Detection of Cocaine and its Metabolites in Amniotic Fluid and ...

Journal of Analytical Toxicology, Vol. 21, March/April 1997

Detection of Cocaine and its Metabolites in Amniotic Fluid and Umbilical Cord Tissue Ruth E. Winecker 1, Bruce A. Goldberger 1,*, lan Tebbett 2, Marylou Behnke3, Fonda Davis Eyler3, Michael Conlon 3, Kathy Wobie 3, Janet Karlix 2, and Roger L. Bertholf4 1University of Florida College of Medicine, 2310 SW 13th Street, Gainesville, Florida 32608; 2University of Florida College of Pharmacy, Gainesville, Florida 32610; 3University of Florida College of Medicine, Gainesville, Florida 32610; and 4University Medical Center, 655 W. 8th Street, Jacksonville, Florida 32209

Abstract ] The increased use of cocaine by women of child-bearing age has left many health care scientists searching for improved methods of detecting prenatal cocaine exposure. To that end, a study of the determination of cocaine and its metabolites in amniotic fluid and umbilical cord tissue was undertaken. Amniotic fluid (n -- 32) and umbilical cord tissue (n -- 70) specimens were collected from pregnant subjects admitted to labor and delivery at Shands Hospital at the University of Florida (Gainesville, FL). Subjects were interviewed regarding drug use during each trimester. Subjects reporting cocaine use were designated as target subjects, and those denying use were control subjects. The specimens were subjected to solid-phase extraction and analyzed for cocaine and its metabolites by gas chromatography-mass spectrometry. Cocaine analytes (predominantly benzoylecgonine) were detected in 28.1 and 18.5 % of the amniotic fluid and umbilical cord tissue specimens, respectively. Other cocaine analytes frequently detected included ecgonine methyl ester and m-hydroxybenzoylecgonine in amniotic fluid specimens and ecgonine methyl ester, norcocaine, and m-hydroxybenzoylecgonine in umbilical cord tissue specimens. This study has shown that cocaine and its metabolites are readily detected in specimens of maternal and fetal origin.

Introduction Cocaine use by pregnant women has become a public health tragedy, particularly in urban areas where surveys have shown an increase in the number of newborns testing positive for cocaine and other drugs (1-3). Cocaine adversely affects the fetus by causing vasoconstriction and uterine contractility, presumably by a catecholamine-mediatedprocess. These effects can result in severe health consequences for the mother, including premature rupture of the amniotic membranes, abruptio placenta, decreased placental blood flow,spontaneous *Author to whomcorrespondenceshould be addressed.

abortion, preterm delivery,and a preeclampsia-like syndrome, which is characterized by hypertension, proteinuria, and edema (1,2,4,5). Cocaine-exposedinfants are susceptible to myocardial calcification (6), renal vascular abnormalities (7,8), intrauterine growth retardation (2,9), congenital abnormalities (2), stroke (2), and increased risk of sudden infant death syndrome (2,10,11). Detection of drug use during pregnancy by immunoassay analysis is usually performed on urine specimens obtained from the mother or the infant. Although urine may be the most convenient specimen, reliable detection of cocaine metabolites (predominantly benzoylecgonine) in urine is limited because of the relatively short half-livesand the variability in elimination of cocaine and its metabolites. It has been suggested that results of meconium analysis may be a better indicator of cocaine exposure based on the theory that drugs present in maternal-fetal circulation accumulate in meconium from the time it is first produced, which is about week 18 of gestation (12). However,some investigators have been unable to confirm this hypothesis. In these studies, cocaine was not detected in meconium specimens unless drug use occurred within three weeks of delivery (13). Alternative specimens such as amniotic fluid and umbilical cord tissue have not been studied as thoroughly as meconium and maternal and neonatal urine. Amniotic fluid is present throughout gestation, and it has been suggested that large amounts of cocaine and its metabolites may accumulate, exposing the fetus through oral and transdermal routes (14-16). Umbilical cord tissue is also present during gestation, but there have been little data published regarding this tissue (16). The comprehensive analysis of cocaine and its metabolites in these samples, and perhaps other maternal and fetal fluids and tissues, may help further identify and characterize neonatal drug exposure. This report describes the analysis of cocaine and its metabolites in amniotic fluid and umbilical cord tissue from subjects participating in a study designed to determine the sensitivity of several maternal and neonatal matrices for the detection of prenatal cocaine exposure.

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Materials and Methods Subjects Subjects (n = 90) were recruited from women admitted to the labor and delivery department at Shands Hospital at the University of Florida (Gainesville, FL). Subjects admitting to cocaine use during the current pregnancy were asked to participate in this study as target subjects (n = 45). Subjects denying cocaine use were recruited to participate as control subjects (n = 45). All maternal subjects were at least 18 years of age. Participants were interviewed confidentially to assess the amount and timing of drug use throughout the pregnancy. Selection and exclusion from a pool of potential subjects was done by a physician investigator who was blinded to drug history. Excluded from the study were subjects admitting to consuming alcohol at > 40 g of absolute alcohol per day or using any of the following drugs chronically: azathioprine, amphetamines, benzodiazepines, barbiturates, cyclosporine, opiates, methadone, methaqualone, or steroids. Also excluded was any woman with a chronic disease known to affect the immune system or a condition that might affect mental status such as mental illness or retardation. The study protocol was approved by the University of Florida Institutional Review Board, and informed consent was obtained from all subjects. Specimens All biological specimens were labeled with a unique research identification number, and the analyst was blinded to drug history. Thirty-two amniotic fluid specimens (5 mL) were collected during parturition. A random, 3-cm segment of the umbilical cord was collected from 70 subjects immediately following parturition and placed into a sterile container. All specimens were stored at -20~ for up to two months until analysis. The stability of the cocaine analytes under these conditions was investigated previously by this laboratory and was found to be sufficient for the purposes of this study (17).

chased from Pierce Chemical Company (Rockford, IL). All other reagents were reagent grade and were purchased from Fisher Scientific (Orlando, FL).

Standard Preparation Calibration standards were prepared by fortifying the appropriate blank matrix with aliquots of standard solutions to form a calibration curve in the range of 25-750 ng/mL or ng/g. Control samples prepared at 0, 200, and 600 ng/mL or ng/g were included in each analytical run. In addition, a 500-ng/mL cocaine standard was included in each run to monitor hydrolysis of cocaine to benzoylecgonine or ecgonine methyl ester or both during extraction and subsequent analysis. All standards and control samples were prepared immediately before extraction. Solid-phase extraction The solid-phase extraction (SPE) of cocaine and its metabolites and subsequent analysis by gas chromatography-mass spectrometry (GC-MS) was adapted from the procedure previously described by Cone et al. (18). Umbilical cord tissue. Bupivacaine (1000 ng) was added as an internal standard to 1.0 g of umbilical cord tissue. The tissue was homogenized with 3 mL of phosphate buffer (0.025M, pH 4) using an Eberbach ConTorque homogenizer (Ann Arbor, MI). Homogenates were centrifuged for 10 min at 1000 rpm, and the supernatants were decanted and applied to Clean Screen| SPE columns (ZSDAU020; United Chemical Technologies, Horsham, PA) that had previously been conditioned with elution solvent (1 x I mL), methanol (1 x 3 mL), deionized water (1 x 3 mL), and 0.025M (pH 4) phosphate buffer (1 x 2 mL). The specimens were followedwith a wash of deionTable II. Limit of Detection (LOD), Limit of Quantitation (LOQ), Recovery, and Precision of Cocaine Analytes in Amniotic Fluid and in Umbilical Cord Tissue* Analyte

Chemicals Benzoylecgonine, cocaine, ecgonine methyl ester, cocaethylene, and norcocaine were purchased from Radian Corporation (Austin, TX). Bupivacaine was obtained from Sigma Chemical Company (St. Louis, MO). Ecgonine ethyl ester and m-hydroxybenzoylecgonine were gifts from Edward Cone. Methyl-(tertbutyldimethylsilyl)-trifluoroacetamide (MTBSTFA)was purTable I. Quantitation and Qualifier Ions for Each Cocaine Analyte and the Internal Standard Analyte

Target ion (m/z)

Ecgoninemethyl ester Ecgonineethyl ester Norcocaine Cocaine Cocaethylene Benzoylecgonine m-Hydroxybenzoylecgonine Bupivacaine

98

256 270 168 182 196 346 282 141

Qualifierions (m/z) 182, 282 196, 282 289 272, 303 272, 317 282,403 533, 82 140, 84

LOQ

Recovery(%)

Precision(%CV)

Amniotic fluid EME+ 5 EEE 50 NCOC 5 COC 5 CE 5 BE 5 MOHBE 10

10 50 10 10 10 10 25

76 79 89 106 111 120 83

16 20 9 8 8 10 17

Umbilical cord tissue EME 5 EEE 25 NCOC 10 COC 2.5 CE 2.5 BE 2.5 MOHBE 5

I0 50 25 5 5 2.5 10

90 90 83 91 97 95 110

15 17 9 9 10 7 6

IOD

* Concentrations noted in ng/mL (amniotic fluid) or ng/g (umbilical cord tissue). * Abbreviations are as follows: ecgonine methyl ester = EME; ecgonine ethyl ester = EEE; norcocaine = NCOC; cocaine = COC; cocaethylene = CE; benzoylecgonine = BE; m-hydroxybenzoylecgonine = MOHBE.


isopropanol-concentrated aqueous ammonium hydroxide, 80:20:2, v/v/v). The extracts were evaporated to dryness at 40~ under a gentle nitrogen stream. The extracts were derivatized by adding MTBSTFA(30 IJL)and heating at 90~ for 60 rain. Amniotic fluid. The viscosity of amniotic fluid required the

ized water (1 x 2 mL) and 0.1M HCI (1 x 2 mL). The columns were then air dried at full vacuum for 2 rain. A methanol wash (1 x 6 mL) and a second 2-rain drying step completed the washes. The analytes were then collected in culture tubes by eluting with 8 mL of the elution solvent (methylene chlorideAbundance 400000 a

TIC:

A

002SP002.D

ARTIFACT

BUPI

350000

300000

250000

EME 200000

COC

CE

150000

NCOC I00000

I BE

l

50000 I'

.~_?;.,I

Time,

MOHBE

EEE

fi 6.oo

' ~ ~.'5o

oo

"I.'o0

Abundance 1.6e+07

8.'56 ~ '~9'.'oo' '9.'56'

TIC:

'1o'.0o To15'o i~.oo

B

061SP042.D

BE

1.4e+07

1.2e+07

le+07

8000000

6000000

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4000000

2000000

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FigureI. Total ion chromatogramsof extractspreparedfrom (A) a 500 nglmL amniotic fluid cocaine analyte standardand (B) an amniotic fluid specimen obtained from an admittedcocaineuser.Ecgonine methyl ester (EME), ecgonine ethylester(EEE),norcocaine (NCOC), cocaine (COO), cocaethylene (CE), bupivacaine (BUPI), benzoylecgonine (BE), and m-hydroxybenzoylecgonine (MOHBE) are readilydetectedin the standard.

99


use of the high flowXtrackT| SPE column (XRDAH515;United Chemical Technologies).To 1.0 mL of amniotic fluid, bupivacaine (1000 ng) was added as an internal standard, followedby 3 mL of phosphate buffer (0.025M, pH 4). The specimens were then applied to the SPE columns in the same manner as described previously.

GC-MS analyses GC-MS analyses were performed with a Hewlett-Packard (Little Falls, DE) 5890 series II Plus GC equipped with a Hewlett-Packard 7673A automated liquid sampler and interfacedwith a Hewlett-Packard5972Amass selectivedetector.The GC and detector were controlled by an HP data system. The GC column was a HP-5 MS (30 m x 0.25-ram i.d. x 0.25-1Jm film thickness). Helium was the carrier gas, programmed at a constant flow rate of 1.0 mL/min. The injection port was fitted with a 2-ram silanized glass liner. The injection port temperature was 275~ and the detector temperature was 290~ The initial oven temperature of 90~ was maintained for 1 rain, programmed to 220~ at 30~ and held for 0.5 rain, then programmed to 330~ at 20~ and maintained for 1 rain. The total run time was 12.33 min. The GC-MS was operated in the selected ion monitoring (SIM) mode with a dwell time of 20 ms/ion, and quantitation was based upon ion peak-area ratios of analyte to internal standard. The quantitation and qualifier ions for each cocaine analyte and the internal standard are listed in Table I.

Results The calibration curves for each matrix were found to be linear over the calibration range by use of the standard error of the estimate statistic (i.e., all calibration points were within two standard deviations of the predicted regression line). Limit of detection (LOD) was defined as the concentration corresponding to a signal-to-noise ratio of 3. The limit of quantitation (LOQ) was determined by the analysis of a series of

decreasing standards and defined as the lowest standard that did not deviate from the target concentration by more than 20%. The LOD, LOQ, recovery, and precision for each analyte in each matrix are presented in Table II. Recoveryand precision were measured at two concentrations, and the data presented in the tables represent an average determination. The coefficients of variation (CV) for several analytes were high; this imprecision was probably due to the low abundance of the quantitation ion and low recovery. Figure 1 illustrates an SIM chromatogram of a 500 ng/mL extracted standard (Panel A) and a positiveamniotic fluid specimen obtained from a target subject (Panel B). The subject specimen was found to contain benzoylecgonine (152,288 ng/mL), ecgonine methyl ester (11,879 ng/mL), ecgonine ethyl ester (335 ng/mL), and trace amounts of cocaine and mhydroxybenzoylecgonine. Table III summarizes the range of analyte concentrations found in amniotic fluid and umbilical cord tissue. In addition, the number of positive specimens in each category is presented. Cocaine and cocaine metabolite results for each positive specimen in amniotic fluid and umbilical cord tissue are presented in Figure 2. In general, amniotic fluid contained the highest concentrations of analytes,with benzoylecgonine being the most common analyte detected,followedby ecgonine methyl ester. In umbilical cord tissue, benzoylecgoninewas the most common analyte, followed by ecgonine methyl ester. Trace amounts of cocaine, norcocaine, and m-hydroxybenzoylecgoninewere found in amniotic fluid, and one specimen was positive for ecgonine ethyl ester. In umbilical cord tissue, small amounts of cocaine, norcocaine, ecgonine methyl ester, and m-hydroxybenzoylecgoninewere detected. Cocaethylene was not detected in either matrix.

Discussion The SPE and GC-MS methods described in this study differ from those described by Cone et al. (18) in several significant

Table III. Concentration Range of Cocaine Analytes in Amniotic Fluid and Umbilical Cord Tissue Specimensand the Number of Positive Specimens in Each Subject Category Matrix

Total number of subjects

EME*

EEE

NCOC

COC

CE

BE

MOHBE

19 13 32

0-11,879 1 4 5

0-335 0 1 1

0-trace 0 1 1

0-trace 0 1 1

0 0 0 0

0-152,288 2 7 9

0-trace 0 3 3

42 28 70

0-52 0 5 5

0 0 0 0

0-172 1 2 3

0-trace 0 1 1

0 0 0 0

0-1237 1 12 13

0-trace 0 3 3

Amniotic fluid (ng/mL) Controls Targets Total*

Umbilical cord tissue (ng/mL) Controls Targets TotaP

* Abbreviations are as follows: ecgonine methyl ester = EME;ecgonine ethyl ester = EEE;norcocaine = NCOC; cocaine = COC; cocaethylene= CE; benzoylecgonine= BE; m-hydroxybenzoylecgonine= MOHBE. "~Although 90 maternal subjectswere recruited, amniotic fluid and umbilical cord tissuespecimenswere not availablefor every subject.

100


A ~"

-1000000

- 100000

- 10000

"1000 Log ng/mL -100 -10

~E

9

NCOC

B '----I0000

-I000

-100

-10

13

COC

Figure 2. Cocaine analyte results for all positive specimens in (A) an amniotic fluid specimen and (B) umbilical cord tissue.The large variation in quantitative results required conversion of the raw data to log values in order to provide meaningful graphs. All values below log 10 ng/mL or ng/g are lessthan the limit of quantitation and are designated as trace.

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of cocaine and its metabolites in alternate maternal and infant specimens will provide valuable information regarding cocaine disposition in the maternal-fetal unit. Amniotic fluid may prove to have several advantages over maternal or infant urine for the detection of prenatal cocaine exposure. Although amniotic fluid is present throughout the gestational period, its composition changes and viscosity decreases as pregnancy progresses because of fetal urination (20,21). The result is the formation of a pool of fetal urine into which cocaine and metabolites are recirculated by fetal swallowing of the amniotic fluid (15). There is also evidence that cocaine and its metabolites may accumulate in the amniotic compartment (14), which results in the prolonged presence of cocaine and metabolites in amniotic fluid and an increased detection window. Finally, amniotic fluid is easier to collect at birth than neonatal urine because urine collection requires the use of uncomfortable and often unreliable urine collection bags that irritate infant skin and dislodge easily. The appearance of cocaine, benzoylecgonine, and ecgonine methyl ester in amniotic fluid in this study is consistent with previously published data on human amniotic fluid (13-16,22,23). Data from previous studies and the current study are summaTable IV. Summary of Amniotic Fluid Cocaine Analyte Concentrations (ng/mL) rized in Table IV. In the study by Moore et al. Reported in the Literature and the Present Study (23), eight amniotic fluid specimens were Investigator Subject COC* BE EME Others obtained either during amniocentesis or transvaginally at birth from women who Mooreet al. (23), 1992 1 trace 230 NA were known to or thought to have abused 2 trace 40 NA cocaine during pregnancy. All eight speci3 250 3060 NA mens were found to contain benzoylecgo4 ND 1980 NA nine or cocaine or both by high-perfor5 ND 1650 NA 6 ND 750 NA mance liquid chromatography. 7 14 39O NA In a continuation of the study by Jain et 8 70 290 NA al. (15), amniotic fluid specimens were obtained from 23 subjects with documented 1 18 836 34 Ripple et al. (22), 1992 cocaine use one day to eight weeks before 2 trace 113 trace delivery. Cocaine or benzoylecgonine or Trace CE 3 11 277 11 both was detected by high-performance 4 19 552 17 liquid chromatography in 74% of the spec5 24 51 trace imens. Concentrations of benzoylecgonine and cocaine ranged from 400 to greater 1 ND 909 115 Casanova et al. (13), 1994 than 5000 ng/mL and from trace to 250 2 ND 925 40 ng/mL, respectively. 3 ND 143 ND In the study conducted by Ripple et al. (22), 450 amniotic fluid specimens were obEEE335, Trace Current study 1 trace 152288 11879 tained during amniocentesis and screened MOHBE for benzoylecgonine by fluorescence polarTrace MOHBE 2 ND 1435 115 ization immunoassay (FPIA) in order to 3 ND 555 104 Trace MOHBE evaluate the incidence of cocaine exposure 4 ND 335 trace Trace NCOC in a low-risk population. Five out of the 450 5 ND 91 ND specimens (1%) screened positive by FPIA 6 ND trace trace and were subsequently confirmed positive 7 ND trace ND for cocaine, benzoylecgonine, and/or ecgo8 ND trace ND 9 ND trace ND nine methyl ester by GC-MS. In the study by Casanova et al. (13), six * Abbreviationsare as follows: ND = not detected;NA = not analyzed;ecgoninemethylester= EME;ecgonine amniotic fluid specimens were obtained ethyl ester = EEE; norcocaine = NCOC; cocaine = COC; cocaethylene = CE; benzoylecgonine = BE; from admitted cocaine users and analyzed m-hydroxybenzoylecgonine= MOHBE. by GC-MS for cocaine and its metabolites.

ways. First, the high-flow XtrackT SPE columns were used in order to overcome the viscosity of the amniotic fluid specimens. The second difference was the use of the derivatizing reagent, MTBSTFA,which produced higher molecular weight derivatives and more improved chromatographic separation than possible with more traditional silyl derivatives. Finally, the volume of elution solvent was increased from 6 to 8 mL. This may have resulted in an improved recovery of ecgonine methyl ester from the 40% reported by Cone et ai. (18) to 76% in amniotic fluid and 90% in umbilical cord tissue reported here. This modified method was found to have excellent sensitivity for the detection of cocaine and its metabolites in amniotic fluid and umbilical cord tissue and is a satisfactory technique for toxicological testing of these matrices. The need to investigate new methods for the detection of prenatal cocaine exposure is evidenced by the poor performance of current methods in identifying cocaine-exposed infants. In some studies, the use of urine and meconium to screen for intrauterine cocaine exposure has resulted in falsenegative rates as high as 50% (3,15,19). In addition, the study

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Three of the six specimens (50%) were found to contain benzoylecgonine or ecgonine methyl ester. In the current study, 53.8% of the amniotic fluid specimens from admitted cocaine users and 10.5% of specimens from those denying use were found to contain cocaine or its metabolites or both. Despite differences in objectives and detection schemes, data from all studies indicate that benzoylecgonine is detected in the greatest amounts with much smaller amounts of cocaine and ecgonine methyl ester. The consequences to the fetus of being exposed to amniotic fluid containing cocaine and metabolites throughout gestation are highly speculative.There is, however, evidence for increased risk to the fetus from this type of exposure for a variety of reasons. First, the fetus is exposed continually to cocaine and its metabolites transdermally during the first half of pregnancy before keratinization of the skin occurs (20), and the fetus will continue to be exposedduring the last half of pregnancy through fetal swallowing of amniotic fluid and reabsorption of these compounds in the gut (14). In addition, benzoylecgonineand its n-desmethyl metabolite, benzoylnorecgonine, have demonstrated powerful vasoconstrictor and convulsant activity, and exposure could result in serious harm to the fetus (24,25). Finally, because cocaine metabolites apparently accumulate in the fetal compartment as a result of slow equilibrium with adjacent compartments, the fetus that is continuously and consistently exposed to cocaine during gestation will be bathed in ever increasingamounts of these deleterious metabolites (14,22). The significanceof the concentrations of cocaine and metabolites found in umbilical cord tissue in this study is unknown at this time, but certainly analytes found here are representative of and proportional to tissue and blood concentrations in the neonate at the time of birth. It is interesting that high concentrations of norcocaine were found in this matrix. This may be an indication of fetal metabolism of cocaine, which is thought to produce more of the n-desmethyl metabolites (3).

Conclusion In summary, cocaine and its metabolites are readily detected in amniotic fluid and umbilical cord tissue specimens from women and neonates exposed to cocaine. In addition, the current data indicate that amniotic fluid is an appropriate specimen for detecting prenatal cocaine exposure. Finally, additional studies are needed to determine the clinical usefulness of umbilical cord tissue.

Acknowledgments The authors would like to thank Dr. Edward J. Cone (Addiction Research Center, NIDA) for supplying several of the cocaine metabolites used in this project. This research was supported by NIDAgrant 1ROIDA03926-01 and a grant from the University of Florida Division of Sponsored Research.

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Journal of Analytical Toxicology, Vol. 21, March/April 1997 21. R.A. Brace. Amniotic fluid dynamics. In Maternal-Fetal Medicine, Principals and Practice. R.K. Creasy and R. Resnilk, Eds. 3rd ed. W.B. Saunders, Philadelphia, PA, 1994, pp 106-11. 22. M.G. Ripple, B.A. Goldberger, u Caplan, M.G. Blitzer, and S. Schwartz. Detection of cocaine and its metabolites in human amniotic fluid. J. Anal. Toxicol. 16:328-31 (1992). 23. C. Moore, S. Browne, I. Tebbett, A. Negrusz, W. Meyer, and L. Jain. Determination of cocaine and benzoylecgonine in human arnniotic fluid using flow solid-phase extraction columns and HPLC. Forensic Sci. Int. 56:177-81 (1992).

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24. J.A. Madden and R.H. Powers. Effect of cocaine and cocaine metabolites on cerebral arteries in vitro. Life Sci. 47" 1109-14 (1990). 25. B.A. Erickson, R.J. Konkol, and J.A. Madden. A comparison of the epileptogenic potential of cocaine and benzoylecgonine following intraventricular administration in nonanesthetized rats. FASEB J. 4: 746A (1990). Manuscript received June 3, 1996; revision received September 18, 1996.