Journal of Analytical Toxicology,Vol. 22, July/August1998
Ag-THCMetabolites in Meconium: Identification of
11-OH-Ag-THC, 813,11-diOH-Ag-THC,and 11-nor-ASTHC-9-COOH as Major Metabolites of A9-THC
Mahmoud A. EISohlyand Shixia Feng
EISohly Laboratories, Incorporated (EL/), 5 Industrial Park Drive, Oxford, Mississippi 38655
I Abstract [
Gas chromatography-mass spectrometry (GC-MS) analysis of
meconium specimensscreening positive for cannabinoidsby the
EMIT 20 Assayshoweda low confirmation rate for 11-nor-9carboxy-A%tetrahydrocannabinol (THCCOOH). A study was
designedto investigatethe possiblecontribution of other ~9.
tetrahydrocannabinol (THC) metabolites, including glucuronides,
to the overall responseof the EIA. Ag-THC-glucuronide was
synthesizedin order to develop the most efficient procedure for
hydrolysis of glucuronides in meconium. Procedureswere
developed for the extraction and GC-MS analysis of Ag-THC,
11-OH-Ag-THC, 8a- and 813-OH-A%THC, 8~,11-diOH-Ag-THC,
and THCCOOH, after enzymatic hydrolysisof meconium extracts.
It is concluded that enzymatic hydrolysisof meconium extracts is
necessary for efficient recovery of AS-THC metabolites; Ag-THC
and its 8-OH metabolite(s) are basically absent in meconium
specimens; and 11-OH -Ag-THC and 8[3,11-diOH-Ag-THC
contribute significantlyto the immunoassayresponseof meconium
extracts. Analysisof several meconium specimensthat screened
positive for cannabinoids but failed to confirm for THCCOOH
showed significantamounts of 11-OH-Ag-THC and 8J],11-diOHAg-THC. Therefore, GC-MS confirmation of cannabinoids in
meconium should include analysis for these two metaholites in
addition to THCCOOH.
Introduction
It has become apparent that use and abuse of drugs by pregnant mothers results in babies born with symptoms of addiction. Although urine and blood are the most obvious specimens
to collect for detection of prenatal exposure to drugs, they
only provide information about recent drug exposure (two to
three daysbefore birth) (1,2). Data collected on the analysis of
meconium in infants born to drug-abusing mothers showed
that meconium is superior to urine and blood because it
extends the window of detection to approximately the last 20
weeks of gestation (3,4).
The majority of the publications on the detection of drugs in
meconium focused on the use of immunoassayswith only a few
reports using gas chromatography-massspectrometry (GC-MS).
Most of the publishedGC-MSconfirmationprocedures,however,
describe the determination of cocaine and its metabolites, with
only a few of the reports covering the cannabinoids (5-7). All of
these GC-MS confirmations of cannabinoids in meconium were
directed toward the major THC metabolite commonly found in
urine and blood (specifically, 11-nor-9-carboxy-A9-tetrahydrocannabinol [THCCOOH]).The confirmation rate for this
compound was usually low. One investigation failed to confirm
any of the specimens screened positive by EMIT (5), whereas
another report (6) showedan 80% confirmation rate by TDxat a
25-ng/g cutoff concentration. In a recent, large-scale study,
E1Sohlyet al. (7) reported on the use of EMITfollowedby GC-MS
for analysis of 2270 meconium specimens for cannabinoids,
cocaine,amphetamine, opiates, and PCP.In that study, 190 specimens screened positive for cannabinoids, but only 26% of these
were confirmed by GC-MS.
These data suggested that the major tetrahydrocannabinol
(THC) metabolites found in meconium may be different from
those in urine and blood. The metabolic profile of THC is very
complex. Allylic hydroxylation produces ll-hydroxy-A9-THC
(8,9) which is the precursor of THCCOOH. Further hydroxylation of 11-hydroxy-Ag-THC at the 8-position gives either
8[3,11-dihydroxy-Ag-THCor 8a, 11-dihydroxy-Ag-THC.Among
the two dihydroxy- metabolites, the 8l],11-dihydroxy-A9-THC
is the more predominant (10). It is also possible for the parent
drug, Ag-THC,or its glucuronic acid conjugate to be present
in meconium. These compounds, with the exception of Ag_
THC-glucuronide, were found to have significant cross-reactivity in THC assays using the EMITsystem at low cutoff levels
(11). The presence of small amounts of any or all of these
other metabolites might explain the high percent of unconfirmed positive screening results for the THCCOOH assay in
meconium. It is possible that the cannabinoids in meconium
contain one or more of the compounds described above, which
contribute to the overall immunoreactivity of meconium
extracts.
The purpose of this study was to investigatethe elimination of
Ag-THC metabolites in meconium specimens collected from
Reproduction(photocopyin8)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.
329
Journal of Analytical Toxicology,Vol. 22, July/August1998
neonates exposed to marijuana prenatally. Because Ag-THC-glucuronide is not commercially available, this metabolite was synthesized for inclusion in this study.
Bacterial ~-glucuronidase (E. Coli, types IX-A), N,Obis(trimethylsilyl) trifluoroacetamide (BSTFA),acetic anhydride,
and pyridine were purchased from Sigma-Aldrich (St. Louis, MO).
All solvents were HPLC grade and purchased from Fisher Scientific (Pittsburgh, PA). Glusulase was obtained from DuPont Chemical Company (Wilmington, DE).
Experimental
Specimens
Meconium specimens were collected from neonates born to
mothers with a self-reported history of marijuana use. No identification on the specimens was provided to the laboratory. However, the proper consent forms were signed by the mothers.
For negative control meconium, a pool of meconium was
collected from unmarked diapers from a local hospital. The pool
was certified to be negative by immunoassay testing at a low
cutoff level (< 20 ng/g cannabinoids).
Equipment
A Varian VXR-300FT NMR spectrometer was used to record
the proton spectra at 300 MHZ. Liquid chromatography-mass
spectrometry (LC-MS) data were obtained using a Vestec model
201 thermospray MS system interfaced with a Waters' Associates
model 600-MS solvent delivery system. High-performance liquid
chromatography (HPLC) separations were carried out using a
Waters model 6000A solvent delivery system with a U6K injector
and a IN detector operated at 227 nm.
Synthesisand characterization of Ag-THC-glucuronide
Ag-THC-glucuronide was synthesized according to an analogous procedure (12) described for the synthesis of AS-THC-glucuronide. Ag-THC (1.23 g, 3.9 retool) and 1.35 g (7.8 retool) of
cadmium carbonate were added to 60 mL of dry toluene. A portion of the solvent (30 mL) was distilled off over 1 h, and a
solution of 3.1 g (7.8 retool) of methyl 2,3,4-tri-O-acetyl-lczbromo-l-deoxy-D-glucopyranuronate in 60 mL
of dry toluene was added in drops. The mixture
Table I. Retention Times and Ions Monitored for Ag-THC and its Neutral
was then refluxed for 35 h. The solid was filtered
Metabolites Analyzed as the Acetate Derivatives and for the Acidic
off, and the solvent was evaporated under
Metabolites Anaylzed as the TMS Derivatives
vacuum. The residue was subjected to silica gel
column chromatography and eluted with
Compound
Rt
Derivative Ions Monitored
hexane/ethyl acetate (4:1). The latter fraction,
which contained more polar material (600 rag),
A9-THC-d~ (I.S.)
5.77
acetate
306,322
was further chromatographed with the same solA9-THC
5.83
acetate
297,313
vent to give 240 mg of yellowish solid. Methyl A980~-and 8[3-OH-A%THC
8.35
diacetate
312,354
THC-l-yl-2,3,4-tri-O-acetyl-13-D-glucopyranosid11-OH-A9-THC
9.63
diacetate
312,354
uronate was isolated from this material by prepar8ff,11-diOH-bg-rHC
9.27
TMS
369,459,562
ative HPLC (Spherisorb) ODS 5 1Jm and eluted
11-nor-Ag-THC-9-COOH
9.35
TMS
371,473,488
with methanol/water/acetic acid (80:20:0.01) as
11-nor-Ag-THC-9-COOH-d6(I.S.)
9.28
TMS
377,494
a white solid (2.98 rag). The mass spectrum was
obtained by LC-MS (thermospray, NH4OAc). The
Table II. Responseof Ag-THC-Glucuronide to Different Hydrolysis
mass spectra mass-to-charge ratios were 648
Conditions
(100% M+ + NH4§ and 631 (35% M§ + H+).
NMR(MeOH-d4
300 MHz) 8 (ppm) were as folReagent
Amount
Conditions(temp/time) Recoveryof Ag.THC (%)
lows: 0.92 (3H, t, J = 6.9 Hz, terminal aliphatic
[3-Glucuronidase* 5000 units
37~
100 (98-102)I
CH3); 1.06 and 1.40 (2 x 3H, two singlets, geminal
~-Glucuronidase* 10000 units
37~
I00*
CH3); 1.66 (3H, bs, olefenic CH3); 3.77 (3H, s, ~-Glucuronidase* 5000 units
60~ h
76 (72-79) I
COOCH3);2.03, 2.02, and 2.00 (3 x 3H, three sin~-Glucuronidase* 10000 units
60~
91 (86-100)I
glets, 3 • COCH3);2.49 (2H, t, J = 7.0 Hz, benzylic
Glusulase~
100 pL
37~
50*
CH2);and 6.37 (1 H,d, J = 1.6 Hz) and 6.66 (1 H,
5% HCI
] mL
100~ h
0 (degraded)
d, J = 1.6 Hz) for two aromatic protons.
Conc. HCI
1mL
100~ h
0 (degraded)
A solution containing 311 tJg/mL Ag-THC-gluBuffer (pH 6.8)
1mL
rt/24 h
0
curonide
was obtained by hydrolysis of 2 mg of
KOH (2N)
l mL
50~
h
0
methyl Ag-THC-l-yl-2,3,4-tri-O-acetyl-13-D-glu* ~Glucuronidase was from bacterial (E. coli) Type IX-A. The experiments were done in 1 mL 0.1M
copyranosiduronate with 0.5 mL of2N NaOH in
phosphate buffer (pH 6.8).
2
mL of methanol at 50~ for 1.5 h. After hydrol* Mean value and range for three runs.
* One determinatiort.
ysis was complete (monitored by HPLC), the
Glusulase contains about 10,000 units of sulfataseand 9000 units of ~-glucuronidase per milliliter in
mixture was neutralized with 2N HCI, and the
solution of I .I M acetate buffer (I mE, pH 5.2).
final volume was brought to 5.0 mL with
Materials
The following standards and internal standards were obtained
from E1Sohly Laboratories, Inc. (Oxford, MS): ll-nor-Ag-THC-9 COOH, ll-hydroxy-Ag-THC, 8~,11-dihydroxy-Ag-THC,ll-nor-A9THC-9-COOH-d6, Ag-THC-dg, 8or
8[3-hydroxy-Ag-THC,and
Ag-THC-glucuronide. Ag-THC was obtained from the National
Institute on Drug Abuse (NIDA, Rockville, MD).
330
Journalof AnalyticalToxicology,Vol.22,July/August1998
methanol.
Initial extraction and enzymatic hydrolysis of
meconium specimens
One gram of meconiumwas weighed into a 16 x 100-mm culture tube. Internal standards (50 pL of 1-pg/mL solutions of A9.
THC-d9and THCCOOH-d6)were added followedby the addition of
4 mL of methanol. The sample was then homogenized using an
ultrasonic disrupter (Tekmar model TM600 set for 30-s cycles).
Disruption was repeated as necessary for complete homogenization. This was followedby centfifugation at 5000 rpm at-10~ for
10 min, and the supernatant was immediately transferred to
another 16 x 100-mm tube. The solvent was evaporated under
nitrogen at 50~ One milliliter of saturated potassium phosphate
monobasic solution and 10 mL of CHCI3 were added to the
residue, and the sample was shaken for 20 rain with a reciprocating shaker operatedat 60 cycles/rain.The dark-coloredaqueous
phase was discarded and CHCI3 was then evaporated under
nitrogen at 50~ The residue was dissolved in 1 mL of 0.1M
phosphate buffer (pH 6.8) and 0.2 mL of [3-glucuronidase(25,000
units/mL in 0.1M phosphate buffer, pH 6.8) was added, and the
tube was looselycappedand put into a 37~ oven overnight (16 h).
Ion 377.00(376.70to 377.70):$201-011.D
200001
Ion
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16O0O
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Ion 306.00(305.70to 306.70~:S201-037.D
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09.~"
9.2()
9:25
9.30
9.35
9.40
Time(min)
Ion322.001321.70~o322.701:S201"037.D
9.45
20oo
Ion 371.00(370.70to 371.70):$201-011.D
Ion
473.00(472.70t9,~73.70): S201-011.D
Ion488.00(487.70t 4~..70):$201-011.O
It
160001
16000t
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4000L
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+9.~
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Time(mln)
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Ion 354.00~353.70~354.701:S201-037.D
110oo1
18oo~,
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7ooo,
2000
~
9.20
T
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//1
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9.15
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30 5.40 5.,50 5.60 5.70 5.00 5.90 6.00 6.10 6.20 6.30 6.40
12000
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+g . ~ + - 9 . + , i ; " 0 . ~
12000.1
0'
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9.60
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9.80
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Time(mln)
-
.'~...~m-. ~
9:30
9:35
Time (mln)
9.40
9.45
Figure1.SIMchromatogramsofa meconiumspecimencontaining(A)50ng/gTHCCOOH-d6,(B)37ng/gTHCCOOH,(C)18.8ng/g88,11-diOH-THC,(D)50
ng/gTHC-dg,and(E)56ng/g11-OH-THC.
331
Journal of Analytical Toxicology, Vol. 22, July/August 1998
After hydrolysis, the sample was cooled to room temperature.
The mixture was then acidified with 0.5 mL of 1N HCI and 10 mL
of hexane/ethyl acetate (9:1) was added, and the tube was shaken
for 5 rain. The top organic layer was transferred into a 15-mL
centrifuge tube, and 1.5 mL of 1N NaOH was added. The tube
was shaken for 2 rain. The top organic layer containing neutral
analytes was transferred into another 15-mL centrifuge tube
for determination of THC, 11-OH-THC, and 8-OH-THC (both aand IMsomers). The bottom aqueous layer containing acidic
analytes (THCCOOHand 813,11-diOH-THC)was filtered through
a cotton plug to remove the semi-solid material.
Fraction containing neutral analytes
Four milliliters of 0.2N NaOH in methanol solution was added
to the organic extract containing the neutral analytes, and the
tube was shaken for 2 rain. After it separated into two phases, the
top hexane layer was carefully removed and discarded. One
milliliter of 1N HCI and 3 mL of deionized water were added to the
bottom methanol layer. The tube was shaken for 1 rain. The top
organic layer was separated following the addition of acid, and
water was transferred to a 13 • 75-mm culture tube. Three
milliliters of hexane/ethyl acetate (9:1) was added to the bottom
aqueous layer, and the solution was shaken for 1 min. The top
organic layer was transferred to the 13 • 75-ram culture tube
containing the organic phase. The solvent was evaporated under
nitrogen at 50~ The residue was derivatized with 20 IlL of pyridine and 60 ]JL of acetic anhydride at 70~ for 30 rain. The sample
was transferred to a GC vial with an insert, and 2 pL of the sample
was injected on the GC-MS for determination of neutral analytes.
Fraction containing acidic analytes
Two milliliters of 1N HCI was added to the aqueous phase from
the initial extraction containing the acidic analytes. Four milliliters
of hexane/ethyl acetate (9:1) was added and shaken for 2 min.
The top organic layer was transferred to a 13 x 75-mm culture
tube, and the solvent was evaporated under nitrogen at 50~ The
residue was defivatized with 80 I~Lof BSTFAat 70~ for 30 min.
The sample was transferred to a GC vial with an insert and 2 IJL
was injected into the GC-MS for determination of acidic analytes.
GC-MS analysis
GC-MS analysis was performed on an HP 5890 GC interfaced with an HP 5970 MSD in EI (70 ev) mode with SIM monitoring. The electron multiplier voltage was set 200 mV above
the tune value. The GC was equipped with a 25-m x 0.2-mm
(0.33-pro film thickness) DB-5 MS column operated in the
splitless mode with the valve closed for 0.6 rnin. Helium carrier
velocity was 38 cm/s, and the injector and detector temperatures were set at 250~ and 280~ respectively.
For analysis of the neutral fraction containing Ag-THC, 11OH-Ag-THC, and 8a- and 813-OH-Ag-THC, the
column
temperature was initially set at 220~ for
Table III. Effect of Enzyme Hydrolysis on the Concentration of THC and
0.5 min., then raised to 275~ at 30~
where
its Metabolities in Meconium Specimens
it was held for 5.0 rain, and finally raised to 290~
Specimen THCCOOH 8[3,11-diOH-THC 11-OH-THC THC 8-OH-THCt
at 30~
and held for 8.0 min. Table I shows
id*
(ng/g)
(ng/g)
(ng/g)
(ng/g)
(ng/g)
the retention times and ions monitored for all
o
neutral analytes under these experimental condiA-hydro
37.0
18.8
56.0
0
o
tions.
A-nonhydro
39.3
3.4~
11.0
0
0
For analysis of the acidic analytes, the column
B-hydro
4.8
5.3
22.0
0
0
temperature was initially set at 200~ and held for
B-nonhydro
6.4
0
0
0
C-hydro
C-nonhydro
219
150
46.9
8.8
178
10.0
3.0
7.0
7.9~
0
hydro = enzymatically hydrolyzed; nonhydro = without hydrolysis,
8-OH-THC was a combination of 8J3-OH-THC and 8(~-OH-THC.
Below assayestablished LOD but with acceptable ion ratios.
Based on a single ion.
Table IV. Concentrations of THC and its Metabolites in Meconium
Collected from Neonates Born to Mothers with Histories of Marijuana
Use During Pregnancy
Specimen THCCOOH 8~,11-diOH-THC 11-OH-THC
id*
(ng/g)
(ng/g)
(ng/g)
D
E
F
G
H
I
J
259
5.6
4.6
25.2
97.5
60.6
102.9
17.7
0
4.9*
2.8*
17.7
14.9
18.8
25.0
13.0
40.0
17.0
74.0
44.0
81.0
*All specimens were analyzed using the enzymatic hydrolysis step.
t 8-OH-THC was a combination of 8~-OH-THC and 8~-OH-THC.
*Based on a single ion.
332
THC
8-OH-THCt
(ng/g)
(rig/g)
0
0
0
4.0
3.0
0
0
16.7
0
0
0
0
0
0
0.5 min. after injection, then raised to 280~ at
30~
and held for 12 min.
Quantitative GC-MS analysis was performed by
either a single- or multi-point curve containing a
known concentration of each ana]yte spiked in
blank meconium. For THCCOOH and THC, the
curves contained 10, 25, 50, 100, and 200 ng/g of
control meconium. For 11-OH-z~9-THC and
813,1I-diOH-Ag-THC,the curves contained 10, 25,
and 100 ng/g. For 8-OH-THC, a single-point calibrator at 25 ng/g was used.
Results and Discussion
Analysis of THC metabolites in meconium is
complicated by the presence of large amounts of
neutral lipids that can give rise to a high background in GC-MS analysis unless the samples are
subjected to extensive cleanup.
All previous work with meconium has focused
on the analysis of the carboxy-THC, and no other
Journal of Analytical Toxicology, Vol. 22, July/August 1998
metabolites of THC have been measured in this matrix. In addition, meconium specimens were analyzed either unhydrolyzed
or followingbase hydrolysis.In this study, the major metabolites
of THC were investigated both in the free form and after enzyme
hydrolysis to determine if any of the THC metabolites existed as
the ether glucuronides and if enzyme hydrolysis would be justified or needed for future analysis of routine specimens.
Selection of the most efficient enzymatic system
for the hydrolysis of Ag-THC glucuronide
Different hydrolysis conditions were examined for Ag-THC
glucuronide. The amount of A9-THC released from hydrolysis
was quantitated against standard solutions of A9-THCby GC-MS
using A9-THC-d9as internal standard. The results are summarized in TableII. The best hydrolysis conditions were established
in experiments using buffer solutions and not meconium suspensions because quantitation methods were not in place to
monitor the efficiencyof hydrolysis from meconium extracts.
As shown in Table II, bacterial 13-glucumnidase(E. coli, type
IX-A)was the most effectivein hydrolyzingA9-THC-glucuronide.
This is in agreement with the information reported by Kemp et
aL (13).
Quantitative hydrolysis was accomplished by incubation at
37~ for 16 h with 5000-10,000 units of the enzyme. This procedure was then used to hydrolyze meconium extracts.
hydrolyzedA9-THCglucuronide from buffered solution, the efficiency of hydrolysis from extracts of meconium spiked with the
glucuronide was only 10% using 5000 units of the enzyme.
Increasing the amount of enzyme or raising the temperature to
60~ did not significantly increase hydrolysis efficiency. This
was probablydue to other components in the extract from meconium which either deactivated or consumed the enzyme. Therefore, it was necessary to remove such component(s) before
performing the enzymatic hydrolysis.For this purpose, saturated
potassium dihydrogenphosphate (pH 3--4) was added to help
salt out the glucuronides that are usually very hydrophilic. Different extraction solvents were then tried, and CHCI3was found
to be effective in extracting A9-THCglucuronide. Subsequent
enzymatic hydrolysis of the extracted glucuronide was accomplished with substantially increased efficiency of 82%. After
enzymatic hydrolysis, the sample was split into two fractions.
Surprisingly, 8[3,11-diOH-Ag-THC,in addition to THCCOOH,
was also found in the acidic fraction, which was in contrast to
reports by Kemp et al. (13,14) in which this compound was
found in the neutral fraction of extracts obtained from urine and
blood specimens. Under our experimental conditions, 80% or
more of total 81~,11-diOH-A9-THCwas found in the acidic fraction, even in urine specimens.
Although only a partial resolution of the TMS derivatives of
A9-THCCOOHand 8~,11-diOH-A9-THCwas accomplished, there
was at least two mass units difference in the ions monitored for
the two metabolites, and they did not interfere with each other
over the concentration range of all the meconium specimens
analyzed in this study. The average recoveryfor these metabolites
was 48% for THCCOOH and 78% for 8~,11-diOH-A9-THCat
the 50-ng/g level.
The neutral fraction contained large amounts of neutral lipids
which caused much interference in the GC-MS analysis. It has
been reported that back extraction of THC into alkaline
methanol from the hexane layer helped separate THC from the
neutral lipid in the blood assay (15-18). This method was then
used and found to be effective in eliminating the interfering
peak for the quantitation ion of 11-OH-THC (ion 312). As a
Effect of enzymatic hydrolysis of meconium extracts on the
concentration of the total metabolites of Ag-THC in
meconium
In order to determine if an enzymatic hydrolysisstep is needed
during the analysis of meconium specimens for A9-THCmetabolites, three meconium specimens that screened positive by
immunoassay were analyzed both hydrolyzed (for total metabolites) and non-hydrolyzed (for free metabolites). Table III shows
the results of the analysis of these specimens identified as A-C.
It is clear, even from this limited number of specimens, that the
concentration of the total hydroxylated metabolites was significantly higher than that of the free metabolites. Specimen B, for
example, showed significant levels of ll-OH-AgTHC and 813,11-diOH-A9-THCafter hydrolysisbut
I Table V. Concentrations of THC and its Metabolites in Meconium
none without hydrolysis. On the other hand, the
Specimens Positive by EMIT 20 Cannabinoids Assay but Failed to
effect of enzyme hydrolysis on the concentration
Confirm by GC-MS for THCCOOH in Earlier Work
of the carboxylic acid metabolite was less draSpecimen THCCOOH 813,11-diOHoTHC 11-OH-THC THC 8-OH-THC t
matic. Although no substantial differences were
id*
(ng/g)
(ng/g)
(ng/g)
(ng/g)
(ng/g)
observed for Specimens A and B, Specimen C
showed an almost 50% increase in the A9-THCK
2.3
0
7.0
0
0
COOH concentration as a result of the hydrolysis.
L
9.1
4.7'
259
0
0
Therefore, it was concluded that an enzymatic
M
7.4
4,1 ~'
22.0
0
0
hydrolysis step is required for the recovery of all
N
4,1
15.9
70.0
0
0
Ag-THCmetabolites in meconium specimens.
O
2.7
0
75.0
0
0
Specimensanalysis
The first step in the extraction process was
accomplished by sonic disruption of meconium
with methanol to recover all metabolites. Evaporation of the methanol resulted in the crude
extract that is to be hydrolyzed. Although the 8glucuronidase enzyme from E. coti quantitatively
P
Q
R
S
9.8
0
26.3
0
5.3
32.2
4.3*
68.6
204
929
34.0
144
0
0
2.4
0
0
0
0
0
*All specimenswereanalyzedusingthe enzymatichydrolysisstep.
tg-OH-THC was a combinationof 81]-OH-THCand 8a-OH-THC.
*BelowassayestablishedLOD but with acceotableion ratios.
~Basedon a singleion.
333
Journal of Analytical Toxicology, Vol. 22, July/August 1998
consequence, the extraction recoveries for the neutral analytes
were relatively low (average 39% for THC, 36% for 11-OH-THC,
and 41% for 8-OH-THC).
Even though extensive cleanup steps were carried out, interference problems still existed for the qualifying ions of Ag-THC.
The acetate derivatives of neutral analytes were selected over
other derivatives tested (propionate, TMS, trifluoroacetate,
pentafluoropropionyl, methyl, and TBDMS) because they provided minimum interference. 813-OH-THCand 8a-OH-THC had
the same retention time with the same ions and ion ratios as the
acetate derivatives; consequently, these two metabolites were
analyzed as one compound (8-OH-THC).
Although much effort was exerted to obtain a single extract
that included all analytes, all efforts led to serious difficulty in
GC-MS analysis.
GC-MS assay performance
A9-THCCOOH and 813,11-diOH-A9-THCin the acidic fraction
had a 2 and 5 ng/g limits of detection (LOD), respectively. The
LOD of 11-OH-THC and 8-OH-THC in the neutral fraction were
10 and 15 ng/g, respectively. The LOD was defined as the lowest
concentration that still produced satisfactory ion ratios (• 20%
of calibrators). For A9-THC,the qualifying ion below 50 ng/g was
difficult to use because of interference. However, the quantitation ion (ion 297) was distinguishable from the baseline at 5
ng/g. The curve for Aa-THC based on the quantitation ion was
linear from 10 ng/g up to 200 ng/g. Because the internal standards for ] 1-OH-THC, 813,11-diOH-A9-THC,and 8-OH-THC were
not their directly deuterated compounds, the curves varied from
batch to batch because of the variation in extraction efficiency of
the internal standards versus the analytes. Figure 1 shows typical chromatograms for all analytes from Specimen A of Table III.
Analysis of meconium specimens collected from infants
born to mothers with a history of marijuana use
With the analytical method established for A9-THCand four of
its metabolites and their glucuronides, the objective was to establish which of these metabolites are abundant in meconium specimens to indicate prenatal exposure to marijuana. Seven
specimens (D-J) were collected from neonates born to mothers
with histories of marijuana use during pregnancy. These specimens were then analyzed for all metabolites with enzymatic
hydrolysis, and the results are shown in Table IV. It is evident from
these data that the major Ag-THC metabolites in meconium are
Ag-THCCOOH, 11-OH-A9-THC, and 813,11-diOH-A9-THC, and
their glucuronides. It is also evident that A9-THC and 8-OH-A9THC and their glucuronides are not present in most specimens
and, therefore, will most likely not play a significant rote in the
GC-MS confirmation process of A9-THC metabolites in meconium.
Confirmation of previously unconfirmed specimens
screening positive by EMIT 20
In a previous study carried out for NICHD, meconium specimens were screened by EMIT immunoassay at a 20-ng/g cutoff,
and positive specimens were subjected to GC-MS analysis for the
free ~9-THCCOOH with only a 26% confirmation rate (7).
Having established that other A9-THC metabolites, including
334
glucuronides, do exist at significant levels in meconium specimens of neonates prenatally exposed to marijuana, the objective
of this effort was to re-analyze some of the specimens that previously failed to confirm for the free Ag-THCCOOHby GC-MS.
Table V shows the results of the analysis of nine such specimens. Several significant observations were made: 1. All specimens were confirmed for the presence of THC metabolites
supporting the positive EMIT screening results. 2. Seven of the
nine specimens showed some level of THCCOOH that could be
the result of enzyme hydrolysis. 3. All specimens were positive
for 11-OH-A9-THC, most of them with substantial levels (> 30
ng/g). 4. The two specimens with no THCCOOH (Q and S)
showed high levels of 11-OH-Ag-THC (929 and 144 ng/g, respectively). 5. Seven of the nine specimens were positive for 813,11diOH-Ag-THC, but the levels were much lower than those for
ll-OH-z~9-THC in all cases. 6. Almost none of the specimens
were positive for Ag-THC or its 8-OH-metabolite(s). 7. The ratio
of the hydroxylated metabolites to the carboxy-THC was different for the different specimens. This could be the result of the
difference in metabolism among neonates or mothers or the
result of differences in time of exposure.
The fact that all specimens screening positive at 20 ng/g by
EMIT were confirmed for one or more ofTHC metabolites suggests that these metabolites have high cross-reactivity to THCCOOH at this low level. Experiments carried out in our previous
unpublished work showed cross-reactivity rates of 31, 161, 74,
and 83% for Ag-THC, 11-OH-Ag-THC, 8[3-OH-Ag-THC, and
813,11-diOH-Ag-THC, respectively. The glucuronide of Ag-THC,
however, had no cross-reactivity up to 400 ng/g.
Conclusion
In conclusion, two additional metabolites of THC, namely
11-OH-THC and 8!3,11-diOH-Ag-THCwere found to be present
in significant quantities in meconium specimens of neonates
prenatally exposed to THC. These metabolites predominantly
exist as the glucuronide conjugates. It is, therefore, necessary to
hydrolyze meconium samples enzymatically when analyzing for
the metabolites. Analysis of these two compounds, along with
THCCOOH, will drastically increase the confirmation rate for
cannabinoids in meconium specimens which screen positive
for marijuana at a relatively low cutoff (20 ng/g). It is also concluded that the parent drug A9-THCand its 8-OH metabolite are
either not detected or present in low concentrations in meconium specimens screening positive for cannabinoids.
Acknowledgment
This work was supported in part by the National Institute on
Drug Abuse (SBIR Contract No. N43DA-6-7057). The authors
also gratefully acknowledge the support of Dr. Henrietta Bada of
the E.H. Crump Hospital, Memphis, TN, and Dr. Linda Wright,
NICHD, National Institutes of Health, for making available
meconium specimens from the MLS Study.
Journal of Analytical Toxicology,Vol. 22, July/August1998
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