Journal of Analytical Toxicology, Vol. 16, September/October 1992
Detection of Cocaine and Its Metabolites in Human Amniotic Fluid M a r y G. Ripple, B r u c e A. G o l d b e r g e r , and Yale H. C a p l a n *
Department of Pathology, School of Medicine, University of Maryland M i r i a m G. Blitzer and Stuart S c h w a r t z
Division of Human Genetics, Departments of Pediatrics and Obstetrics & Gynecology, School of Medicine, University of Maryland
l Abstract The dramatic rise in maternal drug abuse and the Incidence of positive drug findings during neonatal testing has Increased the need for prenatal toxicological testing for drugs of abuse. Human amnlotlc fluid samples collected after 13-39 weeks of pregnancy were screened for cocaine metabolite (benzoylecgonlne) by fluorescence polarization immunoassay (FPIA). All positive samples, as well as any accompanying maternal serum, were confirmed by gas chromatography/mass spectrometry (GC/MS) for cocaine and Its metabolites. Five samples out of 450 were positive for cocaine, benzoylecgonine, and ecgonlne methyl ester by GC/MS. In addition, one sample was also positive for cocaethylene. Two maternal serum samples were positive for benzoylecgonine end ecgonlne methyl ester. The presence of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in the amniotic fluid suggests that the fetus is exposed to cocaine and its metabolites through maternal circulation. The impact of this exposure on the health of the newborn is unknown.
Introduction
Cocaine use has increased dramatically during the past decade. It has been estimated that 50 million Americans have used cocaine at least once and approximately 8 million use it regularly (1). Roughly one-third of addicts are women and 10% of pregnant women use cocaine at least once during pregnancy (2,3). In addition, one in 10 fetuses is exposed to cocaine through maternal drug abuse (4). During the past 10 years, virtually every major urban center has reported an increase in the incidence of positive drug findings during neonatal testing (1,5). Maternal cocaine abuse during pregnancy may affect the fetus either directly, causing teratogenic effects or fetal death, or indirectly, by pharmacologically affecting maternal circulation and uterine activity (3,6). Cocaine administered to pregnant ewes resulted in greater fetal distress than direct intra-amniotic administration to the fetal lamb. Thus, decreased placental per*Author to whom correspondence should be addressed: YaleH. Caplan,cJoNationalCenter for Forensic Science, 1901 Sulphur Spring Road, Baltimore. MD 21227
328
fusion and subsequent anoxia in the fetus may be more deleterious to the fetus than direct effects of the cocaine (7). Cocaine blocks catecholamine reuptake at nerve terminals, causing an increase in circulating norepinephrine which produces vasoconstriction and tachycardia leading to hypertension. Studies have shown that cocaine use during pregnancy produces maternal and fetal hypertension, reduced uterine blood flow, fetal tachycardia, and fetal hypoxemia. Other maternal problems include complications during delivery, such as abruptio placentae, premature labor and delivery, ruptured ectopic pregnancy, and spontaneous abortion. Other neonatal effects from exposure to cocaine include an increased incidence of congenital anomalies, fetal growth retardation, acute hypoxic-ischemic encephalopathy, withdrawal syndrome, low birth weight, and cerebral hemorrhage or infarction. Long term effects may include sudden infant death syndrome (SIDS) and behavioral abnormalities with developmental delay. The obvious adverse effects of cocaine on the developing fetus have underscored the importance of prenatal toxicological testing for drugs of abuse (1-14). Screening of 450 human amniotic fluid samples collected after 13-39 weeks of pregnancy for cocaine metabolite (benzoylecgonine) was performed by fluorescence polarization immunoassay (FPIA). This study reports a method for screening cocaine metabolite by FPIA applied to amniotic fluid. All positive samples, as well as any accompanying maternal sera, were confirmed by gas chromatography/mass spectrometry (GC/MS). The assay was used to determine the incidence of cocaine exposure in a relatively low-risk population.
M a t e r i a l s and M e t h o d s
Chemicals. Benzoylecgonine (BE), deuterated cocaine (d5COC), and deuterated benzoylecgonine (d3-BE) were provided by Research Triangle Institute courtesy of the National Institute on Drug Abuse. Cocaine (COC) was purchased from Mallinckrodt Chemical Co. and ecgonine methyl ester (EME), cocaethylene (CE), and deuterated ecgonine methyl ester (d3-EME) were purchased from Radian Corporation. Derivatizing reagents were obtained from Lancaster Synthesis and all other reagents (reagent grade) were purchased from Fisher Scientific.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Analytical Toxicology, Vol. 16, September/October 1992
0.10-gm film thickness). Helium was used as the carrier gas at a flow rate of 1.5 mL/min. The injection port was fitted with a 2-mm silanized glass liner. The injection AmnioticFluid Maternal Serum port temperature was 250~ and the deSubject GC/MS GC/MS rector temperature was 280~ The oven number FPIA COC BE EME CE COC BE EME CE temperature was maintained at 100~ for 159 860 18 836 34 ND ND 205 22 ND 1 min, p r o g r a m m e d at 3 0 ~ to 184 160 T 113 T ND NS NS NS NS 280~ and then held at 280~ for 3 min. 223 320 11 277 11 T NS NS NS NS The GC/MS was operated in the SIM 311 630 19 552 17 ND ND 151 19 ND mode with the following ions (m/z) mon373 40 24 51 T ND ND ND ND ND itored for each compound at their respective retention times (R,): d5-COC, 308, 9Abbreviations: COC, cocaine; BE, benzoylecgonine; EME, ecgonine methyl esler: CE, cocaethylene; T, trace; ND, none detected; and NS, no sample R t = 5.11 min; COC, 303, R t = 5.12 min; d3-BE, 334, R t = 5.54 min; BE, 331, R t = 5.55 min; d3-EME, 324, R t = 5.10 min; Specimens. Amniotic fluid samples were obtained from the EME, 321, R t = 5.10 min; CE, 196, 317, Re - 5.29 min. The ion Cytogenetics Laboratory at the University of Maryland Medical m/z 196 and d5-COC were used for quantitation of CE. QuantiCenter from 450 pregnant women (13-39 weeks gestation) betation was based upon ion peak-area ratios of analyte to corretween May and October 1991. The majority of pregnant women sponding deuterated analog. The limits of detection and quanwere referred to the laboratory for genetic evaluation because of titation for all analytes were 5 and 10 ng/mL, respectively. increased fetal risks due to advanced matemal age, abnormal maternal serum alpha-fetoprotein, or abnormal ultrasonographic findings. A total of 5-30 mL of amniotic fluid was obtained during amniocentesis from these women. Upon receipt, the amResults niotic fluid was centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant fluid was removed and frozen at Detection and measurement of cocaine metabolite by FPIA. -20~ until FPIA screening and the cells were used for cytoThe accuracy of the FPIA was evaluated by comparing fortified genetic studies. No preservatives were added to the supernatant. urine and amniotic fluid with Abbott calibrator material. FortiThe samples were randomized by the Cytogenetics Laboratory fied urine and amniotic fluid samples containing concentrations and patient information was blinded. The pH of 25 randomly seof BE ranging from 25-1000 ng/mL were assayed for cocaine lected human amniotic fluid samples was between 8 and 9. metabolite by the FPIA assay and compared to the manufacFluorescence polarization immunoassay for cocaine metaboturer's standards (50-5000 ng/mL). The average percent error for lite. Cocaine rnetabolite was measured by FPIA with an Abbott the urine, amniotic fluid, and manufacturer's standards were 13, TDx | analyzer and TDx Cocaine Metabolite reagents (Abbott 8, and 9%, respectively. The precision of a fortified amniotic Diagnostics). The assay was performed according to the manufluid sample containing 100 ng/mL BE (n = 5) showed a coeffifacturer's recommended procedure. Results were reported in cient of variation of 8% with a mean + SD of 95 + 8 ng/mL. Acng/mL of BE. The sensitivity of the assay for BE reported by the cording to the manufacturer's specifications, the lowest concenmanufacturer was 30 ng/mL. tration of BE in urine that can be distinguished from zero with Fortified urine and amniotic fluid specimens containing BE at 95% confidence by the FPIA is 30 ng/mL. An amniotic fluid concentrations of 0, 25, 50, 100, 250, 500, and 1000 ng/mL sample fortified at 25 ng/mL demonstrated a percent error of 4%. were prepared by adding the appropriate amount of standard Samples contaminated with blood typically produced slightly elmaterial. Blank amniotic fluid was obtained by combining alievated results, and thus, a liberal approach was used to select quots of negative amniotic fluid. probable positive samples for GC/MS confirmation. A practical GC/MS assay for cocaine, benzoylecgonine, ecgonine methyl limit of detection for BE in human amniotic fluid was established ester, and cocaethylene. GC/MS standard curves were prepared at 50 ng/mL. in blank amniotic fluid at concentrations of 0, 5, 10, 25, 75, Confirmation by GC/MS. COC, BE, EME, and CE standard t50, 300, 500, and 10O0 ng/mL for aii anaIytes by fortification curves prepared in arnniotic fluid were linear with correlation cowith an appropriate amount of standard material. The deuterated efficients greater than 0.995. Five samples were confirmed by internal standard (IS) concentration was 250 ng/mL. GC/MS for cocaine and its metabolites. Refer to Table I for The GC/MS measurement of COC, BE, EME, and CE in amFPIA and GC/MS results. Sample 223 was positive for CE; the niotic fluid and serum was performed by the method of lsensample was therefore tested for ethanol using automated schmid et al. with minor modifications to increase assay sensiheadspace gas chromatography with n-propanol as IS. The limit tivity (15). Briefly, amniotic fluid and maternal serum containing of detection of this method for ethanol was 0.0025% w/v (16). deuterated analogs of COC, BE, and EME were extracted by The sample contained ethanol at a concentration of 0.004% w/v. pouring over a Chem-Elut column (Varian) and eluting with a Three accompanying maternal serum samples were also anachloroform-isopropanol (9:1) solution. The eluent was collected lyzed by GC/MS. Maternal serum accompanying sample 159 and evaporated to dryness. BE was derivatized to n-propyl COC was collected at the same time as amniocentesis and contained and EME was derivatized to p-fluoro-COC. BE and EME at concentrations of 205 and 22 ng/mL, respecGC/MS analysis was performed with a Hewlett-Packard tively. Maternal serum accompanying sample 373 was collected 5890A Series II GC interfaced with the Hewlett-Packard 5971A at the same time as amniocentesis but did not contain cocaine or mass selective detector and a Hewlett-Packard 7673A auto-samits metabolites. Maternal serum accompanying sample 311 was pler. The column was a Restek RTx-5 (15 m x 0.25 mm i.d. x Table I. Concentrations (ng/mL) of Cocaine and Its Metabolites in Human Amniotlc Fluid and Maternal Serum*
329
Journal of Analytical Toxicology, Vol, 16, September~October1992
collected one week prior to amniocentesis and contained BE and EME at concentrations of 151 and 19 ng/mL, respectively. No cocaine was detected in any of the accompanying maternal sera. The inability to detect cocaine in the maternal sera could have been a consequence of decreased assay sensitivity due to limited sample volume, spontaneous degradation to BE because of lack of preservation, or the absence of cocaine in these samples. The average ratio of BE:COC and BE:EME in amniotic fluid was 26:1 and 27:1, respectively. The average ratio of BE:EME in maternal serum was 8.6:1. For sample 159, the ratios of BE in amniotic fluid:maternal serum and EME in amniotic fluid:maternal serum EME were 4.1:1 and 1.5:1, respectively.
Discussion The FP[A was found to be hi.ghly sensitive and accurate for the detection and measurement of benzoylecgonine in human amniotic fluid. The average percent error for the amniotic fluid samples fortified with concentrations of BE from 25-1000 nglmL was 8% and the precision of a fortified amniotic fluid sample containing 100 ng/mL BE, as measured by the coefficient of variation, was 8%. The FPIA results for BE exhibited excellent correlation with the GC/MS assay results (y = 0.96x -20.5, r = 0.992). The only problem encountered with the FPIA was that samples contaminated with blood typically produced slightly elevated results (eg., greater than 25 ng/mL, but less than 50 ng/mL). A limit of detection for BE in human amniotic fluid in this study was established at 50 ng/mL. The GC/MS assay presented was also sensitive and accurate for the confirmation of cocaine and its metabolites. Standard curves prepared in amniotic fluid for all analytes were linear with correlation coefficients greater than 0.995. Therefore, FPIA is a suitable screening technique for detection of BE in human amniotic fluid, and in conjunction with GC/MS, is a acceptable tool for prenatal toxicological tesling ~br cocaine and its metabolites. The presence of COC. BE, and EME in the amniotic fluid corresponds with human and animal data. Studies have indicated that the fetus is exposed to cocaine and its metabolites through the maternal circulation and through placental and fetal metabolism (4,17) Further, cocaine crosses the placenta by simple diffusion to distribute between maternal and fetal blood (1). Data from guinea pig.~ and pregnant ewes suggest that cocaine does not accumulate in the fetal blood compartment (17,18). However, according to a recent study by Sandberg and Olsen, cocaine accumulates in guinea pig amniotic fluid. Chronic cocaine administration to pregnant guinea pigs resulted in cocaine concentrations in the amniotic fluid that were 3 to 4 times greater than in fetal and maternal plasma. Further, it was determined that cocaine in guinea pig amniotic fluid had an extremely long in-vitro half-life (17). Pharmacokinetically, amniotic fluid represents a very deep compartment and equilibrium with adjacent compartments is slow (19). The Sandberg and Olsen data suggest that the placenta and fetal liver may have a limited capacity to metabolize cocaine and that amniotic fluid serves as a compartment from which the fetus can be exposed to drug and/or its metabolites. In addition, drug and drug metabolites may accumulate in fetal blood because of ion trapping and underdeveloped renal function. Normally, water-soluble drugs and drug metabolites accumulate in the amniotic fluid more than in any other compartment (19). Although BE is water soluble, results of Sandberg and Olsen's guinea pig study indicate that BE does not accumulate in
330
the fetal plasma or amniotic fluid. Instead, benzoylnorecgonine (BN) was the major metabolite of cocaine in the fetal guinea pig and was present in higher concentrations in the fetal plasma and amniotic fluid than maternal plasma ( t 7). High concentrations of this metabolite have also been found in the urine of pregnant cocaine users (20). These results suggest that pregnant females and their fetuses metabolize cocaine via N-demethylation to a greater degree than nonpregnant adults (17,20). In contrast, the high BE amniotic fluid:maternal serum ratio (4.l: 1) in subject 159 suggests that BE might concentrate in human amniotic fluid. However, cocaine may have spontaneously converted to BE, artificially elevating the concentration of BE. Nonetheless, the above data suggests that both cocaine and its melabolites may be found in human amniotic fluid, potentially producing deleterious fetal effects. The accumulation of cocaine and its metabo)ites in amniotic fluid poses an increased risk to the developing fetus for the following reasons: (a) BE and BN have been found to be potent convulsants and vasoconstrictors (17,21); (b) during the first half of pregnancy, drugs may diffuse through the fetal skin, and thus, amniotic fluid might be an extension of the fetal extracel[ular fluid space through which the fetus can be re-exposed (22); (c) the human fetus swallows amniotic fluid and is exposed to cocaine and its metabolites through absorption in the gut (17,19); (d) the cholinesterase activity is relatively low in pregnant women, fetuses, and newborns, which could result in elevated levels of parent cocaine and toxic metabolites (1,4); and (e) uterine artery vasoconstriction in pregnant ewes does not appear to restrict placental transfer of cocaine (6,18,23). In order to fully understand the implications of detecting cocaine and its metabolites in amniotic fluid, data from human and animal tissue distribution and amniotic fluid studies were surveyed. Toxic effects of cocaine have been noted with blood concentralions in the range of 250-5000 ng/mL and fatalities have octuned with concenlrations of 800 ng/mL or more (24,25). Apple and Roe presented the amniolic fluid concentrations of COC and BE in a t6-week-otd fetus wh~se mother had recentIy used cocaine prior to death The amniotic fluid COC and BE concentrations were 3300 and 16(1(I nghnL, respectively (26), Animal studies provide the only other information on the presence of cocaine in amniotic fluid. COC and BE have been detected in the amniotic fluid of pregnant guinea pigs in an HPLC methodology paper by Sandberg and Olsen (27). COC and BE have also been detected in the amniotic tluid of pregnant rats in a pharmacokinetic study by Ferko et al. (28). Also, Sandberg and Olsen chronically administered pregnant guinea pigs cocaine at a dose of 6 mg/kg for 10 days and detected cocaine, benzoylecgonine, and benzoylnorecgonine in the amniotic fluid at mean concentrations of 1062. 72. and 248 ng/mL, respectively (17). It is difficult to establish conclusions based upon the wide range of concentrations of cocaine and its metabolites found in the human amniotic fluid. In lhis study, the details regarding the route, dose, and time of administration in relation to amniotic fluid sampling and the presence of other confounding variables such as maternal health were unknown. Finally, the consequences of fetal exposure to cocaine in these cases is also unknown.
Acknowledgements The authors express their appreciation to Abbott Diagnostics for kindly providing the FPIA reagents.
Journal of AnalyticalToxicology,Vol. 16, September/October1992
References 1. S.D. Dixon. Effects of transplacental exposure to cocaine and methamphetamine on the neonate. West. J. Med. 150:436-42 (1989). 2. L. Jariwala and R.F. Shaw. Clinical, pathological and toxicological findings of licit and illicit drugs in stillboms, newborns, and infants. Calif. Assoc. ToxicoL Newsletter (Winter) 22-25 (1990). 3. E.H. Roland and J.J. Volpe. Effect of maternal cocaine use on the fetus and newborn: Review of the Literature. Pediatr. Neurosci. 15:88-94 (1989). 4. D.A. Roe, B.B. Little, RE. Bawdon, and L.C. GUstrap. Metabolism of cocaine by human placentas: Implications for fetal exposure. Am. J. Obstet. GynecoL 163:715-18 (1990). 5. S.J. Kharasch, D. Glotzer, R. Vinci, M. Weitzman, and J. Sargent. Unsuspected cocaine exposure in young children. Am. J. Dis. Children 145:204-206 (1991 ). 6. M.A. Plessinger and J.R. Woods, Jr. The cardiovascular effects of cocaine use in pregnancy. Reprod. ToxicoL 5:99-113 (1991). 7. J.R. Woods, Jr., M.A. Plessinger, and K.E. Clark. Effect of cocaine on uterine blood flow and fetal oxygenation. J. Am. Med. Assoc. 257:957--61 (1987). 8. I.J. Chasnoff, W.J. Burns, S.H. Schnoll, and K.A. Burns. Cocaine use in pregnancy. N. Engl. J. Med. 313:666-69 (1985). 9. B.B. Little, L.M. Snell, V.R. Klein, and L.C. Gilstrap. Cocaine abuse during pregnancy: maternal and fetal implications. Obstet. GynecoL 73:157-60 (1989). 10. M.R. Spence, R. Williams, G.J. DiGregorio, A. Kirby-McDonnell, and M. Polansky. The relationship between recent cocaine use and pregnancy outcome. Obstet. GynecoL 78:326-29 (1991). 11. S.R. Kandall. Perinatal effects of cocaine and amphetamine use during pregnancy. Bull. N.Y. Acad. Med. 87:240-55 (1991 ). 12. I.J. Chasnoff, D.R. Griffith, S. MacGregor, K. Dirkes, and K.A. Burns. Temporal patterns of cocaine use in pregnancy. J. Am. Med. Assoc. 261:1741-44 (1989). 13. B.B. Little and LM. Snell. Brain growth among fetuses exposed to cocaine in utero: Asymmetrical growth retardation. Obstet. Gynecol. 77:361-64 (1991 ). 14. G.A. "l']chenor, S.S. Ahn, and J. Lazerson. Cocaine exposure in a newborn. Hosp. Pract. 31-32 (1991). 15. D.S. Isenchmid, B.S. Levine, and Y.H. Caplan. A method for the simultaneous determination of cocaine, benzoylecgonine, and ecgonine methyl ester in blood and urine using GC/EIMS with
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derivatization to produce high mass molecular ions. J. Anal ToxicoL 12:242-45 (1988). Standard Operating Procedure Manual. Toxicology Laboratory, Office of the Chief Medical Examiner, State of Maryland. J.A. Sandberg and G.D. Olsen. Cocaine and metabolite concentrations in the fetal guinea pig after chronic maternal cocaine administration. J. Pharmacol. Exper. Therap. 260:587-91 (1992). J.R. Woods, Jr., M.A. Plessinger, K. Scott, and R.K. Miller. Prenatal cocaine exposure to the fetus: A sheep model for cardiovascular evaluation. Ann. NYAcad. Sci. 562:267-79 (1989). F. Reynolds. Distribution of drugs in amniotic fluid. In Amniotic Fluid and its Clinical Significance, M Sandier, Ed., Marcel Dekker, New York, 1981, pp. 261-75. I.J. Chasnoff and D.E. Lewis. Cocaine metabolism during pregnancy. Pediatr. lies. 23: 257A (1988). B. 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). T. Lind. The biochemistry of amniotic fluid. In Amniotic Fluid and its Clinical Significance, M. Sandier, Ed., Marcel Dekker, New York, 1981, pp. 1-25. T.R. Moore, J. Sorg, L. Miller, T.C. Key, and R. Resnik. Hemodynamic effects of intravenous cocaine on the pregnant ewe and fetus. Am. J. Obstet. GynecoL 155:883-88 (1986). A.C. Moffat. Clarke's Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1986, pp. 489-90. A. Poklis, M.A. Mackell, and M. Graham. Disposition of cocaine in fatal poisoning in man. J. Anal ToxicoL 9:227-29 (1985). F.S. Apple and S.J. Roe. Cocaine-associated fetal death in utero. J. Anal ToxicoL 14:259-60 (1990). J.A. Sandberg and G.D. Olsen. Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography. J. Chromatogr. 525:113-21 (1990). A.P. Ferko, E.J. Barbieri, G.J. DiGregorio, and E.K. Ruch. Determination of cocaine and its metabolites in parotid saliva, plasma, urine and amniotic fluid in pregnant and nonpregnant rats. Res. Comm. Subst. Abuse 12:1-17 (1991). Manuscript received March 17, 1992; revision received June 9, 1992.
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Detection of Cocaine and Its Metabolites in Human Amniotic Fluid M a r y G. Ripple, B r u c e A. G o l d b e r g e r , and Yale H. C a p l a n *
Department of Pathology, School of Medicine, University of Maryland M i r i a m G. Blitzer and Stuart S c h w a r t z
Division of Human Genetics, Departments of Pediatrics and Obstetrics & Gynecology, School of Medicine, University of Maryland
l Abstract The dramatic rise in maternal drug abuse and the Incidence of positive drug findings during neonatal testing has Increased the need for prenatal toxicological testing for drugs of abuse. Human amnlotlc fluid samples collected after 13-39 weeks of pregnancy were screened for cocaine metabolite (benzoylecgonlne) by fluorescence polarization immunoassay (FPIA). All positive samples, as well as any accompanying maternal serum, were confirmed by gas chromatography/mass spectrometry (GC/MS) for cocaine and Its metabolites. Five samples out of 450 were positive for cocaine, benzoylecgonine, and ecgonlne methyl ester by GC/MS. In addition, one sample was also positive for cocaethylene. Two maternal serum samples were positive for benzoylecgonine end ecgonlne methyl ester. The presence of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in the amniotic fluid suggests that the fetus is exposed to cocaine and its metabolites through maternal circulation. The impact of this exposure on the health of the newborn is unknown.
Introduction
Cocaine use has increased dramatically during the past decade. It has been estimated that 50 million Americans have used cocaine at least once and approximately 8 million use it regularly (1). Roughly one-third of addicts are women and 10% of pregnant women use cocaine at least once during pregnancy (2,3). In addition, one in 10 fetuses is exposed to cocaine through maternal drug abuse (4). During the past 10 years, virtually every major urban center has reported an increase in the incidence of positive drug findings during neonatal testing (1,5). Maternal cocaine abuse during pregnancy may affect the fetus either directly, causing teratogenic effects or fetal death, or indirectly, by pharmacologically affecting maternal circulation and uterine activity (3,6). Cocaine administered to pregnant ewes resulted in greater fetal distress than direct intra-amniotic administration to the fetal lamb. Thus, decreased placental per*Author to whom correspondence should be addressed: YaleH. Caplan,cJoNationalCenter for Forensic Science, 1901 Sulphur Spring Road, Baltimore. MD 21227
328
fusion and subsequent anoxia in the fetus may be more deleterious to the fetus than direct effects of the cocaine (7). Cocaine blocks catecholamine reuptake at nerve terminals, causing an increase in circulating norepinephrine which produces vasoconstriction and tachycardia leading to hypertension. Studies have shown that cocaine use during pregnancy produces maternal and fetal hypertension, reduced uterine blood flow, fetal tachycardia, and fetal hypoxemia. Other maternal problems include complications during delivery, such as abruptio placentae, premature labor and delivery, ruptured ectopic pregnancy, and spontaneous abortion. Other neonatal effects from exposure to cocaine include an increased incidence of congenital anomalies, fetal growth retardation, acute hypoxic-ischemic encephalopathy, withdrawal syndrome, low birth weight, and cerebral hemorrhage or infarction. Long term effects may include sudden infant death syndrome (SIDS) and behavioral abnormalities with developmental delay. The obvious adverse effects of cocaine on the developing fetus have underscored the importance of prenatal toxicological testing for drugs of abuse (1-14). Screening of 450 human amniotic fluid samples collected after 13-39 weeks of pregnancy for cocaine metabolite (benzoylecgonine) was performed by fluorescence polarization immunoassay (FPIA). This study reports a method for screening cocaine metabolite by FPIA applied to amniotic fluid. All positive samples, as well as any accompanying maternal sera, were confirmed by gas chromatography/mass spectrometry (GC/MS). The assay was used to determine the incidence of cocaine exposure in a relatively low-risk population.
M a t e r i a l s and M e t h o d s
Chemicals. Benzoylecgonine (BE), deuterated cocaine (d5COC), and deuterated benzoylecgonine (d3-BE) were provided by Research Triangle Institute courtesy of the National Institute on Drug Abuse. Cocaine (COC) was purchased from Mallinckrodt Chemical Co. and ecgonine methyl ester (EME), cocaethylene (CE), and deuterated ecgonine methyl ester (d3-EME) were purchased from Radian Corporation. Derivatizing reagents were obtained from Lancaster Synthesis and all other reagents (reagent grade) were purchased from Fisher Scientific.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Analytical Toxicology, Vol. 16, September/October 1992
0.10-gm film thickness). Helium was used as the carrier gas at a flow rate of 1.5 mL/min. The injection port was fitted with a 2-mm silanized glass liner. The injection AmnioticFluid Maternal Serum port temperature was 250~ and the deSubject GC/MS GC/MS rector temperature was 280~ The oven number FPIA COC BE EME CE COC BE EME CE temperature was maintained at 100~ for 159 860 18 836 34 ND ND 205 22 ND 1 min, p r o g r a m m e d at 3 0 ~ to 184 160 T 113 T ND NS NS NS NS 280~ and then held at 280~ for 3 min. 223 320 11 277 11 T NS NS NS NS The GC/MS was operated in the SIM 311 630 19 552 17 ND ND 151 19 ND mode with the following ions (m/z) mon373 40 24 51 T ND ND ND ND ND itored for each compound at their respective retention times (R,): d5-COC, 308, 9Abbreviations: COC, cocaine; BE, benzoylecgonine; EME, ecgonine methyl esler: CE, cocaethylene; T, trace; ND, none detected; and NS, no sample R t = 5.11 min; COC, 303, R t = 5.12 min; d3-BE, 334, R t = 5.54 min; BE, 331, R t = 5.55 min; d3-EME, 324, R t = 5.10 min; Specimens. Amniotic fluid samples were obtained from the EME, 321, R t = 5.10 min; CE, 196, 317, Re - 5.29 min. The ion Cytogenetics Laboratory at the University of Maryland Medical m/z 196 and d5-COC were used for quantitation of CE. QuantiCenter from 450 pregnant women (13-39 weeks gestation) betation was based upon ion peak-area ratios of analyte to corretween May and October 1991. The majority of pregnant women sponding deuterated analog. The limits of detection and quanwere referred to the laboratory for genetic evaluation because of titation for all analytes were 5 and 10 ng/mL, respectively. increased fetal risks due to advanced matemal age, abnormal maternal serum alpha-fetoprotein, or abnormal ultrasonographic findings. A total of 5-30 mL of amniotic fluid was obtained during amniocentesis from these women. Upon receipt, the amResults niotic fluid was centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant fluid was removed and frozen at Detection and measurement of cocaine metabolite by FPIA. -20~ until FPIA screening and the cells were used for cytoThe accuracy of the FPIA was evaluated by comparing fortified genetic studies. No preservatives were added to the supernatant. urine and amniotic fluid with Abbott calibrator material. FortiThe samples were randomized by the Cytogenetics Laboratory fied urine and amniotic fluid samples containing concentrations and patient information was blinded. The pH of 25 randomly seof BE ranging from 25-1000 ng/mL were assayed for cocaine lected human amniotic fluid samples was between 8 and 9. metabolite by the FPIA assay and compared to the manufacFluorescence polarization immunoassay for cocaine metaboturer's standards (50-5000 ng/mL). The average percent error for lite. Cocaine rnetabolite was measured by FPIA with an Abbott the urine, amniotic fluid, and manufacturer's standards were 13, TDx | analyzer and TDx Cocaine Metabolite reagents (Abbott 8, and 9%, respectively. The precision of a fortified amniotic Diagnostics). The assay was performed according to the manufluid sample containing 100 ng/mL BE (n = 5) showed a coeffifacturer's recommended procedure. Results were reported in cient of variation of 8% with a mean + SD of 95 + 8 ng/mL. Acng/mL of BE. The sensitivity of the assay for BE reported by the cording to the manufacturer's specifications, the lowest concenmanufacturer was 30 ng/mL. tration of BE in urine that can be distinguished from zero with Fortified urine and amniotic fluid specimens containing BE at 95% confidence by the FPIA is 30 ng/mL. An amniotic fluid concentrations of 0, 25, 50, 100, 250, 500, and 1000 ng/mL sample fortified at 25 ng/mL demonstrated a percent error of 4%. were prepared by adding the appropriate amount of standard Samples contaminated with blood typically produced slightly elmaterial. Blank amniotic fluid was obtained by combining alievated results, and thus, a liberal approach was used to select quots of negative amniotic fluid. probable positive samples for GC/MS confirmation. A practical GC/MS assay for cocaine, benzoylecgonine, ecgonine methyl limit of detection for BE in human amniotic fluid was established ester, and cocaethylene. GC/MS standard curves were prepared at 50 ng/mL. in blank amniotic fluid at concentrations of 0, 5, 10, 25, 75, Confirmation by GC/MS. COC, BE, EME, and CE standard t50, 300, 500, and 10O0 ng/mL for aii anaIytes by fortification curves prepared in arnniotic fluid were linear with correlation cowith an appropriate amount of standard material. The deuterated efficients greater than 0.995. Five samples were confirmed by internal standard (IS) concentration was 250 ng/mL. GC/MS for cocaine and its metabolites. Refer to Table I for The GC/MS measurement of COC, BE, EME, and CE in amFPIA and GC/MS results. Sample 223 was positive for CE; the niotic fluid and serum was performed by the method of lsensample was therefore tested for ethanol using automated schmid et al. with minor modifications to increase assay sensiheadspace gas chromatography with n-propanol as IS. The limit tivity (15). Briefly, amniotic fluid and maternal serum containing of detection of this method for ethanol was 0.0025% w/v (16). deuterated analogs of COC, BE, and EME were extracted by The sample contained ethanol at a concentration of 0.004% w/v. pouring over a Chem-Elut column (Varian) and eluting with a Three accompanying maternal serum samples were also anachloroform-isopropanol (9:1) solution. The eluent was collected lyzed by GC/MS. Maternal serum accompanying sample 159 and evaporated to dryness. BE was derivatized to n-propyl COC was collected at the same time as amniocentesis and contained and EME was derivatized to p-fluoro-COC. BE and EME at concentrations of 205 and 22 ng/mL, respecGC/MS analysis was performed with a Hewlett-Packard tively. Maternal serum accompanying sample 373 was collected 5890A Series II GC interfaced with the Hewlett-Packard 5971A at the same time as amniocentesis but did not contain cocaine or mass selective detector and a Hewlett-Packard 7673A auto-samits metabolites. Maternal serum accompanying sample 311 was pler. The column was a Restek RTx-5 (15 m x 0.25 mm i.d. x Table I. Concentrations (ng/mL) of Cocaine and Its Metabolites in Human Amniotlc Fluid and Maternal Serum*
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collected one week prior to amniocentesis and contained BE and EME at concentrations of 151 and 19 ng/mL, respectively. No cocaine was detected in any of the accompanying maternal sera. The inability to detect cocaine in the maternal sera could have been a consequence of decreased assay sensitivity due to limited sample volume, spontaneous degradation to BE because of lack of preservation, or the absence of cocaine in these samples. The average ratio of BE:COC and BE:EME in amniotic fluid was 26:1 and 27:1, respectively. The average ratio of BE:EME in maternal serum was 8.6:1. For sample 159, the ratios of BE in amniotic fluid:maternal serum and EME in amniotic fluid:maternal serum EME were 4.1:1 and 1.5:1, respectively.
Discussion The FP[A was found to be hi.ghly sensitive and accurate for the detection and measurement of benzoylecgonine in human amniotic fluid. The average percent error for the amniotic fluid samples fortified with concentrations of BE from 25-1000 nglmL was 8% and the precision of a fortified amniotic fluid sample containing 100 ng/mL BE, as measured by the coefficient of variation, was 8%. The FPIA results for BE exhibited excellent correlation with the GC/MS assay results (y = 0.96x -20.5, r = 0.992). The only problem encountered with the FPIA was that samples contaminated with blood typically produced slightly elevated results (eg., greater than 25 ng/mL, but less than 50 ng/mL). A limit of detection for BE in human amniotic fluid in this study was established at 50 ng/mL. The GC/MS assay presented was also sensitive and accurate for the confirmation of cocaine and its metabolites. Standard curves prepared in amniotic fluid for all analytes were linear with correlation coefficients greater than 0.995. Therefore, FPIA is a suitable screening technique for detection of BE in human amniotic fluid, and in conjunction with GC/MS, is a acceptable tool for prenatal toxicological tesling ~br cocaine and its metabolites. The presence of COC. BE, and EME in the amniotic fluid corresponds with human and animal data. Studies have indicated that the fetus is exposed to cocaine and its metabolites through the maternal circulation and through placental and fetal metabolism (4,17) Further, cocaine crosses the placenta by simple diffusion to distribute between maternal and fetal blood (1). Data from guinea pig.~ and pregnant ewes suggest that cocaine does not accumulate in the fetal blood compartment (17,18). However, according to a recent study by Sandberg and Olsen, cocaine accumulates in guinea pig amniotic fluid. Chronic cocaine administration to pregnant guinea pigs resulted in cocaine concentrations in the amniotic fluid that were 3 to 4 times greater than in fetal and maternal plasma. Further, it was determined that cocaine in guinea pig amniotic fluid had an extremely long in-vitro half-life (17). Pharmacokinetically, amniotic fluid represents a very deep compartment and equilibrium with adjacent compartments is slow (19). The Sandberg and Olsen data suggest that the placenta and fetal liver may have a limited capacity to metabolize cocaine and that amniotic fluid serves as a compartment from which the fetus can be exposed to drug and/or its metabolites. In addition, drug and drug metabolites may accumulate in fetal blood because of ion trapping and underdeveloped renal function. Normally, water-soluble drugs and drug metabolites accumulate in the amniotic fluid more than in any other compartment (19). Although BE is water soluble, results of Sandberg and Olsen's guinea pig study indicate that BE does not accumulate in
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the fetal plasma or amniotic fluid. Instead, benzoylnorecgonine (BN) was the major metabolite of cocaine in the fetal guinea pig and was present in higher concentrations in the fetal plasma and amniotic fluid than maternal plasma ( t 7). High concentrations of this metabolite have also been found in the urine of pregnant cocaine users (20). These results suggest that pregnant females and their fetuses metabolize cocaine via N-demethylation to a greater degree than nonpregnant adults (17,20). In contrast, the high BE amniotic fluid:maternal serum ratio (4.l: 1) in subject 159 suggests that BE might concentrate in human amniotic fluid. However, cocaine may have spontaneously converted to BE, artificially elevating the concentration of BE. Nonetheless, the above data suggests that both cocaine and its melabolites may be found in human amniotic fluid, potentially producing deleterious fetal effects. The accumulation of cocaine and its metabo)ites in amniotic fluid poses an increased risk to the developing fetus for the following reasons: (a) BE and BN have been found to be potent convulsants and vasoconstrictors (17,21); (b) during the first half of pregnancy, drugs may diffuse through the fetal skin, and thus, amniotic fluid might be an extension of the fetal extracel[ular fluid space through which the fetus can be re-exposed (22); (c) the human fetus swallows amniotic fluid and is exposed to cocaine and its metabolites through absorption in the gut (17,19); (d) the cholinesterase activity is relatively low in pregnant women, fetuses, and newborns, which could result in elevated levels of parent cocaine and toxic metabolites (1,4); and (e) uterine artery vasoconstriction in pregnant ewes does not appear to restrict placental transfer of cocaine (6,18,23). In order to fully understand the implications of detecting cocaine and its metabolites in amniotic fluid, data from human and animal tissue distribution and amniotic fluid studies were surveyed. Toxic effects of cocaine have been noted with blood concentralions in the range of 250-5000 ng/mL and fatalities have octuned with concenlrations of 800 ng/mL or more (24,25). Apple and Roe presented the amniolic fluid concentrations of COC and BE in a t6-week-otd fetus wh~se mother had recentIy used cocaine prior to death The amniotic fluid COC and BE concentrations were 3300 and 16(1(I nghnL, respectively (26), Animal studies provide the only other information on the presence of cocaine in amniotic fluid. COC and BE have been detected in the amniotic fluid of pregnant guinea pigs in an HPLC methodology paper by Sandberg and Olsen (27). COC and BE have also been detected in the amniotic tluid of pregnant rats in a pharmacokinetic study by Ferko et al. (28). Also, Sandberg and Olsen chronically administered pregnant guinea pigs cocaine at a dose of 6 mg/kg for 10 days and detected cocaine, benzoylecgonine, and benzoylnorecgonine in the amniotic fluid at mean concentrations of 1062. 72. and 248 ng/mL, respectively (17). It is difficult to establish conclusions based upon the wide range of concentrations of cocaine and its metabolites found in the human amniotic fluid. In lhis study, the details regarding the route, dose, and time of administration in relation to amniotic fluid sampling and the presence of other confounding variables such as maternal health were unknown. Finally, the consequences of fetal exposure to cocaine in these cases is also unknown.
Acknowledgements The authors express their appreciation to Abbott Diagnostics for kindly providing the FPIA reagents.
Journal of AnalyticalToxicology,Vol. 16, September/October1992
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