Umbilical Cord Monitoring of In Utero Drug Exposure to ...

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Journal of Analytical Toxicology, Vol. 34, October 2010

Umbilical Cord Monitoring of In Utero Drug Exposure to Buprenorphine and Correlation with Maternal Dose and Neonatal Outcomes Marta Concheiro1,2, Hendreé E. Jones3, Rolley E. Johnson3,4, Robin Choo5, Diaa M. Shakleya1, and Marilyn A. Huestis1,* 1Chemistry

and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland; 2Departamento Anatomía Patológica y Ciencias Forenses, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 3Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland; 4Reckitt Benckiser Pharmaceuticals, Inc., Richmond, Virginia; and 5Department of Natural Sciences, University of Pittsburgh, Titusville, Pennsylvania

Introduction

Abstract Buprenorphine is under investigation in the U.S. as pharmacotherapy for opioid-dependent pregnant women. Buprenorphine and metabolites were quantified in umbilical cord specimens from women receiving daily buprenorphine doses. Correlations between maternal buprenorphine dose, buprenorphine and metabolite umbilical cord concentrations, and neonatal outcomes were investigated, as well as the ability to identify heroin and cocaine relapse during pregnancy. Umbilical cord concentrations were compared to those of matched umbilical cord plasma and meconium. Buprenorphine metabolites were detected in all cords, but buprenorphine itself was absent. Concentration ranges were 1.2–5.1 ng/g norbuprenorphine, 1.7–4.2 ng/g buprenorphine-glucuronide, and 8.3–23 ng/g norbuprenorphine-glucuronide. Cord concentrations were similar to those in plasma, and lower (16–210-fold), although statistically correlated, than those in meconium. Significant positive correlations were observed for buprenorphine-glucuronide concentrations in umbilical cord and mean maternal BUP daily dose throughout pregnancy and third trimester, but buprenorphine biomarker concentrations did not predict neonatal outcomes. Opiate concentrations were lower (200-fold) in umbilical cord than in meconium, and when cocaine was present in meconium, it was not identified in cord. Umbilical cord can serve as an alternative matrix for identifying prenatal drug-exposure, but is much less sensitive than meconium. Buprenorphine provided a controlled drug administration model for evaluating drug disposition in the maternal-fetal dyad.

* Author to correspondence should be addressed: Marilyn A. Huestis, PhD, Chief, Chemistry and Drug Metabolism, IRP, National Institute on Drug Abuse, National Institutes of Health, Biomedical Research Center (BRC), 251 Bayview Blvd, Suite 200, Room 05A721, Baltimore, MD 21224. Email: [email protected].

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Buprenorphine (BUP) was approved in the U.S. in 2002 for office-based treatment of opioid dependence (1). As popularity of BUP pharmacotherapy increases, high-risk populations such as pregnant women are beginning to receive this treatment (2– 5), emphasizing the need for information on the drug’s disposition within the maternal-fetal dyad. In adults, BUP undergoes phase I metabolism (N-dealkylation) by cytochrome P450 3A4 to norbuprenorphine (NBUP) primarily in the liver (6). BUP and NBUP phase II metabolism to buprenorphine-glucuronide (BUP-Gluc) and norbuprenorphine-glucuronide (NBUP-Gluc), respectively, occurs through the action of uridine diphosphate glucuronosyltransferase (UGT) 1A1 (7) and 2B7 (8). Recently, Rouguieg et al. (9) showed that BUP glucuronidation involves predominantly UGT1A1 and 2B7 isoforms, and for NBUP glucuronidation UGT1A1 and UGT1A3. Toxicological analysis of biological specimens from the mother (e.g., urine, hair, blood, oral fluid, and sweat) and from the newborn (e.g., meconium, hair, and urine) and other alternative matrices available at birth (e.g., umbilical cord blood and tissue, placenta, and amniotic fluid) may identify drug-exposed neonates. Lozano et al. (10) and Gray and Huestis (11) reviewed the advantages and limitations of biological specimens for monitoring in utero drug exposure. In newborns, hair (12) and meconium (13) identify drug consumption, primarily in the third trimester of gestation; however, hair cannot be collected in many cases because specimen amount is low, the mother refuses to cut the child’s hair, or the process is unacceptable for cultural reasons. Meconium also has limitations: there may be a delay in collection for 1–3 days after birth, and specimen amount may be limited or unavailable due to passage prior to birth. However, umbilical cord is considered a waste product and is available at the time of birth in

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adequate amounts for analysis. At term, the umbilical cord is about 50 cm long and 2 cm wide and contains two umbilical arteries and one umbilical vein surrounded by Wharton’s jelly that protects and insulates the umbilical cord vessels (14). Montgomery et al. (15) reported good qualitative correlations between umbilical cord and meconium screening for cocaine, methamphetamine, opiates, and cannabinoids, although most of the specimens were negative for drugs of interest. Moore et al. (16) and Winecker et al. (17) reported cocaine and metabolite concentrations in umbilical cord. No data on BUP parent drug or metabolites in this matrix have been published. BUP controlled drug administration during gestation provides a unique opportunity to investigate BUP and metabolite disposition in the maternal and neonatal dyad. In this research, we compared the sensitivity and specificity of umbilical cord to other biological matrices, umbilical cord plasma and meconium, for identifying prenatal BUP exposure. Unique to this study was the prospective collection of thrice weekly urine specimens throughout pregnancy to capture relapse to heroin and cocaine, and the relative ability of umbilical cord to identify illicit drug use. Additionally, controlled BUP dosing permitted evaluations of correlations between umbilical cord concentrations, maternal BUP dose, and neonatal outcomes, including the neonatal abstinence syndrome (NAS).

Experimental Participants

The Center for Addiction and Pregnancy (CAP) at the Johns Hopkins Bayview Medical Center (Baltimore, MD) recruited participants for a double-dummy, flexible, randomized, stratified, parallel-group controlled study comparing methadone and BUP for the treatment of opioid dependence during pregnancy. Preliminary results of this study and additional methodological details were previously published (18). Maternal BUP dosing

All women received oral methadone for 3–5 days after initially qualifying for the protocol during more extensive screening procedures. Women were randomized and transitioned to study medication with immediate-release morphine in divided daily doses. BUP-maintained women received sublingual BUP HCl (2 mg each, maximum 24 mg/day; ReckittBenckiser Healthcare, Hull, England) and placebo tablets totaling 12 tablets/day, along with 40 mL liquid placebo. All doses were observed in an outpatient clinic. Double-blind medication increases or decreases were determined by physicians based on an a priori protocol that included elements of medication compliance, participant request, urine toxicology results, and participant self-report of opioid withdrawal symptoms or craving (18). Urine drug testing

Urine specimens were assayed three times a week for cocaine, opiates, cannabinoids, and benzodiazepines by im-

munoassay (Dade Behring Diagnostics, Deerfield, IL) with cutoffs of 300 ng/mL for cocaine and opiates, 200 ng/mL for benzodiazepines, and 100 ng/mL for cannabinoids (13). Neonatal outcome measures

Apgar scores at 1 and 5 min, estimated gestational age (EGA) at delivery, birth weight (g), head circumference (cm), length (cm), hospital stay duration from birth until discharge to the research unit (days), and NAS were obtained from medical records. EGA was established by sonogram. NAS was systematically assessed for 10 days using a 19-item modified Finnegan Scale (13,18,19). Time to NAS onset (h) was defined as the time from birth until the first score > 4. The score of 4 was selected as the cutoff on the basis of clinical experience and preliminary blinded-condition comparison data from drug-exposed and nondrug-exposed neonates. Peak NAS score was defined as the highest score obtained and time-to-peak (h) was calculated from time of birth to peak NAS score. NAS duration (h) was defined as the time from first score > 4 to time after which all scores were < 5. Treatment for NAS with morphine solution (0.02 mg/drop) was initiated when a neonate received 2 consecutives scores of 9 or greater. Treatment was reduced by one drop per day if every score for 24 h was ≤ 8, and neonates were discharged following 24 h of no medication and NAS scores ≤ 8 (18). Umbilical cord analysis

Umbilical cord specimens were collected at delivery and frozen at –20°C until analysis. The analyzed segment was rinsed with ultra-pure water to remove maternal blood and biofluids. BUP, NBUP, BUP-Gluc, and NBUP-Gluc were quantified in umbilical cord by a previously published liquid chromatography–tandem mass spectrometry (LC–MS–MS) procedure (20). Briefly, 2 g umbilical cord was homogenized in 8 mL 0.1% perchloric acid in water in the presence of deuterated BUP and NBUP. After centrifugation, the liquid supernatant was subjected to solid-phase extraction (SPE) with Strata-XC cartridges (Phenomenex, Torrence, CA). Reverse-phase separation was achieved with a Synergi Polar column (75 mm × 2.1 mm, 4 µm, Phenomenex) within 20 min under gradient conditions. The assay was linear from 1 to 50 ng/g. Intra- and interassay and total imprecision was < 14.5%, and analytical recovery was between 94.1 and 112.3%. Extraction efficiencies were > 66.3%, and process efficiencies were > 73.4%. Matrix effect ranged from 3.7% to 27.4% (CV < 21.8%, n = 8). The method was specific (no endogenous or exogenous interferences) and sensitive (limit of quantification, LOQ, 1 ng/g). Cocaine, benzoylecgonine (BE), morphine, codeine, 6-acetylmorphine (6AM), methadone, and 2-ethylidene-1,5-dimethyl3,3-diphenylpyrrolidine (EDDP) were quantified in umbilical cord by LC–MS–MS (21). Briefly, 5 mL 0.1% perchloric acid was added to 1 g umbilical cord and homogenized in the presence of the deuterated internal standard solution. After centrifugation, the supernatant was subjected to SPE with mixed mode-reversed phase cartridges Strata-XC (Phenomenex). Chromatographic separation was performed on a Synergi Polar RP column (75 mm × 2.1 mm, 4 μm, Phenomenex) with a gradient of (A) 0.1% formic acid and (B) acetonitrile. Calibration

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curves were linear between 10 and 1000 ng/g for methadone and 2.5 and 500 ng/g for the rest of the analytes. Intra- and interassay imprecision were < 12.7%, and analytical recovery ranged from 85.9 to 112.7%. Extraction efficiency was > 59.2%, and process efficiency > 48.6%. Matrix effect ranged from 4.9 to 39.5% (CV < 22.6%, n = 10). Deuterated analogues were included as internal standards to compensate for these effects. The method was specific and sensitive (LOQ 10 ng/g for methadone and 2.5 ng/g for the rest of the compounds). BUP and metabolite disposition in umbilical cord

BUP and metabolite concentrations were examined in two different locations in each of the five umbilical cords from BUP-maintained pregnant women. These locations were the placental extreme [1] and the fetal extreme [2]. Preliminary statistical analyses

Preliminary statistical analyses were performed with SPSS version 13.0. Pearson correlations evaluated relationships between umbilical cord, meconium and umbilical cord plasma concentrations, and between umbilical cord concentrations and maternal BUP dose, and neonatal outcomes. Statistical probability P ≤ 0.05 was considered statistically significant. The Kolmogorov-Smirnov test was employed to evaluate normal distribution of data. Mean BUP, NBUP, BUPGluc, and NBUP-Gluc concentrations of the two umbilical cord locations are presented.

Table I. Demographics and Self-Reported Drug Use History Over the Past 30 Days Prior to Admission and Buprenorphine (BUP) Doses for Four Opioid-Dependent Pregnant Women Participant

A

B

C

D

Median

Age (years)

30

32

32

28

31

AA*

AA

AA

AA

–

Years education

10

10

12

8

10

Employment

No

No

No

No

–

Race

EGA†

25

20

26

18

22.5

Cocaine use

Yes

Yes

Yes

Yes

–

Opioid use‡

1

2

2

1

1.5

Alcohol use (days)

0

5

0

0

0

Smoking (days)

0

30

30

0

15

No. cigarettes/day

0

3

20

0

1.5

Days in study at delivery

92

118

111

151

114.5

BUP dose at delivery (mg/day)

18

24

18

18

18

Mean daily BUP dose (mg/day)

15.9

18.3

15.9

12.3

15.9

Mean 3rd trimester daily BUP dose (mg/day)

16.9

21.8

17.1

13.3

17.0

Mean last month daily BUP dose (mg/day)

18

24

18

18

18

Cumulative BUP dose (mg)

1464

2160

1760

1850

1805

Cumulative 3rd trimester BUP dose (mg)

1084

1396

1330

1132

1231

558

766

522

522

540

Cumulative last month BUP dose (mg)

* African-American. † Estimated gestational age at admission (weeks). ‡ Opioid use in the past 30 days at admission. 1 ≤ 3/day; 2 ≥ 4/day.

Table II. Thrice-Weekly Urine Tests and Umbilical Cord Results from Four Buprenorphine-Maintained Pregnant Women Cocaine Total % Pos Days Cocaine in % 3rd from last umbilical pos* trimester† pos‡ cord§

Total % Pos Days Opiates in % 3rd from last umbilical pos trimester pos cord#

A Baby-1 Baby-2

32.5

40.7

8

neg neg

75

74.1

7

pos pos

B

35.3

11.1

43

neg

45.1

14.8

55

neg

C

2.1

0

82

neg

8.5

12.5

30

neg

D

23.8

27

54

neg

17.5

16.2

56

neg

Results Participants

One-thousand four-hundred ninety women were screened, and 57 qualified for further evaluation. Most women were ineligible because they were outside the gestational age allowed for treatment in this study, or because they did not arrive for CAP admission (18). Finally, 30 women were randomized, 15 to methadone and 15 to BUP, of which 9 BUP-maintained women completed the study through delivery. Five umbilical cord tissue specimens were available,

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Opiates

Participant

* % Positive urine tests from enrollment to birth. † % Positive urine tests in the 3rd trimester. ‡ Days from last positive urine specimen to birth. § Cocaine testing included benzoylecgonine and cocaine. # Opiate testing included 6AM, morphine, and codeine.


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including a set of monozygotic twins (A Baby-1, A Baby-2), and 3 single births (B, C, and D). Demographics, self-reported drug use history, and BUP dosing are included in Table I. No participant provided a urine specimen that screened positive for cannabis throughout pregnancy. Participant B’s urine speci-

mens were positive for benzodiazepines 22.2% of the time, with her last positive result 43 days before delivery. Cocaine and opiates thrice-weekly urine screening results throughout pregnancy are included in Table II. Neonatal outcomes

Table III. Neonatal Outcomes A Baby-1 Baby-2 Sex

M

B

C

D

Median

M

F

F

M

–

2740

2730

2890

3525

3355

2890

Head circumference (cm)

33

34

31

35

38

34

Length (cm)

49

48

48

53

52

49

Gestational age at delivery (weeks)

Birth weight (g)

Analytical results

37

37

37

39

40

37

1-min Apgar score

8

9

8

8

7

8

5-min Apgar score

9

9

9

9

9

9

Length of hospital stay (days)

5

5

8

4

4

5

Peak NAS score

6

8

9

3

5

6

Time-to-peak NAS score (h)

24

122

167

78

205

122

Time to NAS onset (h)

34

19

29

–

205

Duration NAS (h)

110

149

208

0

6.5

129.5

% NAS scores > 4

14

24

29

0

3.6

19

31.5

Table IV. Neonatal Outcomes Umbilical Cord Tissue (ng/g) Participant

Analyte

Location 1 Location 2

Umbilical Cord Plasma (ng/mL)

Meconium (ng/g)

A Baby-1

BUP NBUP BUP-Gluc NBUP-Gluc

ND* 2.2 2.8 20.6

ND 2.7 2.4 18.6

0.7 0.8 4.4 22.8

59.3 468.4 36.7 79.7

A Baby-2


ND 2.1 3.2 23.0

ND 2.1 2.3 17.3

0.6 0.9 3.8 22.0

59.6 373.4 110.0 234.2

B


ND 4.9 4.2 8.3

ND 4.8 4.2 10.4

ND 1.1 1.6 34.4

240.5 1228.6 56.3 651.6

C


ND 1.2 3.5 13.4

ND 1.2 3.7 17.2

0.3 1.3 13.9 35.8

23.9 730.6 8.2 0

D


ND 5.1 1.7 22.1

ND 5.1 2.2 22.4

0.3 2.9 2.2 39.4

40.4 505.4 51.2 450.2

* Not detected.

Three male and two female normal birth weight infants were delivered fullterm. None required treatment for NAS. Neonatal outcome measures are summarized in Table III.

BUP metabolites were detected in all umbilical cord tissue specimens, but BUP was absent. The predominant analyte was NBUP-Gluc with concentrations ranging from 8.3 to 23.0 ng/g (mean ± SD = 17.3 ± 5.1 ng/g, median = 18.0 ng/g); NBUP concentrations ranged from 1.2 to 5.1 ng/g (3.1 ± 1.6 ng/g, 2.5 ng/g), and BUPGluc from 1.7 to 4.2 ng/g (3.0 ± 0.9 ng/g, 3.0 ng/g). Biomarker concentration variations were less than 23.1% CV in the two extreme sections of each of the five umbilical cords (Table IV). In the monozygotic twins (same placenta, two different umbilical cords), concentrations were similar; NBUP concentrations were 2.5 and 2.1 ng/g, BUP-Gluc 2.6 and 2.8 ng/g, NBUP-Gluc 19.6 and 20.2 ng/g, and no BUP was detected. Meconium (13) and umbilical cord plasma (mixture of vein and arterial blood) (22) concentrations were reported previously. Table IV summarizes umbilical cord and matched umbilical cord plasma and meconium BUP and metabolite concentrations. Cocaine, opiates, and metabolites also were analyzed in one location of each umbilical cord specimen. Only morphine was detected in umbilical cord from participant A twins (Baby-1, 5.9 ng/g; Baby-2, 4.9 ng/g). Opiates tested negative in participants B, C, and D umbilical cord specimens, and cocaine was negative in all of them. Meconium from participant A twins tested positive for opiates and cocaine, and from participants B, C, and D tested negative for these analytes. Morphine meconium concentrations were 1163 ng/g in Baby-1 and 1185 ng/g in Baby-2. With regard to cocaine and metabolites, the predominant meconium analyte was m-OH-benzoylecgonine (m-OH-BE) with concentrations of 124 ng/g (Baby-1), and 122 ng/g (Baby-2). Cocaine was 11 ng/g in Baby-2, but was not

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detected in Baby-1, and BE was 5 ng/g in Baby-1 and 10 ng/g in Baby-2 meconium. Correlation between BUP metabolite umbilical cord concentrations and maternal BUP dose

Pearson correlation coefficients were calculated for BUP dose and BUP metabolite concentrations, total metabolites, and NBUP/NBUP-Gluc ratio in umbilical cord. Dosing parameters included days in the study at delivery, BUP dose at delivery, mean daily dose (from enrollment to delivery), mean third trimester daily dose, mean last month daily dose, cumulative dose (from enrollment to delivery), cumulative third trimester dose, and cumulative last month dose. BUP-Gluc concentration in umbilical cord showed a statistically significant correlation with mean daily dose (P = 0.050, r = 0.879, n = 5), and mean third trimester daily dose (P = 0.031, r = 0.912, n = 5) (Figure 1). Correlation between BUP metabolite umbilical cord concentrations and neonatal outcomes

Pearson correlation coefficients were calculated for BUP metabolite concentrations, total metabolites, and NBUP/NBUPGluc ratio in umbilical cord, and Apgar scores at 1 and 5 min, length of hospital stay (days), peak NAS score, time-to-peak NAS score, time to NAS onset, duration of NAS, and % NAS scores > 4. Because weight, length, head circumference, and EGA at delivery can be altered by twin status, correlations be-

tween BUP biomarkers’ concentrations in umbilical cord and weight, length, head circumference, and EGA at delivery could not been performed because of the low n (n = 3, without twin data). No statistically significant correlations were found between BUP or metabolite concentrations in umbilical cord and neonatal outcomes. Correlation between BUP metabolite concentrations in different matrices

Pearson correlation coefficients were determined for BUP metabolite concentrations in umbilical cord, umbilical cord plasma, and meconium. There were no significant correlations between umbilical cord and umbilical cord plasma concentrations. NBUP-Gluc concentrations in meconium correlated positively with NBUP in umbilical cord (P = 0.033, r = 0.908, n = 5), and a statistically significant negative correlation was found between NBUP concentrations in meconium and NBUP-Gluc in umbilical cord (P = 0.014, r = –0.949, n = 5) (Figure 1).

Discussion BUP metabolites were identified in the five umbilical cord specimens collected from BUP-maintained pregnant women. The predominant analyte was NBUP-Gluc, followed by BUP-

Figure 1. Statistically significant correlations between umbilical cord norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) concentrations, maternal dosing, and meconium concentrations.

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Gluc and NBUP. BUP was not detected in any specimen. Mean and median umbilical cord concentrations were similar for these pregnant opioid-dependent women receiving similar mean third trimester daily BUP doses. Concentrations were in the low nanogram-per-milligram range when mean third trimester daily BUP doses were 13.3–21.8 mg. The distribution and concentrations of analytes in umbilical cord tissue and umbilical cord plasma were of similar magnitude, although BUP < 1 ng/mL was measureable in some plasma specimens. BUP was detected in umbilical cord plasma but not in cord tissue, which was most likely due to the lower LOQ in plasma (0.1 ng/mL) than in tissue (1 ng/g). NBUP concentrations tended to be higher in cord tissue than plasma, suggesting accumulation in the tissue, but glucuronide concentrations were higher in plasma. Most likely BUP analytes passively diffuse into umbilical cord from plasma, as analyte distribution was the same. Glucuronide diffusion is expected to be lower due to the molecules’ higher polarity and molecular weight > 500Da, perhaps producing higher concentrations in umbilical cord plasma than tissue. BUP biotransformation in umbilical cord tissue is not suggested based on these data. Furthermore, marginal CYP3A enzymatic activity that could oxidize BUP to NBUP was shown in umbilical vein endothelial cells (23), and no data about glucuronidation capability in this tissue are available. Although umbilical cord tissue and plasma displayed a similar BUP metabolite distribution, there were no significant correlations for analytes in these matrices. Umbilical cord plasma could better reflect current drug concentrations, whereas cord tissue may suggest drug accumulation over time. BUP-Gluc concentrations in umbilical cord showed a statistically significant correlation with mean daily dose throughout pregnancy and during the third trimester but, surprisingly, no correlation with mean daily dose during the last month. These apparent contradictory results could be due to the low number of participants (four mothers, five babies) and the small range of maternal BUP dose (three received 18 mg/day BUP in the last month of the study, and the other one 24 mg/day) yielding a highly homogeneous group (Table I). In the literature, only two articles describe correlations between BUP and metabolite concentrations in meconium and neonatal outcomes (13,24), with the present work first describing such a correlation for umbilical cord. Marquet et al. (24) reported that meconium from infants who experienced withdrawal (n = 9) tended to have higher BUP concentrations than those who did not (n = 6), but no relationship was noted for NBUP. In a study including 10 newborns from BUP-maintained women (4 of whom were included in the present study), Kacinko et al. (13) observed a statistically significant positive correlation for %NAS scores > 4 and total BUP, total BUP/total NBUP ratio and BUP/NBUP ratio, and negative correlations between %free NBUP in meconium and time-to-peak NAS, between BUP/NBUP ratio in meconium and time to NAS onset, and between BUP concentration in meconium and head circumference. Only five umbilical cord specimens were available in this study, and no statistically significant correlations were found between BUP metabolite concentrations in umbilical cord and neonatal outcomes.

Comparing umbilical cord and meconium, there were differences in analyte distribution and concentrations. The predominant metabolite in meconium was NBUP at concentrations > 373.4 ng/g, whereas in umbilical cord, the main analyte was NBUP-Gluc at concentrations between 9.4 and 23.0 ng/g. NBUP-Gluc was not detected in meconium from baby C, whereas in umbilical cord, this metabolite was always present. BUP was detected in all meconium specimens (8.2–110 ng/g), but was entirely absent in umbilical cord. The physicochemical properties of these matrices (less lipophilic umbilical cord, more lipophilic meconium), metabolism in the fetus (BUP to NBUP and glucuronide formation), and BUP enterohepatic circulation may influence the differential accumulation in these two matrices. NBUP concentrations in umbilical cord and NBUP-Gluc in meconium correlated positively, and NBUP-Gluc in umbilical cord and NBUP in meconium correlated negatively. Perhaps NBUP concentrations increasing in umbilical cord suggest greater delivery of NBUP to the fetus that could be metabolized to NBUP-Gluc and accumulated in meconium. No data are available on fetal BUP and NBUP glucuronidation, but morphine glucuronides are formed at high rates by UGT2B7 in fetal baboon liver, rivaling adult activity (25). In a similar manner, greater NBUP-Gluc concentrations in umbilical cord could signal delivery of less NBUP to the fetus, and hence, less accumulation in meconium. It is possible that the more polar NBUP-Gluc fails to cross membranes as well as the more lipophilic NBUP, and does not enter the fetus’ systemic circulation as readily. Cocaine and opiates were detected in both meconium specimens from subject A twins (13), whereas the matched umbilical cord specimens were only positive for morphine. Morphine concentrations were much higher in meconium than umbilical cord. Montgomery et al. (15) reported good correlations between umbilical cord and meconium results for cocaine (99.2%), methamphetamine (96.6%), opiates (94.9%), and cannabinoids (90.7%), but unfortunately, no data were reported on metabolites analyzed or measured concentrations. In the case of opiates and cocaine, this high correlation reflected mostly negative specimens; out of 118 matched specimens, 105 were negative to opiates and 114 to cocaine in both matrices (15). Moore et al. (16) reported BE at 1200 ng/g in umbilical cord from one cocaine user, and Winecker et al. (17) analyzed 28 umbilical cords from self-reported cocaine users. Thirteen of 28 specimens tested positive for cocaine; the predominant metabolite was BE (0–1237 ng/g), followed by ecgonine methyl ester (0–52 ng/g). No data on drug consumption amount or timing were available. In the present study, urine specimens were tested thrice per week to characterize the timing and relative amounts of illicit cocaine and opiates use, and these results were compared to those in matched umbilical cord specimens. High drug consumption (74.1% opiate-positive urine specimens in the third trimester for participant A) was an important factor influencing incorporation of drugs in umbilical cord (Table II). Additional research with a larger cohort is needed to establish cutoff concentrations and windows of detection for drugs in umbilical cord. In the present study, umbilical cord and meconium specimens were stored at –20°C prior to analysis. There are no published data on long-term sta-

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bility of buprenorphine, cocaine, opiates, and metabolites in umbilical cord or meconium; however, published short-term stability data for 16–24 h at 22°C, 72 h at 4°C, and after three freeze/thaw cycles showed losses of < 20% for all analytes in umbilical cord and meconium, except 6AM in meconium (20,21,26,27). Similar concentrations were found for morphine (5.9 vs. 4.9 ng/g) and for BUP metabolites (Table IV) in umbilical cord specimens from the monozygotic twins. Matched meconium specimens had similar concentrations of morphine, BUP, and NBUP, whereas glucuronide concentrations were lower in Baby-1 than in Baby-2. Boskovic et al. (28) analyzed hair or meconium from 1 monozygotic and 11 dizygotic twins who were exposed in utero to cocaine or cannabinoids. The monozygotic pair of twins had almost identical levels of cocaine, whereas the six dizygotic pairs had large disparities in either cocaine or cannabinoid concentrations. These authors suggested that the placenta had a major role in modulating the amounts of drug reaching the fetus. The different BUP and NBUP glucuronide concentrations in meconium but similar concentrations in umbilical cord plasma and tissue from monozygotic twins, suggest that other factors, such as fetal metabolism, also may affect the disposition of drug and metabolites in the fetus. In this preliminary study, correlations were found between umbilical cord BUP-Gluc concentrations and maternal BUP daily dose throughout pregnancy and in the third trimester, but not between umbilical cord BUP biomarkers’ concentrations and neonatal clinical outcomes. NBUP-Gluc was the main metabolite detected in umbilical cord, albeit in low concentrations. Umbilical cord and umbilical cord plasma BUP metabolite concentrations were similar, but of much lower magnitude than those found in meconium. Umbilical cord tissue could be useful for the identification of recent drug consumption in cases where umbilical cord plasma is not available, as analysis of plasma is simpler than tissue. Although umbilical cord tissue is an interesting matrix available in adequate amounts immediately at delivery, its usefulness to detect in utero illicit drug exposure and to predict neonatal outcomes appears to be limited as compared to other available matrices.

Acknowledgment This research was supported by the Intramural Research Program of the National Institute on Drug Abuse (NIDA), National Institutes of Health, and NIDA grant RO1 DA12220.

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