Maternal Methadone Dose, Placental Methadone Concentrations, and Neonatal Outcomes

Maternal Methadone Dose, Placental MethadoneConcentrations, and Neonatal Outcomes

Ana de Castro,1,2 Hendree E. Jones,3 Rolley E. Johnson,3,4 Teresa R. Gray,1 Diaa M. Shakleya,1 andMarilyn A. Huestis1*

BACKGROUND: Few investigations have used placentaas an alternative matrix to detect in utero drug ex-posure, despite its availability at the time of birthand the large amount of sample. Methadone-maintained opioid-dependent pregnant womenprovide a unique opportunity to examine the placen-tal disposition of methadone and metabolite [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)], to ex-plore their correlations with maternal methadone doseand neonatal outcomes, and to test the ability to detectin utero exposure to illicit drugs.

METHODS: We calculated the correlations of placentalmethadone and EDDP concentrations and their correla-tions with maternal methadone doses and neonatal out-comes. Cocaine- and opiate-positive placenta results werecompared with the results for meconium samples and forurine samples collected throughout gestation.

RESULTS: Positive correlations were found between pla-cental methadone and EDDP concentrations (r � 0.685),and between methadone concentration and methadonedose at delivery (r � 0.542), mean daily dose (r � 0.554),mean third-trimester dose (r � 0.591), and cumulativedaily dose (r � 0.639). The EDDP/methadone concentra-tion ratio was negatively correlated with cumulative dailydose (r � �0.541) and positively correlated with peakneonatal abstinence syndrome (NAS) score (r � 0.513).Placental EDDP concentration was negatively correlatedwith newborn head circumference (r ��0.579). Cocaineand opiate use was detected in far fewer placenta samplesthan in thrice-weekly urine and meconium samples, a re-sult suggesting a short detection window for placenta.

CONCLUSIONS: Quantitative methadone and EDDPmeasurement may predict NAS severity. The placentareflects in utero drug exposure for a shorter time than

meconium but may be useful when meconium is un-available or if documentation of recent exposure isneeded.

© 2010 American Association for Clinical Chemistry

The placenta exchanges nutrients, oxygen, and wasteproducts; synthesizes hormones, peptides, and ste-roids; and protects the developing fetus by limiting xe-nobiotic transfer from the mother. A drug’s physico-chemical properties determine its transfer across theplacenta (1 ), with passive diffusion being the maintransfer mechanism for lipophilic compounds. Placen-tal metabolism may modify the amounts and types oftransferred xenobiotics (2 ), but placental enzymeshave low activities compared with those in maternal orfetal liver (1 ). For ethical and safety reasons, few in vivostudies have assessed the transfer and disposition ofdrugs in human placenta (2 ). Although placental dis-tribution is known from animal models for somedrugs, the results are difficult to extrapolate to humans(1, 2 ).

Methadone, the primary opioid-dependencepharmacotherapy used during pregnancy, reduces fetalheroin exposure, obstetric complications, and neonatalmorbidity and mortality (3 ). To date, there are nodata on the concentrations of methadone and itsprimary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP),5 in placenta and whethertheir concentrations correlate with the methadonedose. Methadone-assisted pharmacotherapy for opioiddependence provides a useful model for investigatingdrug disposition after in utero drug exposure and therelationship between placental concentrations andneonatal outcomes. The child exposed to a drug inutero may experience neonatal abstinence syndrome

1 Chemistry and Drug Metabolism, National Institute on Drug Abuse, NIH,Baltimore, MD; 2 Institute of Legal Medicine, University of Santiago de Com-postela, Santiago de Compostela, Spain; 3 Department of Psychiatry and Be-havioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD;4 Reckitt Benckiser Pharmaceuticals, Richmond, VA.

* Address correspondence to this author at: Chemistry and Drug Metabolism, IRP,National Institute on Drug Abuse, NIH, 251 Bayview Blvd., Baltimore, MD21224. Fax 443-740-2823; e-mail [email protected].

An abstract on this topic was presented at the International Association of

Forensic Toxicology annual meeting in Bonn, Germany (August 29 to September2, 2010).Received August 13, 2010; accepted November 16, 2010.Previously published online at DOI: 10.1373/clinchem.2010.1548645 Nonstandard abbreviations: EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine;

NAS, neonatal abstinence syndrome; JHBMC, Johns Hopkins Bayview MedicalCenter; BE, benzoylecgonine; mOHBE, m-hydroxybenzoylecgonine; CYP, cyto-chrome P450.

Clinical Chemistry 57:3449–458 (2011)

Drug Monitoring and Toxicology

449

(NAS), due to the abrupt end of exposure at birth (4 ).The identification of neonates at risk for more severeNAS could improve infant care.

Our primary objectives were to assess the placentaldisposition of methadone and EDDP in opioid-dependent pregnant women and to explore the corre-lations with maternal methadone dose, placental meth-adone concentration, and neonatal outcome. To betterunderstand the advantages and limitations of drugmonitoring in placental tissue, we also compared theresults in this unique cohort for methadone, opioid,cocaine, and their main metabolites in placenta withthose in matched neonatal meconium samples andwith thrice-weekly urine drug test results obtainedthroughout gestation.

Methods

HUMAN PARTICIPANTS AND NEONATAL OUTCOMES

Participants were recruited from 2 clinical trials (A and B)conducted by the Center for Addiction and Pregnancy,Johns Hopkins Bayview Medical Center (JHBMC) in Bal-timore, Maryland. The JHBMC and National Institute onDrug Abuse Institutional Review Boards approved thestudies. Participants provided written informed consent.Study A examined the prevalence of mood and anxietydisorders in opioid-dependent pregnant women and theimpact of these disorders on methadone treatment success(5), whereas study B compared methadone- andbuprenorphine-assisted therapy during pregnancy (6 ).Participants initially received 30 mg methadone daily(Mallinckrodt Chemical); the dosage was increased to60 mg/day over 4 days. Further dosing adjustmentswere made as clinically indicated.

Estimated gestational age at birth, birth weight,length, head circumference, and 1- and 5-min Apgarscores were extracted from hospital records. NAS wasassessed for the first 10 days of life with a 19-item mod-ified Finnegan scale (6 ). Time to NAS onset was thetime in hours from birth until the first score �4, timeto NAS peak was the time in hours from birth to peakscore, and NAS duration was the time in hours fromthe first score �4 until all scores were �5.

PLACENTA COLLECTION AND ANALYSIS

Placenta samples were stored at �20 °C until analy-sis for methadone, EDDP, morphine, codeine,6-acetylmorphine, cocaine, and benzoylecgonine (BE) bya validated liquid chromatography–ion trap mass spec-trometry method (7). We initially analyzed 7 placentasamples at 4 different locations (1, 4, 6, and 10 cm fromthe umbilical cord) to assess whether analyte distributionwas homogeneous throughout the matrix and thereforewhether analysis of a single intermediate locationwould accurately represent placental disposition.

MECONIUM COLLECTION AND ANALYSIS

We collected meconium samples from the neonates ofmothers providing placental samples until the appear-ance of milk stool. The samples were frozen until anal-ysis, as for placenta samples, and were analyzed forthe same analytes plus m-hydroxybenzoylecgonine(mOHBE), either in house (8, 9 ) or (for opiates, co-caine, and benzodiazepines in some early samples) bythe United States Drug Testing Laboratories (10 ).

URINE COLLECTION AND ANALYSIS

To document relapse to opioid and cocaine use, wecollected observed thrice-weekly urine samples fromthe mothers who provided placental samples, from thetime of enrollment through birth. Urine samples wereanalyzed immediately on site with the AbuscreenONTRAK rapid assays for drugs of abuse (Roche Diag-nostic Systems), with cutoffs of 300 �g/L for opiates(morphine) and cocaine (BE) (11 ). Positive opiate andcocaine results were confirmed by gas chromatography–mass spectrometry. Methadone dosing was observed toensure compliance.

STATISTICAL ANALYSIS

For statistical analyses, we used SPSS for Windows(version 17.0; SPSS). The Kolmogorov–Smirnov testevaluated the fit of data to a normal distribution, andPearson correlation analysis assessed relationshipsamong placental methadone and EDDP concentra-tions, EDDP/methadone concentration ratios, mater-nal methadone dose, and neonatal outcomes (exceptfor 5-min Apgar scores, for which Spearman correla-tion analysis was applied because of data nonnormal-ity). Pearson correlation analysis was also used to eval-uate the relationship between methadone and EDDPconcentrations, and EDDP/methadone concentrationratios in placenta and meconium. A statistical proba-bility (P) value �0.05 was considered significant.

Results

HUMAN PARTICIPANTS AND METHADONE DOSING

Placenta samples, demographic data, and methadone-dosing data were available for 19 pregnant opioid-dependent women receiving methadone-assisted ther-apy. Placenta samples from 3 additional participantswere collected and used to test methadone and EDDPdistribution across the placenta; however, these sam-ples were not included in further data analysis becausematernal methadone-dosing and neonatal-outcomedata were not available for these participants. Maternaldemographic and methadone-dosing data are shownin Table 1. All pregnancies produced single births, for atotal of 19 infants. Most women entered the study dur-ing the second trimester between weeks 10 and 29. The

450 Clinical Chemistry 57:3 (2011)

Tabl

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81.2

1296

480

40

332

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200

3070

66.3

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1333

561

60

433

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104

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44.7

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4695

4230

532

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Methadone and EDDP Disposition in Human Placenta

Clinical Chemistry 57:3 (2011) 451

mean (SD) estimated gestational age on admission was20.8 (5.5) weeks.

DISTRIBUTION OF METHADONE AND EDDP ACROSS

THE PLACENTA

We measured methadone and EDDP concentrations in4 placental locations across 7 test placenta samples.Methadone and EDDP were distributed homoge-neously across the placenta (see File 1 in the Data Sup-plement that accompanies the online version of thisarticle at http://www.clinchem.org/content/vol57/issue3). Concentrations varied �15% for both analytesin 5 placentas (participants 1, 10, 20, 21, and 22); theyvaried �30% in the other 2 placentas (participants 3and 9), owing to the greater variation in the concentra-tion of 1 of the analytes at 1 location in 1 sample. Illicitdrugs also were evenly distributed in placenta. Cocaineand/or BE were found in all 4 placental locations in 2participants, whereas morphine and codeine wereidentified in all sites in 1 placenta. Because of the ho-

mogeneous distributions in these 7 test placentas, 1intermediate location was analyzed for all the otherplacenta samples.

PLACENTAL METHADONE AND EDDP DISPOSITION AND

CORRELATION WITH METHADONE DOSE

Methadone and EDDP concentrations were measur-able in all placenta samples after chronic daily metha-done dosing (Table 2). The concentrations of metha-done were 10-fold to 20-fold greater than for EDDP,with a mean EDDP/methadone concentration ratioof 0.11 (0.06). Statistically significant positive corre-lations were found for methadone and EDDP con-centrations in placenta (r � 0.685; P � 0.001). Theplacental methadone concentration was signifi-cantly correlated with the cumulative daily metha-done dose (r � 0.639; P � 0.003). Similar significantcorrelations also were found between the placentalmethadone concentration and the methadone doseat delivery (r � 0.542; P � 0.017), the mean daily

Table 2. Methadone (MTD) and EDDP concentrations and EDDP/MTD concentration ratios in 19 placenta and 17matched meconium samples from 19 opioid-dependent pregnant women receiving methadone-assisted therapy.

Participantno.

Placenta Meconium

MTD, ng/g EDDP, ng/g EDDP/MTD MTD, ng/g EDDP, ng/g EDDP/MTD

1 1198.6 95.9 0.09 5235 80 503 15.4

2 1909.0 142.2 0.07 5047 38 675 7.7

3 1985.9 103.9 0.05 9864 33 352 3.4

4 1092.4 105.6 0.10 6373 44 369 7.0

5 2647.0 517.4 0.20 2184 53 142 24.3

6 2627.0 302.0 0.11 —a — —

7 1784.2 78.4 0.04 2548 22 900 9.0

8 405.2 71.7 0.18 85 6375 75.0

9 1441.6 86.9 0.06 2492 13 188 5.3

10 1372.1 105.5 0.07 — — —

11 1724.3 213.5 0.12 18 840 62 600 3.3

12 549.2 96.1 0.18 5481 34 753 6.3

13 308.0 35.9 0.12 2555 10 546 4.1

14 1511.6 120.7 0.08 5785 32 826 5.7

15 1538.2 312.3 0.20 11 334 53 800 4.7

16 1553.9 147.8 0.10 4997 28 628 5.7

17 1048.6 90.4 0.09 5009 64 800 12.9

18 2643.5 189.0 0.07 21 902 57 200 2.6

19 537.3 78.6 0.15 7281 57 600 7.9

Mean 1467.2 152.3 0.11 6883.1 40 897.5 11.8

SD 720.4 115.4 0.05 5804.6 20 922.8 17.2

Median 1511.6 105.5 0.10 5235.0 38 675.0 6.3

a Dashes indicate no testing because matched meconium samples were unavailable.


http://www.clinchem.org/content/vol57/issue3

http://www.clinchem.org/content/vol57/issue3

dose (r � 0.554; P � 0.014), and the mean doseduring the third trimester (r � 0.591; P � 0.008).The mean EDDP/methadone concentration ratiowas also significantly correlated (but negatively)with the cumulative daily methadone dose (r ��0.541; P � 0.017). Although the correlations weresignificant, the correlation coefficients themselvesranged from 0.542 to 0.685; the clinical significanceof these weak correlations is not yet known.

COMPARISON OF METHADONE AND EDDP CONCENTRATIONS IN

MATCHED PLACENTA AND MECONIUM SAMPLES

Methadone was the primary analyte in placenta; EDDPconcentrations were much lower (Table 2). In meco-nium, however, EDDP concentrations were 100- to800-fold greater than in placenta. Methadone concen-trations in meconium exceeded those in placenta in 15of 17 matched samples, and EDDP concentrations al-ways were much higher in meconium than in placenta.Moreover, the EDDP/methadone concentration ratioswere reversed, with ratios �0.2 occurring in placentaand much higher ratios (�2.6) in meconium. Neithermethadone and EDDP concentrations nor EDDP/methadone concentration ratios in placentas were cor-related with those in matched meconium samples.

NEONATAL OUTCOMES

Neonatal outcomes for the 19 infants born to the19 opioid-dependent pregnant women receivingmethadone-assisted pharmacotherapy are shown inTable 3. The majority of infants (52.6%) were deliveredat full term (�37 weeks), with a mean estimated gesta-tional age at birth of 36.3 (3.4) weeks (range, 30.4 – 41.0weeks). Three of the 4 infants considered to be of lowbirth weight (�2500 g) had head circumferences lessthan typical (�32 cm) for healthy newborns, and allinfants with lengths shorter than typical (�45 cm)were preterm. All infants experienced NAS, althoughonly 6 required treatment. Peak NAS scores rangedfrom 6 –19, the time to NAS onset varied from 3.5 to127.8 h, and the percentage of NAS scores �4 rangedfrom 6.3% to 90.1%. Apgar scores at 1 min and 5 minwere within reference values (�7) in all cases.

PLACENTAL METHADONE AND EDDP CONCENTRATIONS AND

CORRELATION WITH NEONATAL OUTCOMES

Placenta EDDP concentration was significantly nega-tively correlated with newborn head circumference(r � �0.579; P � 0.009), whereas the EDDP/metha-done concentration ratio was significantly positivelycorrelated with peak NAS score (r � 0.513; P � 0.030).No other significant correlations were detected. Theclinical significance of these correlations is not yetknown.

OPIATES AND COCAINE IN MATCHED PLACENTA, MECONIUM,

AND THRICE-WEEKLY URINE SAMPLES

Cocaine and opiate results for 19 placenta samples werecompared with those for 15 matched meconium sam-ples and 18 sets of thrice-weekly urine results (Table 4).The small amount of meconium collected for 2 samplespermitted only methadone and EDDP measurements.

In utero cocaine exposure was confirmed in 11meconium samples by the presence of cocaine, BE,and/or mOHBE, whereas cocaine exposure was identi-fied in only 1 placenta sample. Placenta and meconiumBE concentrations were higher than cocaine concen-trations. The range of concentrations for the 11cocaine-positive meconium samples was 12.2–254.0ng/g for mOHBE, 2.4 –969.0 ng/g for BE, and4.4 –233.0 ng/g for cocaine. In the single cocaine-positive placenta sample, the concentrations of BE andcocaine were 458.9 ng/g and 7.3 ng/g, respectively;mOHBE was not included in the placental analyticalpanel for cocaine and metabolites.

In utero opiate exposure was identified in 8 meco-nium samples; however, only a single placenta samplewas opiate positive (Table 4). In meconium, the rangeof morphine concentrations was 10 –2555 ng/g; only 1sample had detectable codeine (16.3 ng/g). Morphineconcentrations exceeded codeine concentrations by�10-fold, and no 6-acetylmorphine was present. Mor-phine and codeine concentrations in the only positiveplacenta sample were 39.8 ng/g and 2.9 ng/g, respec-tively, also with no 6-acetylmorphine detectable. Inter-estingly, no opiates were detected in the meconium ofthis participant’s newborn.

Thrice-weekly urine data for opiates and cocaineduring the second and/or third trimesters of pregnancywere available for 18 of the participants who providedplacenta samples. These data confirmed opiate con-sumption during the second and/or third trimesters ofpregnancy for 17 of the participants and confirmed co-caine consumption for 9 participants. Of particular in-terest is the number of days since the last positive urinetest result. These data provide information on the tim-ing of last drug use, which suggests the window fordetecting drugs in placenta and meconium. Clearly,many placenta samples were negative for opiates andcocaine while the results of urine and meconium testswere indicating drug relapse. We should note that thefact that mOHBE was not included in the placentaanalysis could have contributed to the fewer number ofpositive placenta samples. The urine data confirmed allof the meconium results, except for the detection ofmOHBE in meconium from participants 12 and 15.The lack of positive urine data could have been due toconsumption of low cocaine amounts or missed urinesamples. Interestingly, for the one participant with aplacenta sample positive for opiates, no opiates were



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detected in the matched meconium sample. Unfortu-nately, urine data from the third trimester were un-available for this case.

The remaining 18 placenta samples did not con-tain measurable opiate or cocaine biomarkers, despiteurine screening results indicating drug use by 8 moth-ers during the third trimester, within 2 weeks beforedelivery in some participants (the urine results indi-cated opiate consumption 3 days before delivery by par-ticipant 10, and opiate and cocaine consumption 15and 13 days, respectively, before delivery by participant17). These data suggest that placenta has a muchshorter window of drug detection than meconium.

Discussion

Methadone, EDDP, opiates, and cocaine were homo-geneously distributed in the placenta, suggesting that

only a single sampling is necessary. High interindi-vidual variation in placental methadone and EDDPconcentrations was observed. This finding is not sur-prising, given the differences in length of study enroll-ment, gestational age at admission, and cumulativedose throughout pregnancy. Additional factors, in-cluding the participants’ age and race, also could havecontributed to variations in analyte concentrations.The placental concentrations of methadone (308 –2647ng/g) and EDDP (35.9 –517.4 ng/g) were higher thancommonly observed in maternal plasma (methadone,45–757.8 �g/L; EDDP, 15– 46.5 �g/L) after ranges ofmethadone doses (20 –90 mg) similar to those of ourstudy (30 –110 mg) (12–15 ), results that suggest ana-lyte retention. Placental methadone retention was pre-viously noted in vitro by Nekhayeva et al., who assessedmethadone transfer across artificially perfused pla-centa samples (16 ). In addition, methadone and EDDP

Table 4. Morphine (MOR), codeine (COD), cocaine (COC), BE, and mOHBE concentrations in matched placentaand meconium samples.

Participantno.

Urinea

Meconium, ng/g Placenta, ng/gOpiates COC

2ndTRI, %

3rdTRI, %

Time fromlast

positive,days

2ndTRI, %

3rdTRI, %

Time fromlast

positive,days MOR COD COC BE mOHBE MOR COD COC BE

1 17.6 6.7 34 5.9 6.7 34 37.0 Nb N N 119.0 N N N N

2 3.0 0.0 176 3.0 0.0 176 N N N N 104.0 N N N N

3 —c — — — — — N N N N 68.0 N N N N

4 25.0 0.0 104 25.0 0.0 104 N N N N N N N N N

5 3.0 0.0 99 0.0 0.0 AN — — — — — N N N N

6 0.0 14.7 18 0.0 0.0 AN — — — — — N N N N

7 2.8 0.0 128 0.0 0.0 AN 2281.0 N N N N N N N N

8 0.0 0.0 AN 0.0 0.0 AN — — — — — N N N N

9 94.1 — — 88.2 — — N N 233.0 969.0 254.0 39.8 2.9 7.3 458.9

10 16.7 63.6 3 0.0 0.0 AN — — — — — N N N N

11 55.5 0.0 118 0.0 4.4 62 N N N N 12.2 N N N N

12 STT 10.7 61 STT 0.0 AN 10.0 N N N 26.0 N N N N

13 100.0 0.0 42 33.3 0.0 46 10.2 N N N 24.3 N N N N

14 18.2 0.0 120 0.0 0.0 AN N N N N N N N N N

15 33.3 0.0 75 0.0 0.0 AN 14.0 N N N 12.5 N N N N

16 STT 6.7 27 STT 63.3 25 43.0 N N 4.3 200.3 N N N N

17 25.0 7.1 15 31.2 25.0 13 2555.0 16.3 4.4 62.0 57.3 N N N N

18 18.7 0.0 100 0.0 0.0 AN N N N N N N N N N

19 STT 11.5 31 STT 7.7 31 65.9 N N 2.4 155.1 N N N N

a Percentages of thrice-weekly matched urine test results positive for opiates and cocaine in the second (2nd TRI) and third (3rd TRI) trimesters, and the time fromthe last positive urine sample until birth (Time from last positive) are included for comparison of the detection of illicit drug use in the different matrices.

b N, negative results (i.e., below the limit of quantification); AN, always negative; STT, participant started in third trimester.c Dashes indicate data not available.



concentrations in cord blood (arterial–venous blood:28.7–35.7 �g/L and 8 –9 �g/L for methadone andEDDP, respectively, with 40 mg methadone daily) (12 )and infant plasma (2.2– 8.1 �g/L and 1.9 �g/L formethadone and EDDP, respectively, with 50 –105 mgmethadone daily) (17, 18 ) were lower than in maternalplasma and placenta, indicating a role for the placentain protecting the fetus from methadone exposure. Fur-thermore, Nanovskaya et al. studied the effect of theefflux transporter P-glycoprotein in the transplacentaldistribution of methadone and demonstrated the abil-ity of this transporter to excrete methadone from theplacental tissue back into the maternal circulation(19 ), further suggesting its involvement in protectingthe fetus from xenobiotics.

Our comparison of methadone and EDDP con-centrations in placenta and matched meconium sam-ples revealed differences between these matrices in thedistribution of these analytes. Methadone and EDDPconcentrations were much lower in placenta than inmeconium: median meconium/placenta methadoneratio, 4.8 (range, 0.2–13.6); median meconium/pla-centa EDDP ratio, 293.2 (range, 88.9 – 801.3). Further-more, the 2 matrices differed with respect to the relativeabundance of methadone and EDDP [mean (SD)EDDP/methadone concentration ratio, 0.11 (0.06) inplacenta and 11.8 (17.2) in meconium]. These differ-ences may be explained by fetal methadone metabo-lism and subsequent EDDP accumulation in meco-nium. Several cytochrome P450 (CYP) isoenzymeswere identified in fetal liver, with CYP3A7 accountingfor up to 50% of the CYP content in the fetal liver (20 ).CYP3A7 production decreases during gestation, withmetabolism shifting within days of birth to CYP3A4(20, 21 ), which is, in conjunction with CYP2B6, theprincipal enzyme for methadone biotransformation(22 ).

In this study, we also evaluated the possible corre-lations between methadone and EDDP concentrationsin placenta, maternal methadone doses, and neonataloutcomes. Placental methadone and EDDP concentra-tions were significantly correlated, with methadoneconcentrations far exceeding EDDP concentrations, asis found in maternal plasma (plasma EDDP/metha-done concentration ratios, 0.06 – 0.18) (12, 15 ). Pla-cental EDDP appears to derive primarily from EDDPin the maternal plasma, because perfused placental tis-sue converts �1% of methadone to EDDP (16 ). To ourknowledge, we present the first data for placental meth-adone and EDDP concentrations and their correlationwith maternal methadone dose. Collection of plasmasamples in the last weeks of pregnancy, if medicallyacceptable, might have been useful to evaluate the cor-relations between maternal methadone dose andplasma methadone and EDDP concentrations, and be-

tween plasma and placental concentrations. Plasmawas collected only when medically necessary, however.Thus, an assessment of this correlation was not possiblefor our cohort. Nevertheless, in support of our findingsof significant correlations between placental metha-done concentrations and maternal methadone dose,other authors have also observed a strong correlationbetween the methadone concentration in umbilicalcord blood and the maternal methadone dose (r �0.768; P � 0.0001) (23 ). The relationships betweenmethadone dose and concentration reported for ma-ternal plasma have been inconsistent, however(13, 23–25 ).

Placenta EDDP concentration was negatively cor-related with newborn head circumference (r � �0.579;P � 0.009). The mechanism responsible for this corre-lation is not clear, because exposure to the active meth-adone component is thought to be the agent responsi-ble for this outcome. A possible explanation is thathigher EDDP concentrations simply reflect highermethadone concentrations, followed by metabolism tothe inactive metabolite. Furthermore, the placenta me-tabolizes methadone poorly, as in vitro studies havesuggested (16 ); thus, the majority of EDDP in placentaappears to derive from maternal methadone metabo-lism. The clinical significance of this finding is un-known and requires further investigation. We alsonoted a positive correlation between the placentalEDDP/methadone concentration ratio and the peakNAS score. Our results suggest that the placenta con-centration might predict NAS intensity and therefore aneed for neonatal treatment. The sample size in ourstudy was small, however, and the correlation values(r � 0.513) indicate that the clinical relevance of thisassociation is uncertain and requires further research.In support of our findings, previous studies haveshown NAS of higher intensity to be associated withlower neonatal plasma methadone concentrations(23 ), a greater rate of decline in neonatal methadoneconcentration (13 ), or both (26 ). Mack et al. (25 ),however, found no correlation between the methadoneconcentration in the plasma of newborns and the in-tensity of withdrawal symptoms.

Meconium is considered the matrix of choice todetect in utero drug exposure, and assays of meconiumsamples primarily detect drug consumption in thethird trimester (27 ). Placenta may be an effective alter-native, however, if meconium is unavailable because offetal distress and in utero discharge (28 ). The mainadvantage of placenta is its easy and noninvasive col-lection at birth. Only a few reports on the concentra-tions of illicit drugs in the placenta, however, are avail-able to date (29 –33 ), and the window of drug detectionhas not been defined.


We also analyzed placenta samples for cocaine andopiates. The availability of cocaine and opiate resultsfor matched meconium and urine samples collectedthroughout pregnancy allowed us to compare the use-fulness of these matrices for detecting drug exposure inutero. The results for urine samples confirmed mater-nal opiate and cocaine consumption in the second andthird trimesters in 17 and 9 participants, respectively. Alower urinary BE cutoff concentration of 150 �g/L,proposed for the 2008 revision of the MandatoryGuidelines for Federal Workplace Drug Testing Pro-grams (34 ) may have increased cocaine detection.Among our participants, only 1 placenta sample con-tained opiates and cocaine; unfortunately, the unavail-ability of complete urine data for this participant pre-cluded an evaluation of the placenta’s window of drugdetection. Our results show that the placental windowfor detecting cocaine and opiates is quite short, giventhat several of the women used these drugs within a fewweeks of delivery and that this drug use was not re-flected in the placental samples. Moreover, the placen-tal window appears to be much shorter than for meco-nium, for which positive results were found, even whenthe last day of cocaine and/or opiate intake was duringthe second trimester. Although the inclusion of othermetabolites (e.g., mOHBE) in the analytical panel forplacenta may increase the detection of drug exposure,our data show that meconium and regular urine collec-tion provide more information than placental analysis.Meconium clearly is the preferred matrix for detectingin utero drug exposure. Placenta collection for drug

testing would be justifiable only if meconium were un-available. These preliminary data do suggest, however,that placenta methadone and EDDP concentrationscould be valuable if meconium is unavailable and ifrecent drug exposure is forensically important.

Author Contributions: All authors confirmed they have contributed tothe intellectual content of this paper and have met the following 3 re-quirements: (a) significant contributions to the conception and design,acquisition of data, or analysis and interpretation of data; (b) draftingor revising the article for intellectual content; and (c) final approval ofthe published article.

Authors’ Disclosures or Potential Conflicts of Interest: Uponmanuscript submission, all authors completed the Disclosures of Poten-tial Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: R.E. Johnson, Reckitt BenckiserPharmaceuticals.Consultant or Advisory Role: None declared.Stock Ownership: R.E. Johnson, Reckitt Benckiser Pharmaceuticals(immediate family member).Honoraria: None declared.Research Funding: Intramural Research Program, and ExtramuralGrants DA12220 and DA12403 from the National Institute on DrugAbuse, NIH.Expert Testimony: None declared.Other Remuneration: A. de Castro, Direccion Xeral de Investiga-cion, Desenvolvemento e Innovacion, Consellerıa de Innovacion eIndustria (AA-053), Galicia, Spain.

Role of Sponsor: The funding organizations played no role in thedesign of study, choice of enrolled patients, review and interpretationof data, or preparation or approval of manuscript.

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Maternal Methadone Dose, Placental Methadone Concentrations, and Neonatal Outcomes

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