Download Urinary Excretion of ImmunologicallyReactive

CLIN.CHEM.23/2, 241-244 (1977)
Urinary Excretion of ImmunologicallyReactive
Metabolite(s) after IntranasalAdministrationof
Cocaine, as Followed by Enzyme Immunoassay
Craig Van Dyke,1 Robert Byck,2 Paul G. Barash,3 and Peter Jatlow4
Using the enzyme immunoassay technique (EMIT), we
determined the time course of urinary excretion of benzoylecgonine in 16 surgical patients and three volunteers
who received intranasal cocaine. After doses varying from
13 to 130 mg, the test for benzoylecgonine was positive
in ito 4 h, peaked at about 10 to 12 h, remained positive
for 18 to 27 h, and became negative after about 27 h. This
information should be considered by drug dependency
treatment programs in which the EMIT procedure is used
to screen urines for cocaine use.
AdditIonal Keyphrases: screening for drug abuse
assay
.
drug
Identification
of cocaine use by means of urine drug
screening depends on detection of its polar metabolite,
benzoylecgonine
(BE) (1-3). Although other methods
for detection of BE in the urine have been published
(2-8), the procedure most widely used for large-scale
urine screening is EMIT.5 Using this procedure, we
determined the time course of urinary excretion of BE
in 16 surgical patients and three volunteers who received
known doses of cocaine. This type of data, not presently
available, provides guidelines for interpreting the results
of this assay technique
when used for drug-abuse
screening.
The EMIT procedure has been well described and
verified (9, 10). This nonisotopic competitive binding
immunoassay
is rapid and yields a semiquantitative
Department
Pharmacology,2
Laboratory
of Psychiatry,’
Departments
Department
of Anesthesiology,3
Medicine,4
Yale University
of Psychiatry
and
and Department
of
School of Medicine, New
Haven,
Conn. 06510.
5
(Enzyme
Multiplied
Immunoassay
Technique)
registered trademark
of Syva, Palo Alto, Calif. 94304.
Received Oct. 20, 1976; accepted
Nov. 26, 1976.
is a
estimate of the concentration
of BE in the urine, although in screening programs it is generally used only
to detect the presence or absence of BE. Common and
important
questions
related
to all urinary
drugscreening
procedures
are:
1. How soon after drug use are results positive?
2. How long do results remain positive?
3. What is the relationship
between dose and test
response?
As part of a larger study, we had the unique opportunity to answer these practical questions as they relate
to cocaine.
Materials and Methods
Sixteen surgical patients (11) received cocaine as a
vasoconstrictor
before nasal intubation or before nasal
surgery. Three of these underwent cardiovascular
surgery (cardiovascular
patients),
11 underwent
dental
surgery (dental patients),
and two underwent
nasal
surgery. The three volunteers were all men who had
used cocaine previously for recreational purposes. Both
the patients and the volunteers were fully informed
about the study and agreed to participate.
A solution
of cocaine hydrochloride
(100 g/liter) was applied topically to the nasal mucosa of the cardiovascular patients,
dental patients, and the volunteers. The nasal surgery
patients
received crystalline
cocaine hydrochloride
applied topically to the nasal mucosa with a cotton applicator wetted with isotonic saline. One additional
volunteer8 received intravenous
cocaine hydrochloride,
6Urjne
Fischman,
samples
Ph.D.,
from this volunteer were provided
Ph.D. (Department
and C. R. Schuster,
by M. W.
of Psychi-
atry, University of Chicago).
CLINICAL CHEMISTRY, Vol. 23, No. 2, 1977
241
Table 1. Time Course of Benzoylecgonine Excretion in the Urine
of
Time
Subject
151
sample
let
Peak
concn,
mg/liter
Last
positive
Time of
let
negative
hours
Last
sample
weight.
Dose,
no.
kg
mg/kg
1
60
1.5
49 F
3
3
2
3
79
1.5
1.6
55 M
25 M
2
2
2
2
4
72
51
1.5
43 M
25 F
3
1
7
3
1
7
12
9
9
9
8
6
8
6
8
12
4.2
9
24
24
36
35
84
83
12
9
3.0
2.9
22
21
34
71
21
7
20
31
17
69
72
23
59
30
32
83
66
80
71
Age,
sex
positive
hours
peak
3
12
15
24
2
24
7
10
23
3
23
13
23
23
23
46
70
1.7
1.5
7
65
1.5
8
68
60
63
1.5
1.5
67 F
19 F
20 F
1.5
28 M
10
10
50
57
1.5
24 F
9
9
1.5
1.5
28 F
21 M
8
11
8
11
17
1.5
1.5
44 M
8
18
22
3.7
1.5
8
18
22
8
21 M
22 M
4.4
5
6
9
10
11
12
13
14
15
80
87
19 F
16
64
64
17a
72
0.38
25 M
2
3
3
17b
17c
18a
72
72
0.75
1.5
25 M
25 M
1
1
2
2
4
2
68
68
0.19
0.38
26 M
6
6
27
68
74
1.5
0.38
26 M
26 M
2
3
4
3
30 M
6
61
61
61
0.26
0.52
0.52
M
M
M
18b
18c
19
20a
20b
20c
46
17
29
55
15
19
18
46
1.3
27
17
3
1.6
9
20
29
23
36
29
42
36
6
6
2.1
18
18
5
5
8
4.0
8
8
1
1
1
1
1
2
4.2
14
17
17
7
10
17
19
Subjects 1-16 were surgical patients undergoing cardiovascular, dental, or nasal surgery; subjects 17-19 were volunteers who received intranasal cocaine;
subject 20 was a volunteer who received intravenous cocaine. Because of the logarithmic relationship in the EMIT technique, concentrations >5.0 mg/liter were
reported to the nearest whole number. Concentrations <1.0 mg/liter were considered negative.
injected over 1 mm. Doses in the surgical patients were
1.5 to 1.7 mg/kg of body weight (total of 75 to 130 mg);
doses in the volunteers ranged between 0.19 and 1.5
mg/kg of body weight (total of 13 to 108 mg). The volunteers received no other drugs. However, the surgical
patients
received diazepam
(10 mg/70 kg of body
weight) and morphine sulfate (10 mg/70 kg of body
weight) intramuscularly
1 h before induction of general
anesthesia. The cardiovascular
patients were given dia.zepam or sodium pentothal and either succinylcholine
or pancuronium
bromide intravenously
for muscle relaxation, while the dental and nasal surgery patients
were given sodium pentothal and succinylcholine
intravenously. All surgical patients received nitrous oxide
and halothane as maintenance
anesthetics.
We obtained double-voided
(i.e., collected 15 mm
after a preliminary
voiding) urine specimens before
cocaine administration
and at intervals afterwards for
as long as was practical, in some patients as long as 84
h. NQ samples were collected from cardiovascular
patients after they were placed on cardiopulmonary
by242
CLINICAL CHEMISTRY, Vol. 23, No. 2, 1977
pass because the associated hemodilution
would have
rendered subsequent data on urinary concentrations
of
BE invalid. For this reason we do not have urine specimens on cardiovascular
patients beyond 3 h. Twentymilliliter samples of urine were collected in glass bottles
and frozen (-15 #{176}C)
immediately afterwards. All samples from each experiment were analyzed on the same
day. We did EMIT analyses according to the manufacturer’s instructions,
using the Syva assembled system, which consists of a Model 300-N spectrophotometer (Gilford Instrument
Laboratories,
Inc., Oberlin,
Ohio 44074) equipped with a thermostated
cuvette
(Model 3017), automatic pipettor-dilutor
(Cavro Scientific Instruments, Los Altos, Calif.), and programmed
calculator. In this procedure urine, a bacterial suspension, lysozyme covalently bonded to BE, and an anti-BE
antibody are used, and the absorbance change at 436 nm
is monitored at 37 #{176}C
for 40 s. If BE is present, enzyme/bacteria
complex is displaced from the antibody
and the turbid bacterial substrate
clears. Standard
curves were prepared over the range of 0-10 mg/liter.
COCAINE
DOSAGE
x--X
0.38mg/kg
o-.-c
0.75mg/kg
#{149}-e1.5mg/kg
E
‘Si
6
z
z
0
a
U
‘Si
We had the opportunity
to analyze urine samples
from a single volunteer who received intravenous
cocaine (Table 1). These data are too limited for valid
comparison
with that after intranasal
application.
However, topical application
of cocaine is the most
common form of illicit use, and so is more relevant to the
concerns of drug-dependency
treatment
programs.
4
Discussion
3-
0
N
z
U
a
0
0
4
8
2
I6
TIME
0
:
(hours)
Fig. 1. Time course of benzoylecgonine excretion in the urine
after intranasal administration of cocaine, demonstrating the
relationshipbetween peak concentrationsand dose (subject
17)
Because the upper limit of linearity varied between
reagent lots, specimens with concentrations greater than
the highest linear standard point were repeated after
dilutions. As the manufacturer
recommends,
concentrations <1.0 mg/liter were considered negative. The
manufacturer-supplied
antibody to BE used in this
study shows only minimal cross reactivity with ecgonine
and cocaine, and measurements
are therefore assumed
here to reflect concentrations of BE, although there may
be traces of other metabolites,
as yet unverified, that
cross react.
Results
The surgical patients (with the exception of dental
patient No. 7) and volunteers who received intranasal
cocaine all showed a similar pattern of urinary excretion
of BE (Table 1). All urines obtained before administration of cocaine were negative for BE. A positive test
for BE occurred by 1 to 4 h and persisted for as long as
27 h after drug administration.
The urines then were,
with a single exception, negative and continued to be
negative when followed for as long as 84 h. In 17 of 19
subjects who received intranasal cocaine, concentrations
of BE in the urine became maximal within the first 12
h after drug administration,
then decreased. In the
surgical patients who received 1.5 to 1.7 mg/kg of cocaine, the average peak concentration was 9 mg/liter (±2
mg/liter SE) and occurred 10.6 h (±0.9 h, SE) after intranasal application.
In the volunteers who received
cocaine intranasally,
peak concentrations
of BE in the
urine were dose dependent (Figure 1). The smallest dose
(0.19 mg/kg; total dose, 13 mg), administered
to volunteer No. 18a, resulted in a low concentration
of BE,
close to the minimal concentration
detectable by the
EMIT method. There was no difference in the pattern
of excretion between males and females. Surgical patient No. 7 differed from the common pattern in that
BE was found in her urine for 46 h after drug administration.
A similar temporal pattern of excretion was reported
by Schneider et al. (12), who used a different assay
technique to study five patients who had received an
unspecified quantity of cocaine.
Using gas chromatography,
Wallace et al. (7) found
high BE concentrations
within 8 h of intranasal application of 250 mg of cocaine to each of 10 patients who
were undergoing nasal surgery. Their total dosage was
much higher than our (or probable illicit “street”)
dosages. Although they said that specimens were also
analyzed by the EMIT technique, the data were not
presented. In any event, the EMIT procedure is by far
the most widely used method of screening urines for
cocaine use, and the relation of findings by this procedure to time and dose should be studied and known.
Our data are subject to a number of limitations.
We
did not have continuous urine samples, and so we inadvertently
may have missed higher peak concentrations, or some urines may have remained positive for BE
a few hours longer than our last positive sample. Although the use of other drugs theoretically
could have
affected the metabolism of cocaine in surgical patients,
they showed a pattern similar to that of our volunteers,
who did not receive other drugs. Our data are also
subject to all the limitations of analyses of constituents
in single casual (i.e., untimed) urine specimens as well
as the semiquantitative
nature of the analytical procedure. Fluctuations
in urinary pH and volume will, of
course, affect the concentrations
of a drug in a specimen
of urine. Conclusions concerning the pharmacokinetics
of cocaine thus cannot be drawn from our data. Rather,
our data are intended as a guide for those involved with
drug-abuse screening programs that rely on the EMIT
analysis for detection of cocaine use. Specimens are
received by such laboratories for purposes of screening
for drugs of abuse as casual urine specimens. No attempt is made to control for urinary pH, creatinine
concentration, or volume. Treatment programs use such
data to determine if a drug has been used, and how recently it has (or has not) been used. They also wish to
know the likelihood of detecting usage relative to the
frequency of testing.
The doses used in both patients and volunteers in our
study are similar to those used illicitly, and our results
are applicable to drug-screening
programs. Our data
indicate that if cocaine is used topically, in a dose range
of 13 to 130 mg, and BE is tested for by the EMIT procedure, results will be positive within 1 to 4 h of a single
application, will peak at about 10 to 12 h, remain positive for 18 to 27 h, and will after about 27 h be negative.
If BE is found it can be concluded with reasonable
CLINICAL CHEMISTRY, Vol.23,No. 2, 1977
243
confidence that cocaine has been used within the last
27 h. Repeated positives over intervals greater than 27
h are more consistent with repeated use than when there
is only residual excretion of metabolites
from a single
dose. These factors should be considered by drug-dependency treatment
programs when results of urine
screening for cocaine use by the EMIT procedure are
3. Fish, F., and Wilson, W. D. C., Excretion of cocaine and its metabolites
in man. J. Pharm. Pharmacol. 21, 1355 (1969).
4. Javaid, J. I., Dekirmenjian,
H., Brunngraber,
E. G., and Davis, J.
M., Quantitative
determination
of cocaine and its metabolites
zoylecgonine
and ecgonine by gas-liquid chromatography.
J.
matogr.
110, 141 (1975).
ben-
Chro-
5. Koontz, S., Besemer, D., Mackey, N., and Phillips, R., Detection
of benzoylecgonine (cocaine metabolite) in urine by gas-liquid chromatography.
J. Chromatogr.
85,75
(1973).
evaluated.
6. Misra, A. L., Pontani, R. B., and Mule, S. J., Separation of cocaine,
some of its metabolites
and congeners
on glassfiber sheets. J. Chromatogr.
81, 167 (1973).
We thank G. L. Hammond, M.D., V. B. Khachane,
M.D., C. T.
Sasaki, M.D., and H. R. Sleeper, D.D.S., of Yale University, for providing urine samples from surgical patients; Marian W. Fischman,
Ph.D. and Charles R. Schuster, Ph.D., of the Department of Psychiatry, University of Chicago, for providing urine samples from the
subject receiving intravenous cocaine; and Joel Radding, Ann Locniskar, Marc Notrica, and Barbara Clinton, R.N. for their technical
assistance.
This study was supported by National Institute on Drug
Abuse contract
ADM 45-74-164,
in part by grant NIDA 10294, and
by USPHS grant RR 00125 to the Clinical Research
Center at Yale
University. R. Byck is a Burroughs
Weilcome Scholar in clinical
7.
pharmacology.
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