Clinical Mass Spectrometry xxx (xxxx) xxx–xxx

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Clinical Mass Spectrometry

journal homepage: www.elsevier.com/locate/clinms

Detection of in utero Exposure to Cannabis in Paired Umbilical Cord Tissue

and Meconium by Liquid Chromatography-Tandem Mass Spectrometry
Triniti L. Jensenb, Fang Wuc, Gwendolyn A. McMillina,b,
⁎

a
Department of Pathology, University of Utah, Salt Lake City, UT 84132, United States
b
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States
c
Department of Laboratory Medicine and Pathology, Saskatchewan Health Authority & University of Saskatchewan, SK, S7W 0S1, Canada

ARTICLE INFO ABSTRACT

Keywords: Understanding levels of in utero drug exposure is important to properly customize the immediate, as well as
In utero drug exposure ongoing, medical and social management needs of affected newborns. Here, we present the development of a
Cannabinoids liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of 4
Meconium cannabinoid analytes in two neonatal matrices. The analytes targeted were Δ9-tetrahydrocannabinal (THC), 11-
Umbilical cord tissue
nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN). The matrices analyzed were
LC-MS/MS
umbilical cord tissue and meconium. A fifth analyte, cannabidiol (CBD), was also detected uniquely in meco-
nium. Extracts were analyzed by LC-MS/MS in negative electrospray ionization mode.
Paired meconium and umbilical cord samples (i.e., one specimen from each matrix collected from each single
birth, n = 46 pairs) were tested to evaluate concentration and metabolite profiles. THCA was detected in all
positive (containing one or more analytes) meconium samples (n = 32). CBN, THC, 11-OH-THC, and CBD were
present in 57% (n = 26), 39% (n = 18), 24% (n = 11), and 20% (n = 9), respectively. Concentrations were
lower in the umbilical cord samples for all analytes (i.e., 0.27–537 ng/g for meconium and 0.1–9 ng/g for
umbilical cord). In umbilical cord THCA was also detected in all positive samples (n = 19) while THC, CBN, and
11-OH-THC were present in 24% (n = 11), 17% (n = 8), and 11% (n = 5), respectively. Testing neonatal ma-
trices for cannabinoids could be used to support studies designed to detect newborns exposed to cannabis in
utero, as well as provide data that could be examined for correlations with clinical and social outcomes.

1. Introduction
Cannabis (e.g., marijuana) is widely used in the United States,
where it is now legal for either medicinal or recreational use in a ma-
jority of states, but remains illegal based on federal scheduling. The
national survey of drug use and health report from 2016 showed an
increase of past marijuana use among all individuals over 12 years of
age [1]. Increased admission of marijuana use was also noted in preg-
nant women. For example, approximately 8.5% of pregnant women
aged 18–25 admitted to the use of marijuana during the past month in
the 2016 survey, compared to 6.4% in 2015 [1]. The increased pre-
valence of cannabis use in pregnant women has raised concerns about
the impact of cannabis exposure to the fetus, and potential outcomes.
The cannabis leaf contains more than 100 cannabinoids and over
500 other chemicals, with THC being the major psychoactive
phytocannabinoid that has been identified [2]. THC is commonly used
for its intoxicating effects, including euphoria and sedation. During
pregnancy, cannabis has been used to decrease nausea, pain, anxiety,
insomnia, and to stimulate appetite. A study from Vancouver, Canada
found that up to 77% of medicinal cannabis use during pregnancy was
related to nausea and over 50% reported cannabis use to treat lack of
appetite, general pain, insomnia, anxiety, depression and fatigue [3].
Evidence shows that cannabinoids freely cross the placenta and directly
reach the fetus [4–6]. Cannabinoids act on the endocannabinoid system
that is important to the development of the embryonic brain from early
neural stem cell survival, to the differentiation of neuronal cells, and
synaptic function [7–9]. During early stages of brain development,
cannabinoids may affect sensory input, control of body temperature,
and higher order cognitive processes including memory, learning, an-
xiety, depression, pain, and decision making [10,11]. The Ottawa

Abbreviations: CBN, cannabinol; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; NaOH, sodium hydroxide;
NH4OH, ammonium hydroxide; RT, retention time; THC, Δ9-tetrahydrocannabinal; THCA, 11-nor-9-carboxy-THC (THCA); 11-OH-THC, 11-hydroxy-THC (11-OH-
THC
⁎
Corresponding author at: ARUP Laboratories, 500 Chipeta Way, MS-115, Salt Lake City, UT 84108, United States.
E-mail addresses: triniti.scroggin@aruplab.com (T.L. Jensen), fang.wu@saskhealthauthority.ca (F. Wu), gwen.mcmillin@aruplab.com (G.A. McMillin).

https://doi.org/10.1016/j.clinms.2019.01.002
Received 18 October 2018; Received in revised form 23 January 2019; Accepted 23 January 2019
2376-9998/ © 2019 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V. All rights reserved.

Please cite this article as: Jensen, T.L., Clinical Mass Spectrometry, https://doi.org/10.1016/j.clinms.2019.01.002
T.L. Jensen et al.
prenatal prospective study (approximately n = 180) annually mon-
itored individuals that were exposed to cannabis in utero until the age of
6 and then less frequently thereafter until the age of 9. Despite several
limitations of the study design, results suggest behavioral problems,
reduced performance on visual perception tasks, language compre-
hension issues, sustained attention deficiencies, and memory difficulties
at 6–9 years of age that may be related to pre-natal cannabis exposure
[12].
Testing of neonate specimens for the presence of drugs and drug
metabolites, as well as their concentration profiles, can provide con-
vincing evidence of pre-natal drug exposure and a better understanding
of thresholds that can predict newborns who are at risk of future
medical and social complications [13–16]. That said, positive results do
not indicate the timeframe, level, or duration of drug exposure, nor
does a negative result rule out in utero exposure. Analysis of meconium
has been used to assess in utero exposure to drugs, including cannabi-
noids, for more than 20 years [17–20]. Meconium is the first stool
passed by the newborn, and it begins to form and collect in the intestine
between the 12th and 16th week of gestation. Collection of meconium
is unpredictable due to retention for several days after birth or passage
prior to, or during, delivery. Meconium is preferred over hair, urine,
amniotic fluid, and blood due to the broad time window to exposure it
provides that reflects approximately the last trimester of a full term
birth [21–24]. In recent years, umbilical cord tissue has become an
alternative matrix [24]. The umbilical cord is the life line between the
fetus and mother and is formed in approximately the 5th to 6th week of
gestation. Although not as well understood as a site for drug deposition,
analysis of umbilical cord has proven to be sensitive to exposure for
many drugs over the last trimester, and exhibits several advantages
over meconium (e.g., ease of collection at birth, and ample sample
volume). Moreover, umbilical cord testing can avoid misleading results
caused by administration of medications to the newborn directly after
birth, prior to collection of meconium.
Traditional algorithms for drug testing include screening, often by
immunoassay, followed by confirmation testing, typically performed by
a chromatographic method coupled to mass spectrometric detection.
Our laboratory has previously evaluated methods for detection of ex-
posure to cannabis based on targeting a single analyte [25,19], and
proposed that a direct targeted approach to drug testing would be more
efficient, and potentially more sensitive, than the traditional screen-
with-reflex approach [26]. Here, we described a method designed to
detect and quantify 4 cannabinoids in both the meconium and umbilical
cord samples. The cannabinoids targeted include Δ9-tetra-
hydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-
THC (11-OH-THC), and cannabinol (CBN). Cannabidiol (CBD) was de-
tected uniquely in meconium. In addition, 46 paired authentic clinical
samples (both meconium and umbilical cord collected from a single
birth) were analyzed to assess the similarities and differences in me-
tabolite concentrations and abundance profiles between matrices from
the same birth. In addition to identifying in utero exposure to cannabis,
data produced from this method could be correlated with clinical and
social outcomes to help construct prognostic flow charts to assist in
identifying if, and perhaps even when, an individual is likely to need
clinical and/or social support in the future.
2. Methods and materials
2.1. Chemicals and reagents
Ethyl acetate, hexane, methanol and acetonitrile were HPLC grade
and purchased from VWR international (West Chester, PA, USA).
Clinical laboratory reagent water was generated using a Milli-Q in-
tegrated water system by Millipore Sigma (Burlington, MA, USA). The
following reagents were purchased from Sigma-Aldrich (St. Louis, MO,
USA): glacial acetic acid, ammonium bicarbonate, ammonium hydro-
xide (NH4OH), and sodium hydroxide (NaOH). Stock standards and
2
Clinical Mass Spectrometry xxx (xxxx) xxx–xxx
matched deuterated standards for all analytes were purchased from
Cerilliant (Round Rock, Texas, USA).
2.2. Preparation of meconium calibration standards and quality control
(QC) solutions
A primary Cerilliant stock solution containing THC, 11-OH-THC,
THCA, CBN, and CBD, each at 100 µg/mL, was prepared in methanol.
The stock solution was diluted to create four calibration solutions at
10 µg/mL, 6.5 µg/mL, 3.5 µg/mL, and 50 ng/mL. A calibration curve
was generated by fortifying blank (previously verified by an existing
ELISA method to be free of cannabinoids) meconium with stock solu-
tions to create matrix-matched calibrators with the following con-
centration: 5, 350, 650, and 1000 ng/g. QC stock solutions were pre-
pared independently of the calibration standards and included one high
control where THCA-glucuronide was added as an indicator of hydro-
lysis and the free drug was excluded. QC solutions were used to fortify
blank meconium for a final concentration of 7 and 750 ng/g, respec-
tively. Primary stock solutions of THC-d3, THCA-d3, 11-OH-THC-d3,
CBN-d3, and CBD-d3 were diluted in methanol to produce a mixed
internal standard solution of 5000 ng/mL. All stock and working stan-
dards were aliquoted and stored in −80 °C in amber glass vials.
2.3. Preparation of umbilical cord calibration standards and quality control
(QC) solutions
A primary Cerilliant stock solution containing THC, 11-OH-THC,
THCA, and CBN at 50 µg/mL was prepared in methanol. The stock so-
lution was diluted to create four calibration solutions at 400, 200, 20,
and 8 ng/mL. A calibration curve was generated by fortifying blank
pooled umbilical cord (previously verified to be free of cannabinoids)
with stock solutions to create matrix matched calibrators with the fol-
lowing concentration 0.2, 0.5, 5.0, and 10.0 ng/g. QC stock solutions
were prepared independently of the calibration standard and included
one high control where THCA-glucuronide was added as an indicator of
hydrolysis and the free drug was excluded. QC solutions were used to
fortify blank umbilical cord for a final concentration of 0.25 and 7.5 ng/
g, respectively. Primary stock solutions of THC-d3, THCA-d3, 11-OH-
THC-d3, and CBN-d3 were diluted in methanol to produce a mixed
internal standard solution of 100 ng/mL. All stock and working stan-
dards were aliquoted and stored in −80 °C in amber glass vials.
2.4. Sample extraction procedure: Meconium
Blank meconium samples, for the calibrators and controls, were
weighed out (0.25 ± 0.01 g) into 2 mL Eppendorf polypropylene tubes.
Patient samples were weighed out (0.25 ± 0.01 g) into pre-labeled
2 mL tubes. All patient samples and QC samples were spiked with 25 µL
of the deuterated internal standard solution and 1.3 mL of methanol.
QC and calibrator samples were spiked with 25 µL of the appropriate
stock solution and all samples received ½ scoop of 0.5 mm stainless
steel beads (Next Advance, NY, USA). Tubes were capped and samples
were homogenized using a Bead Rupter (Biotage, NC, USA) for 4 cycles,
each 10 s long, at speed 3.1 with a dwell time of 10 s. Homogenates
were centrifuged for 10 min at 14,000 rpm (20,598×g) and 0–4 °C.
Each supernatant was transferred to a 10 mL culture tube. Base hy-
drolysis was performed by adding 1.3 mL 0.5 mol/L NaOH. Basic su-
pernatants were incubated at ambient temperature (23–25 °C) and
continuously shaken for 20 min on a VWR advanced multi-tube vor-
texer at 230 V with a speed 700 rpm. Following hydrolysis, solid phase
extraction was performed using Evolute AX SPE columns (60 mg/3 mL,
Biotage). SPE sorbents were washed and equilibrated with 1 mL me-
thanol and 1 mL water using a positive pressure manifold (SPEware
Incorporated, San Pedro, CA) applying pressure to achieve a 1 drop/3 s
rate. After conditioning, calibrators, controls, and patient specimens
were loaded by gravity onto the SPE columns. If samples did not load by
T.L. Jensen et al.
gravity pressure was applied to achieve a 1 drop/5 s rate. SPE sorbent
were washed with 1 mL 1% NH4OH in 85:15 water: acetonitrile fol-
lowed by 1 mL 50:50 hexane: ethyl acetate at 1 drop/s. Analytes were
eluted into silanized conical bottom LC vials with two 600 µL fractions
of 2% acetic acid in 90:10 hexane: ethyl acetate. Samples were con-
centrated at 40 °C under a gentle nitrogen stream at 30 psi with a Turbo
Vap LV (Biotage, Charlotte, NC, USA) for approximately 15 min.
Reconstitution was completed by the addition of 150 µL of water: me-
thanol (40:60) and autosampler vials were capped with pre-slit cap
prior to instrument analysis.
2.5. Sample extraction procedure: Umbilical cord
Sample extraction procedures followed for umbilical cord were
previously published with minor changes described here, including
sample pre-treatment, homogenization, and hydrolysis changes [27].
Blank umbilical cord samples were weighed (1.0 ± 0.1 g) for the ca-
librators and controls into 7 mL Eppendorf polypropylene tubes with a
screw top. Following the calibrator and control weigh out, patient
samples were placed in a weigh boat and cut into ½ cm cubes with a
disposable razor blade and 1.0 ± 0.1 g was weighed into pre-labeled
7 mL tubes. All patient samples and QC samples were spiked with 25 µL
of the deuterated internal standard solution and 2.5 mL of methanol.
QC and calibrator samples were spiked with 25 µL of the appropriate
stock solution and all samples received six 5.6 mm UFO stainless steel
beads (Next Advance, NY, USA). Tubes were capped and samples were
placed in a −70–80 °C freezer for 10 min prior to homogenization.
Samples were homogenized using a Bead Rupter (Biotage, NC, USA) for
4 cycles, each 30 s long, at speed 5.0 with a dwell time of 30 s.
Homogenates were centrifuged for 10 min at 14,000 rpm (20,598×g)
and 0–4 °C. Supernatants were transferred to 10 mL culture tubes; base
hydrolysis was performed in the same manner as meconium but with
2.5 mL 0.5 mol/L NaOH. Basic supernatants were incubated at ambient
temperature (23–25 °C) and continuously shaken for 20 min on a VWR
advanced multi-tube vortexer, 230 V at speed 700 rpm. Following hy-
drolysis, solid phase extraction was performed using Evolute AX SPE
columns (60 mg/3mL, Biotage), as described previously (28).
2.6. LC-MS/MS conditions for meconium and umbilical cord
Chromatography, analysis parameters and associated instrumenta-
tion were identical for both the extracted meconium and umbilical cord
samples. LC separation was performed on a Poroshell 120 EC-C18
column (2.1 × 50 mm, 2.7 µm particle size) paired with an in-line filter
(Agilent, Santa Clara, CA). Solvent A (5 mM NH4OH in nanopure water,
pH 9.5) and solvent B (methanol) were used to develop a gradient: 60%
B for 0.50 min, linear gradient from 60% B to 80% B between 0.50 and
1.20 min, hold at 80% B for 1.10 min, and a linear gradient from 80% B
to 95% B between 2.30 and 3.20 min, and hold at 95% B for 50 s fol-
lowed by re-equilibration to initial condition (total run time 4.7 min).
The LC eluent was diverted to waste for the first 0.5 min and the final
0.5 min. LC flow rate was 0.75 mL/min with an injection volume of 5 µL
for meconium and 50 µL for umbilical cord (Fig. 1 for chromatographic
separation). The column and autosampler were held at 28 °C and 4 °C,
respectively. MS/MS analysis was performed by AB SCIEX Triple Quad™
5500 mass spectrometer interfaced with CTC PAL HTC-xt-DLW auto-
sampler and Agilent 1260 infinity series binary pump, degasser and
column oven. Samples were ionized in negative electrospray mode and
data were collected using multiple reaction monitoring, see parameters
in Table 1. MS/MS parameters were optimized via direct infusion of a
mixture of standards followed by a split tee injection with LC flow
(curtain gas 30 psi, collision gas 10 psi, ion spray voltage −3500 v,
600 °C, nebulizer gas 20 psi, and heater gas 30 psi). AB SCIEX Analyst
software (version 1.6.1) was used for instrument control. Two transi-
tion ions were monitored for each drug analyte and its respective
deuterated standard, as shown in Table 1. Ion ratios were calculated by
3
Clinical Mass Spectrometry xxx (xxxx) xxx–xxx
dividing the peak area of the qualifier ion by the peak area of the
quantifier ion. An average ion ratio was calculated using the data ob-
tained with calibrators and controls; ion ratios of positive patient
samples were monitored and were considered acceptable if results fell
within ± 30% of the established average. Quantitation was performed
using MultiQuant™ 3.0.2 software (AB Sciex, Foster City, CA) in-
tegrating with the MQ4 algorithm. Calibration curves were constructed
via linear least-squares regression with 1/x weighting factor.
2.7. Method validation
Validation experiments included within- and between-run impreci-
sion, accuracy, patient correlation, linearity, sensitivity, SPE recovery,
method recovery, matrix effect, carryover and on-board stability of
extracts using blank meconium and umbilical cord fortified with non-
deuterated and deuterated standards.
Within-run, between-run and total imprecisions of the assay (percent
coefficient of variation, %CV) were evaluated by extracting samples
across the analytical measurement range (AMR) in triplicate over five
days. Accuracy was determined using calibrators (prepared in matrix)
tested in triplicate over five days where the experimental concentra-
tions were compared to the expected concentration. The linearity of the
assay was assessed by evaluating the data generated by a four-point
calibration curve run in triplicate for 3 days alongside 5 AMR samples.
Samples were prepared with drug-free meconium and umbilical cord
fortified with an appropriate concentration of the analytes. The lower
limit of quantitation (LLOQ) was defined as the lowest calibrator, and the
validation was designed to evaluate performance with a signal to noise
ratio (S/N) ≥ 10, as well as all qualitative and quantitative parameters,
including Gaussian chromatography, retention time (RT) within ± 2%
of the target RT, all monitored ions present and all ion mass ratios
(calculated by dividing the area of quantifier ions by the area of qua-
lifier ions) within ± 30% of the mean ion mass ratios of calibrators and
controls, accuracy within 85–115% of the target concentrations and
imprecision ≤20%. The limit of detection (LOD) for each analyte was not
specifically determined, but performance of the method at concentra-
tions equivalent to half of the LLOQ was verified to ensure that the
acceptance criteria defined above and a S/N of ≥3 were met for each
analyte. The upper limit of quantitation (ULOQ) was the highest con-
centration at which all samples were accurate within 85–115% of the
target concentrations with imprecision ≤20%. Solid phase extraction
(SPE) recovery was evaluated by analyzing the same patient samples in
triplicate over five days. Deuterated standard was added to a set of
patient samples before and after SPE extraction. Patient sample re-
coveries were calculated based on the difference in concentrations be-
tween the pre-extracted and post-extracted samples. Method (analytical)
recovery was determined by spiking a set of drug-free samples with a
known amount of standard. Method recovery was evaluated by how
well the method accurately quantitated the spike samples where the
expected and experimental concentrations were compared. Patient
correlation was determined by analyzing 40 samples previously ana-
lyzed by a validated method along with 30 fortified negative patient
samples. Hydrolysis efficiency was calculated for THCA-glucuronide
using the high control spiked 1.51 times the target concentration due to
mass differences between glucuronidated and free drug. Mass spectro-
metry ion suppression was evaluated by injecting negative meconium or
umbilical cord extracts while infusing a solution containing each ana-
lyte of interest at 250 and 5 ng/g, respectively. Carryover studies were
performed to assess a) carryover due to a contaminated syringe, and b)
septum/injection port/column carryover using samples prepared at
concentrations twice the ULOQ followed by samples prepared with
concentrations near the low end of the AMR (i.e., 7 ng/g). Internal
standard counts were monitored within runs and between runs to as-
sure consistency across a run ( ± 30%). On-board stability of the ex-
tracted analytes was evaluated by re-analysis of a full batch (n = 54)
every 24 h for 3 days. The concentration and area counts at each time
T.L. Jensen et al. Clinical Mass Spectrometry xxx (xxxx) xxx–xxx

Fig. 1. Representative LC separation and extracted ion chromatograms of 1) 11-nor-9-carboxy-THC (THCA), 2) 11-hydroxy-THC (11-OH-THC), 3) cannabidiol (CBD),
4) cannabinol (CBN), 5) Δ9-tetrahydrocannabinal (THC) and matched (deuterated d3) internal standards at the limit of quantitation for meconium (5 ng/g) and
umbilical cord (0.2 ng/g).

4
T.L. Jensen et al. Clinical Mass Spectrometry xxx (xxxx) xxx–xxx

Table 1 average meconium SPE recoveries observed for THC, THCA, 11-OH-
Multiple reaction monitoring transitions (MRM) for non-deuterated and in- THC, CBN, and CBD were 73%, 83%, 74%, 83%, and 27%, respectively.
ternal standards for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC The average umbilical cord SPE recoveries observed for THC, THCA,
(THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol 11-OH-THC, and CBN were 74%, 82%, 58%, and 86%, respectively.
(CBD) with the matched deuterated (d3) internal standards and their respective
Because minimal amounts of CBD could be recovered from umbilical
retention times (RT).
cord, this analyte was excluded from the validated method. Using
Non-deuterated RT (min) MRM transitions for MRM transitions for THCA-glucuronide as a hydrolysis indicator showed that base hydro-
analyte/deuterated non-deuterated deuterated analyte lysis was effective to achieve concentrations within ± 20% of the target
internal standard analyte
concentration for both matrices. Short-term on-board stabilities of ex-
THC/THC-d3 3.8 313.10 → 245.1 316.0 → 248.0 tracted THC, THCA, 11-OH-THC, CBN, and CBD were evaluated using 3
313.10 → 191.1 316.0 → 194.0 concentrations for meconium extracts and 7 concentrations for umbi-
THCA/THCA-d3 1.9 343.1 → 244.9 346.0 → 248.2 lical cord extracts. Expected sample concentrations were within ±
343.1 → 191.0 346.0 → 194.0
20% of the target value with CVs < 20% when maintained at 4 °C in
11-OH-THC/11-OH- 2.9 329.1 → 268.1 332.1 → 271.0
THC-d3 329.1 → 173.3 332.1 → 173.0 the autosampler for 72 h. The analytical measurement range was
CBN/CBN-d3 3.6 309.3 → 222.1 312.0 → 282.0 5–1000 ng/g for THC, THCA, 11-OH-THC, CBN, and CBD in meconium
309.3 → 171.0 312.0 → 171.0 and 0.2–10 ng/g for THC, THCA, 11-OH-THC, and CBN in umbilical
CBD/CBD-d3 3.1 313.0 → 245.1 316.1 → 248.0 cord with r ≥ 0.995. No significant carryover was observed im-
313.1 → 179.0 316.1 → 182.0
mediately after a sample containing 2000 ng/g of each analyte. Matrix
effects in meconium were minimal for THC, THCA, 11-OH-THC, CBN,
point were compared to the initial condition. The ratios of the results and CBD at 78%, 101%, 107%, 109%, and 79%, respectively (a value of
were calculated. EP Evaluator software (Data Innovations, Burlington, 100% indicated no matrix effect). Matrix effects were observed in
USA), Multiquant, and Excel (Microsoft, Redmond, WA) were used for umbilical cord matrices with 93% (THC), 91% (THCA), 94% (11-OH-
data analysis. THC), and 92% (CBN), respectively.
A set of 46 paired samples collected from the same birth were
analyzed. Fig. 2 summarizes the concentrations of each analyte in both
2.8. Authentic clinical specimens matrices. Meconium: 70% (n = 32) of samples were confirmed positive
for one or more analytes. THCA was the most commonly observed
Forty-six paired meconium and umbilical cord samples collected analyte in meconium, present in 100% of the positive samples (n = 32)
from the same birth were evaluated with this method. Samples were with concentrations between 1.4 and 537 ng/g (median = 21 ng/g).
selected for inclusion in this study based on the ordering patterns for CBN was present in 57% of the samples (n = 26) with a concentration
archived clinical specimens [28,29]. Thus, specimens for which both range of 0.27–46 ng/g (median = 6 ng/g). THC was present in 39% of
meconium and umbilical cord testing for THC was requested for the the samples (n = 18) with a concentration range of 0.31–8 ng/g
same newborn were sought. Following procedures approved by the (median = 2 ng/g). 11-OH-THC and CBD were the least frequently
Institutional Review Board of the University of Utah, residual paired identified analytes, being observed in only 24% (n = 11), and 20%
samples (indifferent of THC results) were retrieved prior to scheduled (n = 9) of the samples with concentration ranges of 1.4–4 ng/g
discard dates. The specimens were de-identified, and analyzed with the (median = 3) and 2–53 ng/g (median = 4), respectively. Umbilical cord:
method described here. The concentrations of five cannabinoids in concentrations observed in umbilical cord were an order of magnitude
meconium and four cannabinoids in umbilical cord in authentic spe- lower than those in meconium. Similar to meconium, THCA was the
cimens were analyzed by the same LC-MS/MS method described above most frequently observed analyte in umbilical cord, showing up in 41%
and metabolite profiles, as well as respective concentrations, were as- (n = 19) of the samples with concentrations ranging from 0.1 to 9 ng/g
sessed. Data was analyzed in MultiQuant™ and imported into Excel (median = 2 ng/g). Interestingly, the second most frequently identified
Spreadsheets (Microsoft, Redmond, WA), EP Evaluator, and Rstudio analyte in umbilical cord was THC, present in 24% (n = 11) of samples
using ggplot2 [30] to perform statistical analysis. with a concentration range of 0.1–1 ng/g (median = 0.23 ng/g). CBN
was present in 17% (n = 8) of the samples (range = 0.1–0.15 ng/g,
3. Results and discussion median = 0.14 ng/g) followed by 11-OH-THC in 11% (n = 5) of the
positive (for one or more analytes) samples (range = 0.2–0.33,
In this study, we present a method for the detection and quantita- median = 0.28 ng/g). These results highlight the discrepancies of can-
tion of THC, THCA, 11-OH-THC, CBN and CBD in meconium and a nabinoid concentrations between meconium and umbilical cord. Con-
similar method optimized for umbilical cord to detect and quantitate sidering that little is known about the stability, drug deposition char-
THC, THCA, 11-OH-THC, and CBN. The additional analytes could im- acteristics, and pharmacokinetics of each individual drug and its
prove the sensitivity of analysis for in utero exposure to cannabinoids, metabolites in both matrices, these discrepancies are not surprising.
particularly when mothers are using CBD-containing products instead Fig. 3 shows the positivity rates of the five cannabinoids for the paired
of THC-containing products, and help support a better understanding of sample from the same birth. Again, discrepant positivity rates were
the abundance profiles of cannabinoids in meconium and umbilical observed between the two matrices. Of the 46 paired specimens, 32
cord. meconium specimens were positive for one or more analytes, compared
Tables 2 and 3 detail the results for meconium and umbilical cord, to 19 umbilical cord specimens positive for one or more analytes. This
respectively, for accuracy, within- and between-run imprecision, and discrepancy suggests 13 false-negative umbilical cord results (a 40%
extraction recoveries during validation. For meconium within-run false-positive rate). Upon qualitative assessment, the negative agree-
(n = 15) and between-run (n = 15) imprecision at low control (n = 30, ment between meconium and umbilical cord was 100%, while positive
7 ng/g) ranged from 1 to 3% and 4–6%, respectively, while, at high agreement was 59% between matrices. This anomaly suggests meco-
control (n = 30, 750 ng/g), within- and between-run imprecisions nium is a more sensitive matrix for the detection of in utero exposure to
ranged from 2 to 3% and 3–13%, respectively. For umbilical cord cannabis, likely due to a less efficient deposition of cannabinoids in the
within-run (n = 15) and between-run (n = 15) imprecision at low umbilical cord. Further studies will be needed to correlate these me-
control (n = 30, 0.25 ng/g) ranged from 2 to 3% and 8–20%, respec- tabolite profiles with the risk of adverse outcome. Raw patient data
tively, while, at high control (n = 30, 7.5 ng/g), within- and between- collected by the presented method are shown in the Table 4. Further
run imprecision ranged from 4 to 6% and 8–15%, respectively. The data analysis showed that, of the 13 uniquely positive meconium

5
T.L. Jensen et al. Clinical Mass Spectrometry xxx (xxxx) xxx–xxx

Table 2
Validation results for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD) in
meconium by LC-MS/MS.
Analyte* Average Accuracy (N = 36) Imprecision (%Coefficient of Variation) Recovery

Low (N=15) High (N=15) Method Solid Phase Extraction

7.5 ng/g 750 ng/g

Total Between Run Within Run Total Between Run Within Run

THC 99% 5% 5% 1% 3% 3% 2% 86% 73%

THCA 98% 5% 4% 2% 7% 7% 2% 102% 83%
11-OH-THC 98% 6% 6% 2% 4% 4% 2% 85% 74%
CBN 97% 4% 4% 3% 7% 6% 3% 83% 83%
CBD 97% 6% 5% 3% 13% 13% 2% 94% 27%

* Limit of quantitation and the limit of detection for all analytes in meconium is 5 ng/mL and 2.5 ng/mL, respectively.

Table 3
Validation results for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN) in umbilical cord by LC-
MS/MS.
Analyte* Average Accuracy (N = 36) Imprecision (%Coefficient of Variation) Recovery

Low (N = 15) High (N = 15) Method Solid Phase Extraction

0.25 ng/g 7.5 ng/g

Total Between Run Within Run Total Between Run Within Run

THC 97% 14% 14% 2% 9% 8% 4% 99% 74%

THCA 98% 8% 8% 3% 12% 10% 6% 97% 82%
11-OH-THC 94% 19% 19% 2% 10% 9% 4% 89% 58%
CBN 93% 20% 20% 3% 16% 15% 5% 94% 86%

* Limit of quantitation and the limit of detection for all analytes in meconium is 0.2 ng/mL and 0.1 ng/mL, respectively.

Fig. 2. Concentration distribution (log scale) of results positive for 11-hydroxy-THC (11-OH-THC), cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinal
(THC), and 11-nor-9-carboxy-THC (THCA) in meconium and umbilical cord paired samples from the same birth.

6
T.L. Jensen et al.
Fig. 3. Positivity rates of 11-hydroxy-THC (11-OH-THC), cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinal (THC), and 11-nor-9-carboxy-THC (THCA)
for paired meconium and umbilical cord samples from the same birth.
samples (correlating negative umbilical cord results), 7 samples only
contained THCA, while 3 contained all analytes except CBD. Only one
sample contained all five cannabinoid analytes. A wide range of con-
centrations were detected in these 13 meconium samples, which over-
lapped with the range of concentrations observed in samples, wherein
both meconium and umbilical cord results were positive. The wide
range of concentrations may reflect differences in metabolism, cannabis
use/exposure patterns, or physiological composition (e.g., chemical
composition) of the specimens themselves.
Limitations of this study include (i) poor extraction efficiency for
CBD in the umbilical cord samples, (ii) small numbers of paired spe-
cimens available, and (iii) a lack of information about the associated
pregnancies/births. It is unknown, for example, whether any of the
specimens tested in this study were associated with mothers known to
use drugs, the term of the pregnancies, nor the outcomes of the new-
borns. It will be important that additional samples are analyzed to
better understand the relationship between meconium and umbilical
cord in newborns exposed to cannabis in utero. Although several studies
have reported the agreement of drug screening results between cord
and meconium, among certain common drug classes [31,32], one
published study reported results similar to that reported here [33].
These results accentuate the importance of establishing clinically sig-
nificant cutoffs for these matrices. Relevant questions triggered by this
inconsistency include: What sample type should be submitted for
testing, and what is an appropriate cutoff to reduce false negatives
while reducing the possibility of false positive results that could occur
7
Clinical Mass Spectrometry xxx (xxxx) xxx–xxx
due to passive exposure? How would results change management de-
cisions for the newborn and/or the mother? Is one specimen more likely
to be positive based on the duration of the pregnancy or specific ma-
ternal drug use patterns? Would breast feeding be discouraged due to
continued risk of exposure to cannabinoid analytes?
4. Conclusion
A LC-MS/MS method was developed and validated to detect and
quantify THC, THCA, 11-OH-THC, CBN, and CBD in meconium and
THC, THCA, 11-OH-THC, and CBN in umbilical cord. Concentrations
and profiles of cannabinoid analytes varied considerably between um-
bilical cord and meconium samples. Concentrations of analytes in
umbilical cord were much lower than in meconium, suggesting the
need for different cutoffs to minimize false negative results. Analytes
other than THCA were commonly observed, but the relevance of the
profiles and concentrations of the individual analytes is not yet un-
derstood. These concentration and metabolite profiles could potentially
help indicate time of exposure and characterize the potential impacts of
in utero exposure to cannabis. In summary, the assay we describe here
could help clinicians and researchers answer these and other questions
by mapping clinical outcomes with analyte concentrations and abun-
dance profiles in meconium and umbilical cord collected from can-
nabis-exposed newborns.
T.L. Jensen et al. Clinical Mass Spectrometry xxx (xxxx) xxx–xxx

Table 4
Raw patient data for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD)
collected by LC-MS/MS.
Meconium Umbilical Cord

THCA (ng/g) THC (ng/g) 11-OH-THC (ng/g) CBN (ng/g) CBD (ng/g) THCA (ng/g) THC (ng/g) 11-OH-THC (ng/g) CBN (ng/g) CBD (ng/g)

Sample 1 16.2 4.6 0.0 6.0 0.0 0.5 0.0 0.0 0.0 NA
Sample 2 537.0 8.5 4.0 33.8 53.2 8.7 1.2 0.3 0.0 NA
Sample 3 122.0 3.0 0.0 7.3 0.0 4.1 0.2 0.2 0.2 NA
Sample 4 89.9 4.1 3.7 9.9 11.4 3.3 0.1 0.3 0.2 NA
Sample 5 79.4 3.9 3.3 7.6 0.0 2.7 0.3 0.2 0.1 NA
Sample 6 84.5 2.4 0.0 3.6 0.0 0.8 0.3 0.0 0.0 NA
Sample 7 157.0 2.3 0.0 7.3 0.0 2.0 0.0 0.0 0.2 NA
Sample 8 15.2 1.4 2.2 3.2 0.0 0.0 0.0 0.0 0.0 NA
Sample 9 14.7 1.4 0.0 4.7 0.0 0.0 0.0 0.0 0.0 NA
Sample 10 75.6 1.3 0.0 3.3 3.5 0.7 0.0 0.0 0.0 NA
Sample 11 14.6 0.0 0.0 2.0 0.0 0.1 0.0 0.0 0.0 NA
Sample 12 12.2 1.2 1.8 4.2 2.2 0.0 0.0 0.0 0.0 NA
Sample 13 36.1 1.6 3.6 4.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 14 3.1 0.0 0.0 1.6 0.0 0.0 0.0 0.0 0.0 NA
Sample 15 1.7 0.0 0.0 1.4 0.0 0.1 0.0 0.0 0.0 NA
Sample 16 137.0 2.8 4.3 46.5 11.6 0.6 0.0 0.0 0.0 NA
Sample 17 67.2 2.5 2.5 6.1 2.3 4.2 0.3 0.3 0.1 NA
Sample 18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 19 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 20 7.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 21 1.4 0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0 NA
Sample 22 8.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 23 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 24 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 25 22.4 0.3 3.2 6.9 0.0 0.0 0.0 0.0 0.0 NA
Sample 26 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 27 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 28 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 29 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 30 101.0 2.0 0.0 19.0 3.0 3.0 0.4 0.0 0.1 NA
Sample 31 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 32 6.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 33 41.0 0.0 0.0 4.0 0.0 0.4 0.0 0.0 0.0 NA
Sample 34 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 35 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 36 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 37 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 38 60.0 0.0 1.4 6.8 2.3 2.5 0.1 0.0 0.1 NA
Sample 39 17.0 0.0 0.0 0.3 0.0 0.7 0.0 0.0 0.0 NA
Sample 40 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 41 18.0 0.0 0.0 9.0 0.0 0.2 0.2 0.0 0.0 NA
Sample 42 20.0 2.0 0.0 9.0 0.0 1.9 0.2 0.0 0.1 NA
Sample 43 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 44 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA
Sample 45 370.0 2.0 3.0 20.0 5.0 3.9 0.1 0.0 0.0 NA
Sample 46 38.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA

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