An Analysis of the Duration of Fentanyl and Its Metabolites
in Urine and Saliva
Jeffrey H. Silverstein, MD*, Michael F. Rieders, PhDZ, Matthew McMullin,
Schulman, MD*, and Kenneth Zahl, MD*
M S ~ ,Steven
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*Departmentof Anesthesiology, The Mount Sinai Medical Center, New York, New York and $National Medical Services,
Inc., Willow Grove, Pennsylvania
This study was undertaken to determine if metabolites
of fentanyl might be useful in the detection and monitoring of substance abuse. The presence of fentanyl
and two of its metabolites in the urine and saliva of
seven female patients receiving small doses (110 ? 56
pg) of fentanyl was studied up to 96 h from the time of
administration. Fentanyl and its two metabolites (norfentanyl and despropionylfentanyl) were extracted
from samples and analyzed by gas chromatography/
mass spectrometry. Unchanged fentanyl was detectable in urine in all patients immediately postoperatively and in 3 of 7 patients at 24 h. By 72 h, fentanyl
U
rine testing is presently fundamental to the
detection and monitoring of chemically dependent individuals (1). Fentanyl is the drug
of choice for addicts in the anesthesia workplace (2).
Due to its short elimination half-life (t"P = 219 min)
and approximately 90% metabolism (31, fentanyl is
difficult to detect in urine. Murphy et al. suggested
that norfentanyl would persist in the urine (4), and
Hammargren and Henderson, using a difficult extraction and detection method, were able to demonstrate
the presence of both fentanyl and norfentanyl in urine
(5). The time course of the presence of these compounds in urine or saliva and the feasibility of using
these methods as a monitor for substance abuse have
not been evaluated. We tested urine and saliva for the
presence of fentanyl and two of its metabolites (norfentanyl and despropionylfentanyl) (Figure 1) from
individuals receiving less than 400 pg of fentanyl as a
component of intravenous analgesia for outpatient
surgery. We sought to determine the feasibility of
using this method as a surveillance test or screen for
~
~~~~
~~
Presented in part at the Annual Meeting of the American Society
of Anesthesiologists,San Francisco, California,October 1991.
Accepted for publication September 29, 1992.
Address correspondenceand reprint requests to JeffreyH. Silverstein, MD, Department of Anesthesiology, Box 1010, Mount Sinai
Medicaf Center, I Gustave L. Levy PIace, New York, NY 10029-6574.
was undetectable. Norfentanyl was present in larger
quantities than fentanyl immediately postoperatively
and was detected in all patients at 48 h and in 4 of 7
patients at 96 h. Despropionylfentanyl was not detected in any of the urine specimens tested. Neither
fentanyl nor its metabolites could be detected consistently at any time in saliva. Saliva testing does not appear to be a viable alternative to urine testing based on
this study. Urinary norfentanyl might be considered as
the substance of choicewhen testing for fentanyl abuse.
(Anesth Analg 1993;76:618-21)
chemically dependent individuals and to determine if
saliva would provide a viable alternative for the detection of fentanyl and its metabolites.
Methods
This study was approved by the Institutional Review
Board of The Mount Sinai Medical Center and informed consent was obtained from all participants.
ASA I and I1 patients presenting for ovum retrieval
were included in this study. Seven female patients who
received less than 400 pg of fentanyl for operative analgesia were eligible for participation. The anesthetic
regimen for ovum retrieval consisted of intravenous
sedative/analgesic technique with incremental bolus
doses of fentanyl, midazolam, thiamylal, propofol,
lidocaine, and /or droperidol. Medications used in conjunction with fentanyl were not analyzed in the study,
but were determined not to interfere with the analytical
method (data not shown). Participants were asked to
provide urine (10-mLbottles) and saliva (1teaspoon or
approximately 4 mL) specimens preoperatively, immediately postoperatively, and subsequently at 12,24,48,
72, and 96 h. Urine and saliva specimens were stored
at -4°C (for sanitary reasons) and shipped frozen
(-20°C) to one of the authors for analysis.
Fentanyl and its two metabolites were extracted
from patient samples using pH-controlled acid-base,
01993 by the International Anesthesia Research Society
618
he&
Analg 1993;7661€&21
0003-2999/93/$5.00
ANESTH ANALG
iw3;76:61azi
SILVERSTEIN ET AL.
DURATION OF FENTANYL. METABOLITES IN URINE
Fentanyl and Metabolites
619
Table 1. Method Validation
Fentanyl Norfentanyl
0
Limit of detection (ng/mL)
Reporting limit (ng/mL)
Run-to-run precision at 10
ng/mL (% coefficient of variance)
Accuracy (at 10 ng/mL)
Analvte stabilitv at -10°C
Fentanyl
Norfentanyl
Despropionylfentanyl
Figure 1. Fentanyl is metabolized in the body to norfentanyl and
despropionylfentanyl. Two other metabolites, hydroxyfentanyl and
norhydroxyfentanyl, which are found in lower quantities in human
serum, were not evaluated in this study.
solvent extraction and back extraction (6-8). The extracted internal standard utilized was 8-methoxyloxapine (8-MLOX) (Aldrich Chemical Company,
Milwaukee, WI). Aliquots (2 mL) of standards, controls, blanks, and patient samples were placed into
tubes. One milliliter of 10% sodium hydroxide and 100
pL of 8-MLOX (10 ng/100 pL) were added to each tube.
After equilibration for 5 min, 4 mL of water/
isopropanol (3:2, v/v), plus 6 mL of petroleum ether
were added. The tubes were capped and rotomixed for
10 min at 20 rpm, centrifuged for 5 min at 1400 g, and
allowed to stand for 10 min. The upper layer was transferred carefully to a tube containing 2 mL of 0.05 N HC1.
The tubes were vortexed for 1 rnin, then centrifuged for
5 min at 1400 g. The lower aqueous layer was transferred to a tube containing 0.2 mL of 5%NaOH, and the
tubes were briefly vortexed. Final extraction was
achieved by adding 0.3 mL of dichloromethane, vortexing for 1min, and then centrifuging for 2 min at 1400
g. The bottom organic layer, containing fentanyl and its
metabolites, was transferred to a new tube for analysis.
Norfentanyl and despropionylfentanyl were derivatized by adding 5 pL of a mixture of to1uene:butyric
anhydride:4-dimethylaminopyridine(catalyst) (90:9:1)
to each tube. The tubes were heated at 70°C for 22 min
under a stream of nitrogen to prevent oxidation. The
residue was reconstituted with 30 pL of toluene and
transferred to an auto sampler vial and sealed with a
crimp top. Gas chromatographic (GC) analyses were
performed with a Hewlett-Packard Model 5890A GC
equipped with a nitrogen phosphorus detector (GC/
NP), 7673A liquid auto sampler,and a 3396A integrator.
The injections were made in the splitless mode into a
4-mm glass injection port liner, followed by an analytical 15-m X 0.32-mm internal diameter fused silica capillary column with a bonded 0.15-pm DB-17 film U&W
0.1
0.2
5.9
0.1
0.2
6.3
+8.3%
>6 mo
>6 mo
*9.7%
Scientific, Folsom, CA). The operating conditions were
a helium carrier gas at a head pressure of 10.0 psi, column oven temperature programmed from 150°C to
300°C at 35"C/min, final time 1 min, initial and inlet
purge time were both 2 min, injector and detector temperatures were 270°C and 300"C, respectively. The volume injected on the column was 2 pL. Gas
chromatographic/mass spectral (GC/MS) analyses
were performed on a Hewlett-Packard Model 5988A
system using the same chromatographic parameters
described above. The electron impact mass spectral
data were acquired in the selected ion monitoring
mode (SIM) for the following ions (rn/z>:fentanyl, 146,
189, 245; norfentanyl, 132, 158, 231; despropionylfentanyl, 146, 189, 259; 8-MLOX, 247, 287 (5).
The manufacturer's certified primary standards utilized were: fentanyl citrate (Janssen Pharmaceutica,
Piscataway, NJ), despropionylfentanyl, and norfentanyl (Goldmark Biologicals, Phillipsburg, NJ). Fentanyl,
norfentanyl, despropionylfentanyl, and 8-MLOX demonstrated baseline separation in this chromatographic
system with typical retention times of 6.85, 6.22, 7.01,
and 7.37 min, respectively.
All specimens were analyzed by GC/NP. Those
specimens that were initially positive for fentanyl
and/or its metabolite(s1were analyzed by GC/MS for
confirmation of identity and quantitation. A representative sampling of negative patient specimens were run
as blank controls along with the positive patient samples for both GC/NPand GC/MS. The validation characteristics for the assays of fentanyl and norfentanyl are
presented in Table 1.
Results
A representative GC/NP chromatogram is presented in
Figure 2. Demographicdata and results for each subject
are presented in Table 2. The mean fentanyl dose administered was 110 ? 56 pg. The specimens were collected at the times requested 2 2 h as reported by the
participants. The reporting limit for this assay is 0.2
ng/mL for both fentanyl and norfentanyl. Samples reported as negative represent levels <0.2 ng/mL. Fentanyl was detected in all immediate postoperative urine
specimens, in 3 of 7 specimens at 12 and 24 h, and
620
-TI4
SLVERSTEIN ET AL.
DURATION O F FENTANYL METABOLITES IN URINE
ANALG
1993;7661&21
Table 2. Demographic Data and Results
Urine norfentanyl (ng/mL)
Urine fentanyl (ng/mL)
Other
Age (yr)" Fentanyl ( p g ) b medications' Postop 12 h 24 h 48 h 72 h 96 h Postop 12 h 24 h 48 h 72 h 96 h
35
43
200
170
32
100
38
100
40
100
38
50
42
50
M 4 mg
M 4 mg
D1.25
M 4 mg
L 25 mg
T 250 mg
M 2 mg
P 90 mg
L 50 mg
M 2 mg
D 0.25 mg
T 250 mg
M 4 mg
P 100 mg
D 1.25 mg
M 4 mg
P 150 mg
D 1.25 mg
0.8
2.0
0.2
0
0
0.2
0.9
0
0
0
0
5.8
5.4
13.8
3.7
5.3
1.9
4.7
2.6
1.3
0.2
0.3
0
0.9
0
0
0
0
0
11.5
10.2
3.1
1.7
0.7
0.7
0.5
0
0
0
0
0
10.9
7.6
0
0.5
0
0
4.7
0.4
0.4
0
0
0
10.0
23.0
3.3
0.8
0.6
0.5
1.9
0.3
0.6
0
0
0
8.5
20.0 12.0
0.4
0.7
0
0.8
0
0
0
0
0
10.0
0.6
0.4
0.6
1.1
0
0
, Average age = 38.3 i 3.9 yr.
Average fentanyl dose = 110 i 56 pg.
M = midazolam; P = prupofol; T = thiamylal; D
=
droperidol; L = Lidocaine.
in one specimen at 48 h. Fentanyl could not be detected
at 72 or 96 h. By contrast, norfentanyl was detected in
greater quantities at all times and in 4 of 7 samples at
96 h (Figure 3). Despropionylfentanylwas not detected
in any of the urine specimens. Initial screening of the
preoperative specimen from one patient was positive
by GC/NP for norfentanyl (0.7 ng/mL), but GC/MS
analysis did not confirm the initial positive GC/NP
screen.
The requested quantities of saliva, 5 mL, proved difficult to obtain and quantities of 1-2 mL were typically
produced. Neither fentanyl nor despropionylfentanyl
were detected in any saliva specimens. Norfentanyl
was detected in two saliva specimens from one patient
who received 100 pg of fentanyl, but could not be detected in any of the other 43 specimens.
II
f
Q
l
- I I/
a
Figure 2. Gas chromatographic/nitrogen phosphorus chromatograph showing separation of fentanyl and three metabolites and
8-methoxyloxapine (8-MLOX), the internal standard. The numbers
represent retention times on the chromatographiccolumn.
Discussion
In this study, seven female patients undergoing ovum
retrieval received analgesia with 1200 pg of fentanyl.
Urine and saliva specimens were analyzed for the presence of fentanyl and two of its metabolites, norfentanyl
and despropionylfentanyl. Our results are consistent
with the known disposition of fentanyl in humans
which involves excretion of approximately 90% of an
administered dose as metabolites (5).At 72 h, fentanyl
could not be detected in any urine sample, but norfentanyl was detected in 86% of samples. Despropionylfentanyl was not detected at any time in any
of the urine specimens in this study.
Hammargren and Henderson described a different
extraction and detection procedure for fentanyl and
norfentanyl(5). They evaluated an unspecified number
of individuals, but did not determine the time course
of the presence of these normetabolites in urine.
McClain and Hug (3) and Murphy et al. (4)suggested
that norfentanyl would persist in urine as confirmed by
our results.
The doses of fentanyl used in this study (50-200
pg) are small compared to the quantities that may be
used by a fentanyl addict. Our interest is the monitoring of the recovering addict as well as the detection of the early addict. The doses studied are appropriate to these situations. Fentanyl metabolism and
excretion into urine were highly variable among individuals. There was no discernible relationship between the administered fentanyl dose and the levels
SKVERSTEIN ET AL.
DURATION OF FENTANYL METABOLITES I
N URINE
Duration of Norfentonyl a n d Fentanyl in Urine
20
means i S D
.
18
0 Norfentonyl
16
Fentoriyi
c.1 4
E
\
y
12
c
.? 10
0
c
8
6
4
2
0
I
1
v
~
POST-OP
1 2 HOURS
24 HOURS
48 HOURS
72 HOURS 9 6 HOURS
Collection Time
Figure 3. Utilizing an assay with a reporting limit of 0.2 ng/mL,
norfentanyl was detected in 4 of 7 samples at 96 h. Fentanyl levels
approached the reporting limit by 12 h.
of fentanyl and norfentanyl detected in urine. Some
of the variability may be attributable to differences in
urine specific gravity or creatinine clearance. The effect, if any, of the other coadministered drugs on fentanyl metabolism and excretion is not known. Although the present study included only female
subjects, there are no data to suggest that fentanyl
metabolism is significantly different in males.
Other medications coadministered for sedation (Table 2) were not monitored or analyzed in this study. It
was determined, however, that neither these substances nor other common substances of abuse interfere with the quantitative identification of fentanyl or
its metabolites (data not shown). Because individuals
who abuse drugs frequently abuse more than one substance, the detection of fentanyl or its metabolites
among the mix of other drugs and metabolites represents realistic conditions.
Situations leading to urine fentanyl or fentanyl metabolite concentrations of less than 0.2 ng/mL will result in a false negative finding. False positive results are
unlikely because the method requires that the GC retention time index and the MS ion ratios be within restricted limits. False positives can result, however, from
sample mix up, unexpected carryover ("ghosting")
from a previous high concentration sample, and other
erroneous analytical and handling techniques. The
presence of norfentanyl on the initial screening (by
GC/NP) for one patient highlights the necessity of
621
careful confirmation procedures in urine drug testing.
This patient denied use of fentanyl or other analgesics,
and GC/MS analysis made clear that the substance was
not norfentanyl.She was the same patient for which the
saliva norfentanyl samples also were positive. We have
no explanation for the differences in this patient's results. Although the GC/NP screen will identify a substance that has similar chromatographic characteristics
as, in this example, norfentanyl, the molecular structure was distinctly different than norfentanyl, as resolved by mass spectrometry.
Saliva proved difficult to collect according to the patients studied. Four samples contained volumes insufficient to analyze. Norfentanyl was detected in two saliva specimens from one patient at 12 h (0.6 ng/mL)
and at 72 h (1.9 ng/mL). Fentanyl and despropionylfentanyl were not detected in any saliva sample.
Thus, it would appear that saliva sampling is not a
good alternative to urine sampling.
Norfentanyl is detectable in the urine for a considerably longer period of time than fentanyl. Drug testing
programs either for monitoring recovering addicts or
screening health care professionals with access to fentanyl might consider testing for norfentanyl rather than
fentanyl. The ability to detect norfentanyl from 48 to 96
h postadministration will enhance the quality and utility of monitoring programs. Further, larger scale studies should be undertaken to better understand the implications of urine testing for fentanyl and its
metabolites. Radioimmunoassays under development
will require similar confirmation.
References
1. Canavan DI. Screening: urine drug tests. Md Med J 1987;36:
229-33.
2. Ikeda R, Pelton C. Diversion programs for impaired physicians.
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3. McClain DA, Hug CC Jr. Intravenous fentanyl kinetics. Clin
Pharmacol Ther 1980;28:106-14.
4. Murphy MR, Hug CC Jr,McClain DA. Dose-independent pharmacokinetics of fentanyl. Anesthesiology 1983;59:53740.
5. Hammargren WR, Henderson GL. Analyzing normetabolites of
the fentanyls by gas chromatography/electron capture detection. J Anal Toxicol 1988;12:183-91.
6. Van Rooy HH, Vermeulen MI', Bovill JG.The assay of fentanyl
and its metabolites in plasma of patients using gas chromatography with alkali flame ionisation detection and gas
chromatography-mass spectrometry. J Chromatogr 1981;223:
85-93.
7. Goromaru T, Matsuura H, Yoshimura N, et al. Identification and
quantitative determination of fentanyl metabolites in patients
by gas chromatography-mass spectrometry. Anesthesiology
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8. Gillespie TJ,Gandolfi AJ,Maiorino RM, Vaughan RW. Gas chromatographic determination of fentanyl and its analogues in human plasma. J Anal Toxicol 1981;5133-7.