CLIN. CHEM. 33/6, 806-808 (1987)
Concentrationsof Morphine and Codeine in Serum and Unne after Ingestionof Poppy Seeds
Lyle W. Hayes,1 Wendell G. Krasselt,2 and Paul A. Mueggler
We measured morphine and codeine in commercially available poppy seeds and in serum and urine samples from
healthy adults who had ingested these poppy seeds. Four
brands of black poppy seeds, examined by gas chromatography-mass spectroscopy (GC-MS) with deuterated internal
standards, contained from 17 to 294
of morphine and 3 to
14 g of codeine per gram of seeds. Morphine was detected
by GC-MS in hydrolysates of serum as late as 24 h after
ingestion, with a maximum mean concentration of 100 ng/mL
(range 82-131) measured 2 h after the subjects ingested 25
g of seeds. Opiates were detectable (>300 ig/L) in urine by
enzyme-multiplied immunoassay (EMIT; Syva Co.) and by
radioimmunoassay screening procedures for as long as 48 h
after ingestion. The identity and quantities of morphine and
codeine in poppy seed extracts and in hydrolysates of serum
and urine were confirmed by GC-MS. Therefore a positive
finding of morphine or codeine in blood and urine may
sometimes be due to ingestion of poppy seeds.
The recent increase in demand for drug testing in the U.S.
has made more urgent the need for accuracy in drug
screening, particularly
because confirmatory
tests are not
always carried out and because the rate of false-positive
results is substantial
(1). Morphine being a major metabolite of both heroin and codeine (2), its presence in urine has
been used as an indicator of opiate use (3, 4). However,
recent reports (5,6) from abroad have identified morphine in
urine of persons who had ingested poppy seeds orpoppy-seed
cakes. To confirm these reports and investigate
this possible
source of interference in opiate-screening
tests, we measured the concentrations
of morphine and codeine in poppy
seeds obtained
in the United States and have examined
serum and urine for content of these opiates after our
subjects ingested poppy seeds. We also determined
the
duration of the effect of these concentrations
on three widely
used screening
and confirmatory
tests for opiates: EMIT
(Syva Co., Palo Alto, CA), RIA, and gas chromatographymass spectroscopy (GC-MS).
Materials
and Methods
Materials. Black poppy seeds were obtained from retail
stores in Portland, OR Morphine, codeine, normorphine,
and norcodeine
were obtained from Ailtech Associates/
Applied Science, State College, PA 16804. The deuterated
standards
[N-C2H3]morphine
and [N-C2H3]codeine were
prepared from normorphine
and norcodeine by the method
of Ebbighausen
et al. (7). After alkylation with [2H3]methyl
iodide, mass spectral analysis showed that the deuterated
standards contained <0.5% undeuterated
morphine or codeine. All glassware was pretreated with dichiorodimethyl
silane (AlltecbiApplied
Science)
to prevent adsorption of
‘Department of Health Sciences, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, WI 53201.
2Toxicology Section, Department of Clinical Pathology, Oregon
Health Sciences University,
3181 S.W. Sam Jackson Park Rd.,
Portland, OR 97201.
Received January 21, 1987; accepted March 9, 1987.
806 CLINICALCHEMISTRY, Vol. 33, No. 6, 1987
opiates from aqueous samples. Six healthy adult humans
with no history of opiate use volunteered for the ingestion
studies.
Poppy-seed ingestion experiments. In the first trial, four
healthy adults (two men, two women) each consumed 25 g of
poppy seeds (see Table 1 below, batch 1) with juice or yogurt.
Urine and serum samples were collected for analysis at
intervals up to four days, or until no opiates were detected
by screening or CC-MS. In the second trial, two healthy
adults (one man, one woman) consumed 40 g of poppy seeds
(batch 2). Urine and serum samples were obtained more
frequently than in the first trial, and analyzed.
Hydrolysis of urine and serum and extraction of opiates for
GO-MS. To determine
total morphine
and codeine,
we
incubated 1-mL serum or urine samples for 1 hat 37#{176}C
with
800-1600 U of /3-glucuromdase
(EC 3.2.1.23, Type IX, G
3510; Sigma Chemical Co., St. Louis, MO). To determine
free morphine and codeine, we mixed 1 mL of serum or urine
or 1 mL of serum or urine hydrolysate (from the glucuromdase incubation) with 100 zL of an aqueous internal standard containing 100 ng each of [2H3]morphine and [2H3]codeine and applied the mixture to a dry Chem Elute column
(CE 1103; Analytichem
International,
Harbor City, CA).
After rinsing the sample tubes with 1.5 mL of water and
adding this rinse to the column, the opiates were eluted with
two 4.5-mL applications
of chloroform. The solvent was
removed under a stream of nitrogen, and the residue was
reconstituted
with 0.1 mL each of chloroform and trifluoroacetic anhydride (TFA; Sigma Chemical Co.). After incubation at 60 #{176}C
in capped tubes for 20 mm, the TFA derivatives
were evaporated just to dryness under a stream of nitrogen,
dissolved in 10-20 L of chloroform, and analyzed within 30
mm by CC-MS.
Extraction of opiates from poppy seeds. Using a Polytron
homogenizer (Brinkmann
Instruments,
Westbury, NY), we
homogenized 1 g of poppy seeds in 5 mL of 0.1 mol/L citrate
buffer (pH 4.0). Morphine and codeine were determined by
CC-MS as described for serum and urine samples, except
that hydrolysis was unnecessary
and samples were filtered
before application to the extraction column.
Instrumentation.
For CC-MS we used a Finnigan 4500
quadrupole
instrument
interfaced to an INCOS 2300 data
system (Finnigan MAT, San Jose, CA), injecting 3-fl pL of
the TFA derivatives in chloroform onto a 2mm (i.d.) x 1.3 m
glass column containing 3% OV 1 on 100/200 Supelcoport
(Supelco, Inc., Bellfonte, PA). The chromatograph was operated isothermally at 220 #{176}C
with helium carrier gas at a
flow rate of 25 mL/min. The mass spectrometer was operatodin the electron impact mode at -70 eV. We identified and
quantified the TFA derivatives by multiple ion detection for
the following eight mie values: morphine,
364 and 477;
[2H3]morphine, 367 and 480; codeine, 282 and 395; [2H3]codeine, 285 and 398. Calculations, performed with the standard INCOS quantification program package, were based on
the peak area ratios of the following ions: 364/367 and 477/
480 for morphine; 282/285 and 395/398 for codeine.
The EMIT Opiate procedure (Syva Co.) was performed with
an Autocarousel
II system (Syva Co.) as specified by the
manufacturer
for screening of urine for opiates. The Abuscreen Opiate RIA procedure (Roche Diagnostic
Systems,
Nutley, NJ) was also performed as specified by the manufacturer for urine screening. The cutoff value for a positive
result for both urine screening procedures was 300 ng/mL.
We also used the Abuscreen method without modification to
quantify opiates in serum samples.
Results and Discussion
By GC-MS, poppy-seed samples contained from 17 to 294
g of morphine and from 3 to 14 p.g of codeine per gram of
seeds (Table 1). We confirmed the identity of the CC peaks
by comparing the complete mass spectra with those obtained for codeine and morphine
standards.
Table 2 summarizes the results of the first ingestion trial,
in which the richest source of opiates was used. The 25-g
oral dose of poppy seed provided approximately 7.5 mg of
morphine
and 0.4 mg of codeine. The concentration of
morphine in serum averaged 100 g/L (range 82-131) 2 h
after ingestion, and 6 zg/L (range 3-10) after 24 h. The
concentration of codeine in serum averaged 7 ug/L (range 411) after 3 h (data not shown).
Both EMIT and RIA urine opiate screening procedures
tested positive (above the cutoff of 300 g/L) for 48 h after
ingestion (three of four subjects tested positive at 48 h), with
an average peak concentration
of 1568 pg/L (range 11582100) at 3 h, as measured by RIA. Again, we confirmed the
identity of the morphine and codeine peaks resolved in the
urine extracts, after treatment
with 13-glucuronidase,
as
described for poppy-seed extracts. We could detect morphine
in hydrolysis extracts of urine by CC-MS for up to three
days after ingestion.
The second trial was designed to monitor more closely the
Table 1. Morphine and Codeine Content of Poppy Seed
Morphine
Batch
Source
Codeine
g/g
294
63
1
French’s, lot no. 075
2
French’s, lot no. 175
3
Spice Islands,lot no. 2230780
4
Schilling, lot no. 1329A
ND, nonedetected.Valuesare mean ± SD.
±
±
17±
28±
15
15
7
6
14
4
±
2
±
0.4
3 ± 0.8
ND
Table 2. OpIates in Serum and Urine after Consumption
of Poppy Seeds (Trial 1)
appearance of detectable opiates in serum after oral mgestion of 40 g of poppy seeds that contained approximately
2.5
mg of morphine and 0.16mg of codeine. Peak concentrations
of total morphine in serum were 43 and 51 jtgfL at 1.5 h.
Maximum free-morphine
values were 2.5 and 3.0 1zgfL at 3
h (Figure 1). Urine EMIT results were positive (>300 zg/L)
for as long as 24 h, and CC-MS measurements
of total
urinary morphine exhibited peaks at 3 and 6 h (Table 3).
Peak concentrations
of total morphine were 700 and 2600
or up to approximately
nine times the value suggested
as a positive cutoff by the manufacturers
of the EMIT and
RIA kits.
Concentrations
of uncosijugated opiates in blood are correlated with clinical response, and measurements
in blood or
serum (often by immunoassays
that cross react with conjugated opiates) are used for estimates of impairment
in
medical-legal
situations (8). Therefore it was of interest to
note that morphine and codeine were identified in this study
in serum hydrolysates by CC-MS and also were detected by
the RIA procedure, which cross reacts with the glucuronides. In the serum samples tested, we found morphine to be
preponderantly in the conjugated form (>92%)-as
would be
expected, because morphine reportedly is nearly completely
conjugated to glucuronide in the first pass through the liver
(9). No analgesic or euphoric effect was reported by subjects
in this or in previous reports (5,6) in which similar amounts
of morphine were ingested. Blood and urine samples obtained immediately
before ingestion of poppy seeds contained no detectable opiates as measured by the methods we
used.
Although urine samples contained opiate in concentrations below the positive reference standard cutoff (300 g/L)
two days after poppy seed ingestion, morphine could still be
found in urine hydrolysates
by CC-MS (Table 3). Serum
hydrolysates
contained measurable
concentrations
(2.5 to
10.5 jtgfL) a day afer ingestion, although these values are
near the limit of sensitivity of the method.
The quantities of poppy seed ingested in this study (25
and 40 g) may be expected to be contained
in one or two
servings of poppy-seed cake (5, 6); therefore, poppy seeds
represent a potentially
serious source of falsely positive
results in testing for opiate abuse.
Since there is no consistent agreement
among drugtesting laboratories concerning
values that constitute
a
60
Concn
In serum,
1zgIL
Hours
after
ingestion
Subject
GC-MS
morphine
82
3
102
80
4
131
2
24
48
Urine
RIA
RIA opiates,
opiates
.tg/L
1158
130
156
127
196
‘
EMIT
opiates
1248
2100
+
+
+
1767
+
1
2
3
3
10
ND
ND
432
+
921
+
3
ND
620
+
4
8
ND
906
+
1
2
3
4
ND
ND
ND
629
237
299
ND
415
#{149}
30
q)
20
/0
\:
‘p
- -
10/
I
-
2
3
4
5
6
Time (hours)
+
After hydrolysis with p-glucuronidase.
means concentration exceeded the detection limit of this system, 3
ig/L. ND, none detected.
+“
40
+
+
50
Fig. 1. Morphine concentahons in serum after ingestion of 40 g of
poppy seed (containingapproximately
2.5 mg of morphine)by subject
(#{149})
and subject 6 (0)
Brokenlines: free (unconjugated)morphine; solid lines: total morphine after
hydrolysiswith -glucuronidase as determinedby GC-MS (see text)
5
CLINICALCHEMISTRY, Vol. 33, No. 6, 1987 807
Table 3. OpIates Detected in Urine after Consumption
of Poppy Seed (Trial 2)
Concn in urine, ig/L
Hours
after
Ingestion
1
Subject
Codeine
opiates
5
6
374
ND
+
920
5
700
11
42
+
3
6
6
5
2635
514
45
12
6
1460
134
14
24
GCMSa
5
Morphine
15
10
6
233
76
10
48
5
2
6
73
a Determined
afterhydrolysiswith$-glucuronidase.
“+‘
means >300 j.ig/L.
+
+
+
+
-
+
-
positive opiate result, our evidence indicates that a positive
result might well be reported for urine samples as long as
three to four days after ingestion of poppy seeds, and for as
long as one day for serum. It would seem that yet another
difficulty is present for testing for opiate abuse in this
country. Not only is it difficult to distinguish
heroin or
morphine abuse from therapy with codeine (3) (given the
nonspecificity of the antisera provided for the EMIT and RIA
procedures in common use), but dietary poppy seed can give
a strong positive result for urinary opiate of several days
duration that is confirmed by CC-MS analysis, as we have
shown. A recent report has described the possible use of the
presence of 06-monoacetylmorphine,
a heroine metabolite,
in urine as a specific indicator of heroin use (10), but this
method as reported is hampered by difficulty in detecting
this molecule, and interpretation of the CC-MS results was
not always clear. More-sensitive methods for detecting this
metabolite, or identification and detection of a marker for
808 CLINICALCHEMISTRY, Vol. 33, No. 6, 1987
poppy-seed
ingestion, possibly will provide a means of
heroin abuse from poppy-seed ingestion, but
there is currently
no reliable method in use for urine
screening or confirmation of screening results that can
accomplish this differentiation.
differentiating
We thank Dr. U. Bray for the suggestion that this work be done.
References
1. Hansen HJ, Caudill SP, Boone J. Crisis in drug testing. Results
of CDC blind study. J Am Med Assoc 1985;253:2382-7.
2. Boerner U, Abbott S, Roe RL. The metabolism of morphine and
heroin in man. Drug Metab Rev 1975;4:39-73.
3. Posey BL, Kimble SN. High performance liquid chromatographic study of codeine, norcodeine
and morphine as indicators of
codeine ingestion. J Anal Toxicol 1984;8:68-74.
4. Clarke P, Foltz RL. Quantitative
analysis of morphine in urine
by gas chromatography-chemical
ionization-mass
spectrometry
with [N-C2H3]morphine
as an internal standard.
Clin Chem
1974;20:465-9.
5. Bjerver K, Jonnson J, Nilsson A, Schuberth J, Schuberth J.
Morphine
intake
from poppy seed food. J Pharm Pharmcol
1982;34:798-801.
6. Fi-itschi G, Prescott WR. Morphine levels in urine subsequent to
poppy seed consumption. Forensic Sci hit 1985;27:111-7.
7. Ebbighausen WOR, Mowat JH, Vestergaard P, Kline NS. Stable
isotope method for assay of codeine and morphine by GC-MS. Adv
Biochem Pyschopharmacol 1973;7:135-146.
8. Pearce D, Wiersema 5, Kuo M, Emery C. Simultaneous determination of morphine and codeine in blood by use of selective ion
monitoring and deuterated internal standards. Clin Toxicol
1979;14:161-8.
9. Vandenberghe HM, Soldin SJ, MacLeod SM. Pharmacokinetics
of morphine: a review, In: Moyer TP, Boeckx RL, eds. Applied
therapeutic drug monitoring. Vol. 2, Review and case studies.
Washington, DC: Am Assoc Clin Chem, 1984:321-5.
10. Fehn J, Megges G. Detection of 06-monoacetylmorphine
in
urine samples by GC-MS as evidence of heroin use. J Anal Toxicol
1985;9:134-8.