Cocaine and Opiates Use in Pregnancy: Detection of Drugs in Neonatal Meconium and Urine

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In this study, the case of a newborn with symptoms of hyperexcitability was analyzed. After it was confirmed in the hospital that the mother had consumed drugs during pregnancy using an enzyme multiplied immunoassay technique, samples of the newborn's urine and meconium were sent to our laboratory to observe the evolution in the distribution of cocaine and opiates during the days following birth. For urine analysis, screening was done with an immunoassay technique, and the confirmation was done by gas chromatography-mass spectrometry (GC-MS) according to a published method. A GC-MS method for simultaneous analysis of cocaine, benzoylecgonine, codeine, morphine, and 6-acetylmorphine in meconium is described. GC-MS confirmation of urine and meconium results showed consumption of cocaine and codeine during pregnancy and also showed the levels of drugs gradually declined, totally disappearing by the third day.
In this study, the case of a newborn with symptoms of
hyperexcitability was analyzed. After it was confirmed in the
hospital that the mother had consumed drugs during pregnancy
using an enzyme multiplied immunoassay technique, samples of
the newborn’s urine and meconium were sent to our laboratory to
observe the evolution in the distribution of cocaine and opiates
during the days following birth. For urine analysis, screening was
done with an immunoassay technique, and the confirmation was
done by gas chromatography–mass spectrometry (GC–MS)
according to a published method. A GC–MS method for
simultaneous analysis of cocaine, benzoylecgonine, codeine,
morphine, and 6-acetylmorphine in meconium is described.
GC–MS confirmation of urine and meconium results showed
consumption of cocaine and codeine during pregnancy and also
showed the levels of drugs gradually declined, totally disappearing
by the third day.
Maternal cocaine use during pregnancy is a significant pub-
lic health problem. The number of pregnant women using co-
caine in society is increasing. This is a result of the increase of
cocaine use in the general population due to the easy of avail-
ability of inexpensive cocaine, its addictive quality, and soci-
ety’s permissive attitude towards substance abuse in general.
Prenatal cocaine use is a serious problem in our society. Co-
caine use in pregnancy is associated with obstetric complica-
tions to the mother, fetus, and newborn. These effects depend
on the dose, duration of drug ingestion and gestational age.
Maternal complications of cocaine use in pregnancy include
placental abruption (1–7), preterm labor (4,5,8), and death
(2,5). Complications to the fetus include spontaneous abor-
tion (4,5,8), congenital malformation (4,5,9), intrauterine
growth restriction (IUGR) (2,4,5,10,11), abnormal CTG tracing
(5), preterm delivery (4,5,11,12), and stillbirth (4,5). Effects
on the newborn can include meconium stained liquor (5,10)
and neonatal abstinence syndrome (NAS) (5,8), nevertheless
the presence of NAS after cocaine use is less clear than after
opiate use, with neurobehavioral abnormalities appearing 2–3
days after birth (10).
Consumption of other substances of abuse such as alcohol,
heroin or tobacco together with cocaine may act synergisti-
cally to worsen the adverse effects of cocaine on the newborn,
fetus and mother. Various studies have documented that co-
caine users during pregnancy are more likely than non-users
to use other drugs, such as tobacco, alcohol, and other sub-
stances of abuse (13).
It is frequently difficult to obtain trustworthy information
about the consumption of toxic substances during pregnancy.
For this reason, the suspicion is often clinical and must be
confirmed through biological matrices analysis.
A newborns urine analysis helps identify some drug-exposed
newborns, but not all. Detection of drugs in urine is only use-
ful if exposure occurred in the last 3–7 days prior to delivery
(11). Meconium is an optimal matrix for identifying in uterus
exposure as it is considered to be static once deposited in the
fetal intestine, thus being a preserved record of the ultimate
exposure by the fetus (14). In spite of increased sample prepa-
ration time relative to blood and urine, meconium’s the long
metabolite history, coupled with the ease and wide window of
collection make meconium the ideal matrix for determining
fetal drug use.
Acid and methanolic extractions developed for immunoas-
says can be used to extract cocaine, opiates, and metabolites
from meconium with varying degrees of success (15–17). How-
ever, other extraction procedures with much higher sensitivity
and lower limit of detection (LOD) have been development for
gas chromatography–mass spectrometry (GC–MS) (17) and
high-performance liquid chromatography (HPLC) (18).
Cocaine crosses the placental barrier by passive diffusion
(20). Review of the literature shows several reports dealing
with the analysis of meconium specimens for cocaine and/or
opiates using different immunoassays (15–17,20–24). Confir-
mation of the presence of these analytes was carried out by
techniques such as thin-layer chromatography (TLC) (24),
HPLC (18,22,25), or GC–MS (17,20,23,26).
Cocaine and Opiates Use in Pregnancy: Detection of
Drugs in Neonatal Meconium and Urine
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Journal of Analytical Toxicology, Vol. 33, September 2009
* Author to whom correspondence should be addressed. E-mail:
P. López*, A.M. Bermejo, M.J. Tabernero, P. Cabarcos, I. Álvarez, and P. Fernández
Institute of Legal Medicine, Forensic Toxicology Service, Faculty of Medicine, University of Santiago de Compostela,
San Francisco, s/n, 15782, Santiago de Compostela, Spain
The aim of this work was to validate a method that allows
the determination of cocaine, benzoylecgonine (BE), codeine,
morphine, and 6-acetylmorphine (6AM) in meconium samples
and to apply this method to meconium samples obtained the
first three days of the birth of a newborn with symptoms of
yperexcitability. With the same purpose, the newborn’s urine
will also be analyzed with a previously published method (27)
after a screening test.
Methanol, acetonitrile, hexane, and dicloromethane from
(Darmstadt, Germany) were gradient grade solvents.
Cocaine, BE, codeine, morphine, and 6AM were purchased
from Lipomed
(Mundolsheim, France), and their respective
deuterated-labeled analogues, cocaine-d
, BE-d
, codeine-d
, and 6AM-d
solutions, were purchased from Cer-
(Round Rock, TX). KH
, Na
, C
COOH, ethyl acetate, 2-propanol, NH
OH, trimethyl-
clorosilane (TMCS), and N,O-bis-trimethylsilyltrifluoroac-
etamide (BSTFA) were obtained from Merck.
Sample specimens
Urine (14, 34, 60, 80, and 103 h) and meconium (first, sec-
ond, and third day) of a newborn with symptoms of hyperex-
citability were sent to our laboratory the days following birth
proceeding of the Clinical Hospital of Santiago de Compostela
(Spain) to observe the evolution in the distribution of cocaine
and opiate. Drug consumption in pregnancy was tested previ-
ously in the hospital in mother’s urine by enzyme multiplied
immunoassay technnique (EMIT).
Screening tests were carried out on COBAS INTEGRA 400
(Roche Diagnostics, Basel, Switzerland) and GC–MS analysis
on a Hewlett-Packard
6890 series GC equipped with a mass
selective detector Hewlett-Packard
5973 Inert (Agilent Tech-
nologies, Spain).
Analysis of urine samples by COBAS and GC–MS
A previous screening of urine samples have done using the
immunoassay COBAS. Posterior the confirmation was done to
each sample applying a method previously published for our
laboratory (27) consisting in a liquid–liquid extraction with
Toxitubes A followed by GC–MS analysis.
Analysis of meconium samples by GC–MS
Meconium (0.5 g) with the I.S. (50 μL, 10 ng/µL) was trans-
ferred into 25-mL screw-capped glass tubes and 4 mL of
methanol were added. The samples were shaken for 10 min.
After centrifugation at 2000 rpm for 10 min, the organic layer
was dried under nitrogen. The dried extracts were then dis-
solved in 2 mL phosphate buffer (pH 6) and applied on a Bond
Elut solid-phase extraction (SPE) column (130 mg, 3 mL),
which had been preconditioned with 3 mL of methanol fol-
lowed by 3 mL of distilled water and 2 mL phosphate buffer
pH 6. The cartridge was then washed with 3 mL of distilled
water, 1 mL of sodic acetate buffer (pH 4), 3 mL of ethyl ac-
etate/hexane (1:1), and finally 3 mL of methanol. The analytes
ere eluted with 3 mL of dicloromethane/2-propanol (80:20)
+ 2% NH
OH, and the eluent was evaporated to dryness under
a gentle nitrogen stream.
A 40-μL volume of BSTFA/TMCS (99:1) was added to the
dried extracts. The tubes were tightly closed, vortex mixed (10
s), and heated at 100°C for 20 min. And finally, after cooling at
room temperature, 1 μL was injected into the GC.
GC–MS analysis
GC conditions. A 30-m × 250-µm HP-5 column (0.25-µm
film thickness) was operated with a temperature program of
90°C (1 min) to 190°C (1 min) at 30°C/min, then to 260°C (4
min) at 8°C/min and finally to 290°C (10 min). Helium was
used as a carrier gas at a flow rate of 1 mL/min. The injector
was maintained at 240°C and operated for 2 min in splitless
MS conditions. The mass selective detector was kept at
300°C, the ion source at 250°C, and the quadrupole at 100°C.
The mass analyzer operated by electron impact (70eV) in se-
lected ion monitoring (SIM) acquisition mode. Table I shows
retention times and selected ions for the analytes of interest
and their corresponding internal standards.
Validation of the method
Prior to application the method to meconium samples, it
has been tested using the following criteria.
Selectivity. Eight meconium samples from newborns of
mothers who stated they had not consumed cocaine and/or
opiates were extracted and analyzed for assessment of poten-
tial interferences from endogenous substances. The apparent
response at the retention times of the analytes under investi-
gation was compared to the response of analytes at the limit of
quantitation (LOQ).
Journal of Analytical Toxicology, Vol. 33, September 2009
Table I. Retention Times, Selected Ions of the Analytes,
and the Corresponding Internal Standards
Retention Time Selected Mass-to-Charge
Analyte (min) Ratios*
Cocaine 13.8 182, 198, 303
185, 201, 306
BE 14.9 240, 361
243, 364
Codeine 17.9 178, 234, 371
181, 237, 374
Morphine 18.9 236, 414, 429
239, 417, 432
6AM 20.5 287, 340, 399
290, 343, 402
* Quantifying ions are in bold.
Linearity. Solutions were prepared in the range of 20 to
1000 ng/g. For each standard curve, five different concentra-
ions were used, not including the blank matrix. The regres-
sion line was calculated by the method of least squares, and
linearity was expressed by the correlation coefficient (r
LOD and LOQ. LOD was defined as the concentration with a
signal-to-noise (S/N) ratio of at least 3 and acceptable precision
and accuracy. LOQ was the lowest standard with an S/N ratio of
at least 10 and acceptable precision and accuracy. Both param-
eters were determined empirically by analysis of a series of de-
creasing concentrations of the drug-fortified meconium in five
Precision and accuracy. Precision and accuracy were evalu-
ated over the linear dynamic range for three different concen-
trations: low limit of quantitation (LLOQ), intermediate con-
centration, and upper limit of quantitation (ULOQ). Intraday
precision and accuracy were assessed with five determinations
per concentration in one day, and interday precision and accu-
racy were evaluated by six determinations per concentration in
different days. Precision (coefficient of variation) was calculated
as (standard deviation/mean) × 100, and accuracy was calcu-
lated as (mean concentration quality control nominal con-
centration)/nominal concentration) × 100.
Recovery. Recovery or extraction efficiency (%) for each an-
alyte was determined at low and high concentration levels, and
calculated comparing the areas of peaks after extraction of sam-
ples with the internal standard and the drugs, with those peaks
obtained after extraction of samples only with the internal stan-
dard and subsequently spiked with the drugs at the same con-
Results and Discussion
Urine results
The analysis of mother’s urine done in the Clinical Hospital
of Santiago de Compostela (Spain) was positive for cocaine and
opiates using EMIT, but this result was not confirmed by any
other method.
Table II shows the results of newborn’s urine screening with
COBAS. These results have been confirmed using GC–MS.
This confirmation demonstrates that the positive result for
cocaine was due to the presence of BE and cocaine in minor
measure. In the case of the opiates, urine GC–MS confirmation
Journal of Analytical Toxicology, Vol. 33, September 2009
Figure 1. GC–MS chromatogram of a blank sample of meconium.
able II. Screening Results of Urine Samples
Hour Day Cocaine Opiates
14 1+ +
34 2+ +
60 3–
0 4–
103 5–
Table III. Results of the Calibration Method
Calibration Correlation LOD LLOQ
Compound Curve Coefficient (r
) (ng/g) (ng/g)
Cocaine y = 0.007 + 1.523x 0.996 5 20
BE y = 0.018 + 1.970x 0.996 10 20
Codeine y = 0.083 + 1.274x 0.997 5 20
Morphine y = 0.014 + 1.932x 0.996 10 20
6AM y = 0.010 + 1.155x 0.997 10 20
Table IV. Precision, Accuracy, and Recovery Obtained for
Analytes Investigated
Precision and Precision and
Accuracy Accuracy
Intraday Interday
Concentration Recovery
(n = 5) (n = 5)
Compound (ng/g) (%) CV MRE CV MRE
Cocaine 20 18.1 19.5 20.8 12.2
200 40.6
500 44.9 2.99 6.49 5.37 6.16
1000 5.47 9.26 1.13 1.38
BE 20 14.8 14.1 11.4 10.1
200 40.1
500 42.3 2.98 7.21 2.93 7.50
1000 5.67 7.36 0.60 1.56
Codeine 20 16.1 20.1 6.28 4.81
200 57.8
500 72.8 3.76 2.27 6.28 2.48
1000 5.73 9.36 1.58 0.46
Morphine 20 8.15 17.5 15.0 23.7
200 60.7
500 59.4 3.55 4.57 7.29 5.53
1000 6.08 11.2 1.47 1.38
6AM 20 17.3 16.7 20.4 4.36
200 40.3
500 44.3 3.22 4.14 5.45 4.03
1000 6.05 10.5 1.35 0.92
demonstrates that the positive result was to the presence of
codeine and morphine, but 6-AM was not detected.
Meconium results
Validation of the method
Selectivity. No additional peak due to endogenous substances
that could have interfered with the detection of compounds of
interest was observed in their retention times (Figure 1).
Linearity, LODs, and LOQs. Table III shows the results of
linearity and the LODs and LOQs. Linear calibration curves
were obtained with an average correlation coefficient higher
than 0.99 for the compounds of interest. LODs and LOQs ob-
tained with this method are lower than those obtained previ-
ously in a published paper for our laboratory (28); therefore,
this method of extraction is more sensitive.
Precision, accuracy, and recovery. Table IV provides the re-
sults obtained for recovery and intra- and interday precision
and accuracy. Precision and accuracy of analytes under inves-
tigation at reported concentrations satisfactorily met the in-
ternational established acceptance criteria (29). Higher results
of recovery using Bond Elut cartridges suggested that this car-
tridge is better for extracting the analytes of interest than other
cartridge used in our laboratory for extract the same analytes
(28). Mean recoveries were lower at a low analyte concentra-
tion than at a high analyte concentration.
Application of the method
A representative chromatogram of the sample of meconium
obtained the first day is presented in Figure 2. Figure 3 shows
the table with the results of the GC–MS analyses of the meco-
nium samples and the graphical representation of these re-
sults. Observing the graph, it is possible to see clearly that as
the days passed, the drug concentrations declined, and the
drugs were eliminated by the third day.
On the other hand, as we have seen for the urine, GC–MS re-
sults for the meconium demonstrate the existence of BE, co-
caine, codeine, and morphine in meconium samples, the prin-
cipal compounds being BE and codeine. The presence of
cocaine and BE in meconium was attributed to transfer across
the placenta from the mother (30,31), and the presence of
codeine in higher concentration than morphine in conjunc-
tion with the abstinence syndrome during the first hours after
birth (typical of heroin consumption during gestation) led us
to think that the mother’s consumption was mainly codeine.
The GC–MS method to analyze cocaine and opiates in meco-
nium reported in this paper was validated according to inter-
nationally acceptance criteria and provides a wide window for
the detection of fetal exposure to cocaine and opiates.
The analysis of meconium demonstrated the exposure to co-
caine and codeine during the last 20 weeks of gestation (32),
whereas urine was only able to prove that these drugs had been
consumed in the last hours prior to birth. Therefore, meco-
nium can be considered an alternative for the assessment of
exposure to drugs during pregnancy that cause alterations in
newborns presenting symptoms of hyperexcitability, as was the
case in this study.
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Manuscript received April 16, 2009;
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    • "COC and its metabolites have been detected in different maternal fetus and neonatal matrices including urine, blood, hair samples, neonatal gastric fluid, neonatal nails, meconium, amniotic fluid and umbilical cord blood [9,10]. Urine and meconium are the preferred specimens for analysis, but the detection of COC and metabolites in such samples does not explain the distribution of COC [11,12]. Placenta has also been considered an important alternative biological matrix [13] due to the distinct advantage of its non-invasive and relatively easy collection. "
    [Show abstract] [Hide abstract] ABSTRACT: We encountered evidence of myocardial infarction due to coronary thrombosis in an autopsy of an occasional marijuana smoker. These findings prompted us to perform a narrative review of the literature to determine when post-mortem toxicological tests may support a temporal relationship between marijuana smoking and cardiovascular disease. Toxicological examination showed the presence of Δ-9-tetrahydrocannabinol, its main metabolite and cannabinol in blood and urine. Quali-quantitative analysis revealed that Δ-9-tetrahydrocannabinol was taken within 2 h of the onset of cardiovascular symptoms, according to circumstantial data. Post-mortem toxicological results must take into account the degradation and post-mortem redistribution of analytes. However, for any inference about the specific cardiovascular triggering effect of Δ-9-tetrahydrocannabinol intake, we maintain that cannabinoid analysis in blood samples must be considered an essential requirement to estimate the time of last intake and avoid incomplete documentation. The literature, combined with the present case report, highlights an association between marijuana use and negative cardiovascular events, although few authors have supported their conclusions with toxicological results. Thus, additional research is needed. Copyright © 2015 John Wiley & Sons, Ltd.
    Article · Nov 2015
    • "These positive results were later confirmed with the meconium samples. The method used to obtain this confirmation is the same used in a study published by some of the authors of the present study [31]. None of the newborns included in the study showed remarkable neurological disorders, and the Ballard's test was concordant to their morphological age. "
    [Show abstract] [Hide abstract] ABSTRACT: Alcohol consumption during pregnancy, even when moderate, implies a risk of impaired neurodevelopment, physical impairments and malformations. Its early identification is essential for establishing preventive measures to diminish disabilities among newborns. To determine the frequency of consumption of substance use in pregnant women, we have used the techniques of gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry to detect drugs and markers of chronic consumption of alcohol in meconium. We performed a prospective study during a period of 10 months among 110 infants in our hospital, assessing anthropometry, neuromuscular development and determination of toxic substances in urine and meconium. Furthermore, meconium analysis identified fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg). We also conducted a survey regarding the obstetric history, toxic habits, and employment status of the mothers. According to early detection markers analyzed in meconium (FAEE >1000 ng/g and/or Etg >50 ng/g meconium), 34.65% of pregnant women consumed alcohol during pregnancy, and 17% were positive for both markers. Within the positive cases, 50% of those exceeding a FAEE's value of 5000 ng/g in meconium had low birth-weight children. Only 5/110 mothers (4.5%) admitted to occasional alcohol consumption during pregnancy. Nobody admitted to frequent intake. The cocaine test was positive in three cases; two of them were positive for alcohol as well. As expected, many screening devices do not accurately capture use during pregnancy and supplemental methods such as meconium analysis of biomarkers of chronic alcohol consumption may be warranted.
    Full-text · Article · May 2014
    • "Sometimes the information provided by the mothers in these cases is not reliable. Gas chromatography mass spectrometry (GC/MS) has been in use as a non-invasive method for detection of prenatal drug exposure by screening meconium for several years1234. The aim of this study is to demonstrate that the introduction of alcohol markers into the drug screening program is essential, especially in cases of known drug abuse by the mother or observed APSs in the newborn. "
    [Show abstract] [Hide abstract] ABSTRACT: Alcohol consumption during pregnancy is a widespread problem and can cause severe fetal damage. As the diagnosis of fetal alcohol syndrome is difficult, the implementation of a reliable marker for alcohol consumption during pregnancy into meconium drug screening programs would be invaluable. A previously published gas chromatography mass spectrometry method for the detection of fatty acid ethyl esters (FAEEs) as alcohol markers in meconium was optimized and newly validated for a sample size of 50 mg. This method was applied to 122 cases from a drug-using population. The meconium samples were also tested for common drugs of abuse. In 73 % of the cases, one or more drugs were found. Twenty percent of the samples tested positive for FAEEs at levels indicating significant alcohol exposure. Consequently, alcohol was found to be the third most frequently abused substance within the study group. This re-validated method provides an increase in testing sensitivity, is reliable and easily applicable as part of a drug screening program. It can be used as a non-invasive tool to detect high alcohol consumption in the last trimester of pregnancy. The introduction of FAEEs testing in meconium screening was found to be of particular use in a drug-using population.
    Full-text · Article · Aug 2013
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