. Grant, R. Shah,
A.M. Arria, S.A. Della Grotta, A. Strauss, W.F. Haning, B.M. Lester,
and M.A. Huestis. Identification of prenatal amphetamines exposure by maternal interview and meconium toxicology in the Infant Development, Environment and Lifestyle (IDEAL) study. Ther.
Drug Monit. 31(6): 769–75 (2009).
3. E.R. Harrison, J. Haaga, and T. Richards. Self-reported drug use
data: what do they reveal? Am. J. Drug Alcohol Abuse 19(4):
423–441 (1993).
Manuscript received June 15, 2010;
revision received July 21, 2010.
53