Journal of Analytical Toxicology, Vol. 16, July/August 1992
The Analysisof Cocaineand Benzoylecgoninein Mec0nium G r e g o r y D. Clark*, I. B r u c e R o s e n z w e i g t , and V i d m a n t a s A. R a i s y s
Department of Laboratory Medicine, SB-IO, University of Washington, Seattle, Washington 98195 C h a r l e s M. C a l l a h a n
Department of Pediatrics, RD-20, University of Washington, Seattle, Washington 98195 T h e r e s e M. Grant and A n n P. S t r e i s s g u t h
Department of Psychiatry and Behaviora/ Sciences, GG-20, University of Washington, Seattle, Washington 98195
l Abstract J We describe an assay for measuring cocaine and benzoylecgonine in meconlum of Infants born to mothers suspected of using cocaine during their pregnancy. The assay involves the use of fluorescence polarization immunoassay (FPIA) to screen for benzoylecgonine in a methanolic extract of the meconlum. The FPIA is sensitive to 0.6 I~g benzoylecgonlne per gram meconium. Confirmation of the presence (or absence) of benzoylecgonine and cocaine in meconium samples was performed by solid phase extraction of a second methanolic extract of the meconium, derivatizing using BSTFA, followed by a gas chromatographic/mass spectrometric (GC/MS) analysis, which can detect both cocaine and benzoylecgonine. The GC/MS confirmation was sensitive to less than 0.25 pg cocaine or 0.5 I~g benzoylecgonine per gram meconlum. FPIA, which is commonly used in many toxicology laboratories, is advantageous because it precludes the need to use radioimmunoassays for the initial screen. The confirmation step provides greater certainty for the presence of cocaine and/or benzoylecgonine in meconium.
reprisal when use is admitted (3). Further, when a mother does report substance use, the accuracy of information about the timing of fetal exposure is often subject to poor recall. Unless the fetal exposure to the drug occurred two to three days prior to delivery, neonatal or maternal urine drug screens are often negative (3). A screen for drugs of abuse has been described (7-9) in which radioimmunoassays (RIA) were used for the detection of drugs in the meconium of infants born to drug-using mothers. We describe here an analysis for passive, gestational exposure to cocaine, including screening and confirmation steps. This work has been previously presented in a poster session at the 42nd National Meeting of the American Association of Clinical Chemistry (10, 11). Fluorescence polarization immunoassay (FPIA) is used to screen the meconium for benzoylecgonine (BEG/cocaine metabolite). For this study, all samples were then analyzed for benzoylecgonine and/or cocaine using a gas chromatographic/mass spectrometric (GC/MS) technique involving a second methanolic extraction of cocaine and benzoylecgonine from the meconium, a solid-phase extraction and preconcentration step, and derivatization of the benzoylecgonine with BSTFA. While the immunoassay's sensitivity is somewhat less than a previous report (9), the advantages of this method include the fact that the assay does not require the use of radioisotopes, FPIA, commonly available in many clinical laboratories, is used, and a confirmation step is employed to increase the validity and utility of a positive result.
Introduction
There is growing concern in the medical community over the maternal use of illicit drugs during pregnancy (1). In one study, approximately 11% of women studied in 36 hospitals had used illicit drugs during pregnancy (2). Deleterious effects of maternal use of these drugs, in particular cocaine, during pregnancy have been noted (1, 3-6), including decreased birth weight (3--6), decreased head circumference (3-6), decreased gestational duration (4,6), abruptio placenta (5), meconium staining of amniotic fluid (6), congenital defects (3,6), and in some cases emergent medical problems at birth. Assessing the extent of maternal drug abuse during pregnancy is difficult. Maternal history is often suspect because of fear of
9 Address correspondence to this author at: SmithKline BeechamClinical Laboratory, 1737 Airport Way South, Suite 200, Seattle,Washington 98134. r Current Address: Veterans Administration Medical Center, LaboratoryService (113), 5901 E. 7th St., Long Beach, California 90822.
M a t e r i a l s and M e t h o d s
Apparatus. We used an Abbott TDx and reagents (calibrators, reagents, and control kits) for the fluorescence polarization immunoassay of BEG in meconium (Abbott Labs). Bond Elut Certify columns and a Vac-Elut vacuum system (Analytichem) were used for solid phase extraction. We used a Syva ETS and Syva EMIT reagents, including calibrators, controls, and reagents, for the enzyme multiplied immunoassay of benzoylecgonine in urine (Syva). We used a GC/MS system consisting of a Model 5890 gas chromatograph and a Model 5970 mass spectrometer, both from Hewlett-Packard. An HP-5 methylphenyl capillary column was also used. Chromatography was performed isothermally at 260~ The effluent was monitored using selective ion monitoring (SIM) of the following ions (m/z): 182, 272, and 303 (cocaine); 240, 346,
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
261
Journal of Analytical Toxicology, Vol. 16, July/August 1992
and 361 (trimethylsilyl derivative of benzoylecgonine); and 243, 349, and 364 (trimethylsilyl derivative of da-benzoylecgonine). Reagents. N,O-bis(Trimethylsilyl)trifluoroacetamide(BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from Pierce. Ammonium hydroxide (30%) was obtained from Baker. Benzoylecgonine tetrahydrate (BEG) and cocaine (COC) were obtained from from Alltech. Deuterated benzoylecgonine (d 3BEG) (3-benzoyloxy-8-[methyl-d3-azabicyclo[3.2.1]-octane-2carboxylic acid tetrahydrate) was obtained from Sigma Chemical. All other reagents were nanograde, unless otherwise noted. Isopropyl alcohol, methanol, and methylene chloride were obtained from Mallinckrodt. Samples and standards. Standard solutions in concentrations of 1 lag/mL were prepared in methanol and were used for the preparation of the positive meconium controls. Control samples. Meconium samples were prepared as controis by adding the appropriate amount of the stock drug solution. Pooled meconium from infants whose mothers reported no gestational use of cocaine was used as negative control material for beth the screening assay and confirmation assay. This material was analyzed using both the immunoassay and GC/MS and confirmed to be free of cocaine or metabolites. These negative meconium samples were also used as starting material for the positive controls. The immunoassay uses a single positive control containing 0.6 lag/g BEG. Two similar controls were prepared for each GC/MS determination. The BEG and cocaine concentrations in the low and high control were 0.3 and 1 lag/g, respectively. Meconium samples. Meconium samples were obtained from infants at three Seattle-area hospitals with approval from the human subjects review boards. Meconium was collected from infants whose mothers consented to participate in a clinical follow-up study on the neurological effects of in-utero cocaine exposure. Post-partum mothers participated by answering a questionnaire and being interviewed about their drug use during pregnancy. According to the interviews, 41 women were self-reported cocaine users during their pregnancy, of which 33 acknowledged use or passive exposure during the second and/or third trimester. One additional patient was suspected of cocaine use during the final trimester of her pregnancy. Eighteen women were included who denied use of cocaine during pregnancy. A total of 59 samples were collected. One meconium sample was collected by the nursery staff from each infant in the study within the first three days of life. The samples were submitted to the laboratory, which froze them on receipt (to allow easier handling of the samples). lmmunoassay screening. Meconium samples were screened for the presence of benzoylecgonine. A 0.5-g sample of the meconium was suspended in 5 mL methanol. The sample was vortexed for two minutes to homogenize the sample. The sample was then centrifuged at 500 • g for l0 min with the supernatant filtered through Whatman grade 615 paper. The methanol solvent was evaporated to less than 1 mL at 40~ under a stream of air. Following reconstitution with 0.1M phosphate buffer, pH 7.4, the sample was again centrifuged at 9,500 x g for 4 min. The samples were analyzed on the TDx along with a control meconium and TDx positive control material. The TDx can detect BEG down to 100 tag/L, whereas the cross-reactivity for cocaine with the assay is 0.8%. Urine samples were screened for the presence of BEG with Syva EMIT reagents and the Syva ETS Immunoanalyzer. The screening cutoff in urine for benzoylecgonine is 300 lag/L. GC/MS confirmation. A second sample was prepared for GC/MS analysis by suspending 0.5-0.75 g meconium in 5 mL methanol and vortex mixing for 5 min. Deuterated BEG was
262
added to the sample to a concentration of 1 p.g/g. The sample was centrifuged at 500 • g for 10 minutes. The methanolic supernatant was removed and evaporated to dryness at 40~ under a stream of air. Five mL of 0.1M phosphate buffer, pH 6.0, was used to resuspend the sample, which was then added to a Bond-Elut Certify solid phase extraction (SPE) column (preconditioned sequentially with methanol and 0.1M phosphate buffer [pH 6.0]). The column with adsorbed sample was sequentially washed with water, 0.1M HCI, and methanol. The cocaine and BEG were eluted with 2 mL methylene chloride-isopropyl alcohol (80:20) with 2% ammonium hydroxide. The eluent was dried at 40~ under a stream of air and resuspended in 0.5 mL methylene chloride, which was evaporated again (to remove any trace of water or methanol). The sample was suspended in 25 [xL BSTFA (with 1% TMCS) and heated at 60-70~ for 15 min to form a trimethylsilyl derivative of benzoylecgonine (12). Two I~L of this solution was injected into an HP GC/MS operating in the SIM mode.
Results and Discussion Immunoasny screen
The analytical sensitivity of the technique was determined by adding BEG to the negative control meconium. The analytical sensitivity of FPIA was found to be 0.6 lag BEG per gram fresh weight of meconium. The urine sensitivity is 300 tag/L. The method of Ostrea (8,9) employed an acid extraction. This method was attempted by us initially. However, analytical sensitivity was very poor. Positive results by FPIA (and GC/MS) were obtained only at high (5 mg/g and greater) BEG concentrations. The use of methanol for extraction proved to be the best solvent for optimizing benzoylecgonine recovery for FPIA. When compared with urine, the recovery from a spiked meconium sample was 61%. This led to a concentration cutoff of 0.6 lag/g.
GC/MS confirmation The chromatogram of a positive sample (Figure 1) demonstrated baseline resolution of cocaine and the trimethylsilyl derivative of benzoylecgonine with retention times of 5.91 and 6.77 min, respectively. Additionally, the chromatograms were clean and free of interferences. The sensitivity of the assay was determined by adding cocaine and BEG to negative meconium. The assay was found to be sensitive to less than 0.25 ktg/g cocaine and 0.5 lag/g BEG. Further dilution was not performed, given the sensitivity of the screen to 0.6 lag/g. The prechromatographic extraction efficiency was checked by adding the same amount of cocaine and BEG stock solution to blank meconium and to an empty test tube. The meconium was then subjected to methanolic extraction and solid phase extraction. The cocaine and BEG solution added to the clean test tube was evaporated to dryness at 40~ under a stream of air. Both samples were then reacted with BSTFA (1% TMCS) and analyzed by GC/MS. The extraction efficiency for cocaine was 99%, whereas the extraction efficiency for BEG was 30%. The linearity of the GC/MS assay for cocaine and BEG was found to be linear to 10 lag/g with regression coefficients, r 2, of 0.99 and 0.97, respectively. The within-run precision for 5 I.tg/g COC and BE was found to be 8.1% and 11.2% respectively. FPIA and GC/MS were used to analyze meconium from 59
Journal of AnalyticalToxicology,Vol. 16, July/August1992
samples. The 34 samples from cocaine-exposed infants were measured by FPIA, with 18 positive for BEG and cocaine for a sensitivity o f 52%. GC/MS analysis was performed on 27 samples, yielding an analytical sensitivity of 74% (20/27) for BEG. Cocaine was found in each sample positive for BEG. Cocaine was not found alone. Seven samples had insufficient meconium for GC/MS analysis. Infant urinalysis on 29 samples yielded an analytical sensitivity of 38% (11/29). The GC/MS assay is apparently more sensitive than the FPIA screen. The FPIA was, in turn, more sensitive than the infant BEG urinalysis. Further work is underway to study the efficacy of the use of meconium to determine fetal exposure to cocaine (13). Fluorescence polarization immunoassay, GC/MS confirmation, and maternal and infant hair analysis for cocaine will be compared with maternal interview. However, at this juncture, it can be said that the use of meconium may be useful for the determination of gestational exposure. The advantages of tiffs approach include the use of a nonradioactive immunoassay for the analysis of BEG in meconium, the use of GC/MS for confirmation, and the use of a technology (FPIA) that is available in many toxicology laboratories. Another theoretical advantage is that meconium begins to accumulate in the fetus at approximately 16 weeks gestation (14). Therefore, the "window" of drug exposure that can be detected by a toxicology screen is about 20 weeks, compared with a 3-5 day window of detection for a urine drug abuse screen.
4000 I 8 2 i 5aOO~ 3000 272 3/~3 2090 227 S '] " g I 5" ~ " 3r 2c10 t2~ 24QMI,z20 c~ 28R 3~m 32~I 34D 9s |/Oh m~'.p..~
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.
Figure 1. Total ion chromatogramand selectiveion monitoringspectrum of cocaine(top) and benzoylecg0nine(bottom) of a sampleof an infant who wasexposedto cocainein utero.
Acknowledgments Fellowships from the Department of Laboratory Medicine for GDC and IBR are gratefully acknowledged. This research was funded in part by the Alcoholism and Drug Abuse Institute, Seattle, Washington, and the National Institute on Drug Abuse grant number R01 DA05365 to Ann P. Streissguth. We greatly appreciate both the technical assistance of the medical technologists at Harborview Medical Center and the cooperation of the nursing staff at the University of Washington Medical Center, Valley Medical Center, and Providence Medical Center.
References 1. 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) 2. I.J. Chasnoff. Drug use and women: Establishing a standard of care. Ann. New York Acad. Sci. 562:208-10 (1989). 3. B. Zuckerman, D.A. Frank, R. Hingson, H. Amaro, S.M. Levenson, H. Kayne, S. Parker, R. Vinci, K. Aboagy, K.E. Fried, H. Cabral, R. Timperi, and H. Bauchner. Effects of maternal marijuana and cocaine use on fetal growth. N. Engl. J. Med. 320: 762-68 (1989) 4. A.S. Oro and S.D. Dixon. Perinatal cocaine and methamphetamine exposure: Maternal and neonatal correlates. J. Pediatr. 114:571-78 (1987). 5. A.J. Hadeed and A.J. Siegel. Maternal cocaine use during pregnancy: Effect on the newbom infant. Pediatrics 84:205-10 (1989) 6. I.J. Chasnoff, D.E. Lewis, D.R. Griffith, and S. Willey. Cocaine and pregnancy: Clinical and toxicological implications for the neonate. Clin. Chem. 35:1276-78 (1989). 7. E.M. Ostrea, D. Ascensio, A. Naluz, K. Simkowski, M.G. Subramanian, and E. Abel. The detection of heroin, cocaine and cannabinoid metabolites in the stools of infants of drug dependent mothers. Pediatr. Res. 21:240 (1987). 8. E.M. Ostrea, P.M. Parks, and M.J. Brady. Rapid isolation and detection of drugs in meconium of drug dependent mothers. Clin. Chem. 34:2372-73 (1988) 9. E.M. Ostrea, M.J. Brady, P.M. Parks, D.C. Asensio, and A. Naluz. Drug screening of meconium in infants of drug-dependent mothers: An alternative to urine testing. J. Pediatrics 115: 47477 (1989). 10. G.D. Clark, I. Bruce Rosenzweig, and V.A. Raisys. Analysis of cocaine and benzoylecgonine in meconium of infants born to cocaine dependent mothers. Clin. Chem. 36:1022 (1990). 11. I.B. Rosenzweig, G.D. Clark, N.J. Thiersch, and V.A. Raisys. Neonatal drugs of abuse screening in meconium, a comparison between Abbott TDx and Syva ETS. Clin. Chem. 36:1023 (t990). 12. J. Drozd. Chemical Derivatization in Gas Chromatography, Vo119 in Journal of Chromatography Library, Elsevier, New York, 1981, pp. 69-73. 13. C.M. Callahan, T.M. Grant, P. Phipps, G.D. Clark, A.H. Novack, A.P. Streissguth, and V.A. Raisys. Measurement of gestational cocaine exposure. J. Amer. Med. Assoc. In press. 14. R.E. Behrman and V.C. Vaughan. In Nelson Textbook of Pediatrics, 13th ed., Saunders, Philadelphia, 1987, p. 7. Manuscript received July 2, 1991; revision received December 30, 1991.
263
The Analysisof Cocaineand Benzoylecgoninein Mec0nium G r e g o r y D. Clark*, I. B r u c e R o s e n z w e i g t , and V i d m a n t a s A. R a i s y s
Department of Laboratory Medicine, SB-IO, University of Washington, Seattle, Washington 98195 C h a r l e s M. C a l l a h a n
Department of Pediatrics, RD-20, University of Washington, Seattle, Washington 98195 T h e r e s e M. Grant and A n n P. S t r e i s s g u t h
Department of Psychiatry and Behaviora/ Sciences, GG-20, University of Washington, Seattle, Washington 98195
l Abstract J We describe an assay for measuring cocaine and benzoylecgonine in meconlum of Infants born to mothers suspected of using cocaine during their pregnancy. The assay involves the use of fluorescence polarization immunoassay (FPIA) to screen for benzoylecgonine in a methanolic extract of the meconlum. The FPIA is sensitive to 0.6 I~g benzoylecgonlne per gram meconium. Confirmation of the presence (or absence) of benzoylecgonine and cocaine in meconium samples was performed by solid phase extraction of a second methanolic extract of the meconium, derivatizing using BSTFA, followed by a gas chromatographic/mass spectrometric (GC/MS) analysis, which can detect both cocaine and benzoylecgonine. The GC/MS confirmation was sensitive to less than 0.25 pg cocaine or 0.5 I~g benzoylecgonine per gram meconlum. FPIA, which is commonly used in many toxicology laboratories, is advantageous because it precludes the need to use radioimmunoassays for the initial screen. The confirmation step provides greater certainty for the presence of cocaine and/or benzoylecgonine in meconium.
reprisal when use is admitted (3). Further, when a mother does report substance use, the accuracy of information about the timing of fetal exposure is often subject to poor recall. Unless the fetal exposure to the drug occurred two to three days prior to delivery, neonatal or maternal urine drug screens are often negative (3). A screen for drugs of abuse has been described (7-9) in which radioimmunoassays (RIA) were used for the detection of drugs in the meconium of infants born to drug-using mothers. We describe here an analysis for passive, gestational exposure to cocaine, including screening and confirmation steps. This work has been previously presented in a poster session at the 42nd National Meeting of the American Association of Clinical Chemistry (10, 11). Fluorescence polarization immunoassay (FPIA) is used to screen the meconium for benzoylecgonine (BEG/cocaine metabolite). For this study, all samples were then analyzed for benzoylecgonine and/or cocaine using a gas chromatographic/mass spectrometric (GC/MS) technique involving a second methanolic extraction of cocaine and benzoylecgonine from the meconium, a solid-phase extraction and preconcentration step, and derivatization of the benzoylecgonine with BSTFA. While the immunoassay's sensitivity is somewhat less than a previous report (9), the advantages of this method include the fact that the assay does not require the use of radioisotopes, FPIA, commonly available in many clinical laboratories, is used, and a confirmation step is employed to increase the validity and utility of a positive result.
Introduction
There is growing concern in the medical community over the maternal use of illicit drugs during pregnancy (1). In one study, approximately 11% of women studied in 36 hospitals had used illicit drugs during pregnancy (2). Deleterious effects of maternal use of these drugs, in particular cocaine, during pregnancy have been noted (1, 3-6), including decreased birth weight (3--6), decreased head circumference (3-6), decreased gestational duration (4,6), abruptio placenta (5), meconium staining of amniotic fluid (6), congenital defects (3,6), and in some cases emergent medical problems at birth. Assessing the extent of maternal drug abuse during pregnancy is difficult. Maternal history is often suspect because of fear of
9 Address correspondence to this author at: SmithKline BeechamClinical Laboratory, 1737 Airport Way South, Suite 200, Seattle,Washington 98134. r Current Address: Veterans Administration Medical Center, LaboratoryService (113), 5901 E. 7th St., Long Beach, California 90822.
M a t e r i a l s and M e t h o d s
Apparatus. We used an Abbott TDx and reagents (calibrators, reagents, and control kits) for the fluorescence polarization immunoassay of BEG in meconium (Abbott Labs). Bond Elut Certify columns and a Vac-Elut vacuum system (Analytichem) were used for solid phase extraction. We used a Syva ETS and Syva EMIT reagents, including calibrators, controls, and reagents, for the enzyme multiplied immunoassay of benzoylecgonine in urine (Syva). We used a GC/MS system consisting of a Model 5890 gas chromatograph and a Model 5970 mass spectrometer, both from Hewlett-Packard. An HP-5 methylphenyl capillary column was also used. Chromatography was performed isothermally at 260~ The effluent was monitored using selective ion monitoring (SIM) of the following ions (m/z): 182, 272, and 303 (cocaine); 240, 346,
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
261
Journal of Analytical Toxicology, Vol. 16, July/August 1992
and 361 (trimethylsilyl derivative of benzoylecgonine); and 243, 349, and 364 (trimethylsilyl derivative of da-benzoylecgonine). Reagents. N,O-bis(Trimethylsilyl)trifluoroacetamide(BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from Pierce. Ammonium hydroxide (30%) was obtained from Baker. Benzoylecgonine tetrahydrate (BEG) and cocaine (COC) were obtained from from Alltech. Deuterated benzoylecgonine (d 3BEG) (3-benzoyloxy-8-[methyl-d3-azabicyclo[3.2.1]-octane-2carboxylic acid tetrahydrate) was obtained from Sigma Chemical. All other reagents were nanograde, unless otherwise noted. Isopropyl alcohol, methanol, and methylene chloride were obtained from Mallinckrodt. Samples and standards. Standard solutions in concentrations of 1 lag/mL were prepared in methanol and were used for the preparation of the positive meconium controls. Control samples. Meconium samples were prepared as controis by adding the appropriate amount of the stock drug solution. Pooled meconium from infants whose mothers reported no gestational use of cocaine was used as negative control material for beth the screening assay and confirmation assay. This material was analyzed using both the immunoassay and GC/MS and confirmed to be free of cocaine or metabolites. These negative meconium samples were also used as starting material for the positive controls. The immunoassay uses a single positive control containing 0.6 lag/g BEG. Two similar controls were prepared for each GC/MS determination. The BEG and cocaine concentrations in the low and high control were 0.3 and 1 lag/g, respectively. Meconium samples. Meconium samples were obtained from infants at three Seattle-area hospitals with approval from the human subjects review boards. Meconium was collected from infants whose mothers consented to participate in a clinical follow-up study on the neurological effects of in-utero cocaine exposure. Post-partum mothers participated by answering a questionnaire and being interviewed about their drug use during pregnancy. According to the interviews, 41 women were self-reported cocaine users during their pregnancy, of which 33 acknowledged use or passive exposure during the second and/or third trimester. One additional patient was suspected of cocaine use during the final trimester of her pregnancy. Eighteen women were included who denied use of cocaine during pregnancy. A total of 59 samples were collected. One meconium sample was collected by the nursery staff from each infant in the study within the first three days of life. The samples were submitted to the laboratory, which froze them on receipt (to allow easier handling of the samples). lmmunoassay screening. Meconium samples were screened for the presence of benzoylecgonine. A 0.5-g sample of the meconium was suspended in 5 mL methanol. The sample was vortexed for two minutes to homogenize the sample. The sample was then centrifuged at 500 • g for l0 min with the supernatant filtered through Whatman grade 615 paper. The methanol solvent was evaporated to less than 1 mL at 40~ under a stream of air. Following reconstitution with 0.1M phosphate buffer, pH 7.4, the sample was again centrifuged at 9,500 x g for 4 min. The samples were analyzed on the TDx along with a control meconium and TDx positive control material. The TDx can detect BEG down to 100 tag/L, whereas the cross-reactivity for cocaine with the assay is 0.8%. Urine samples were screened for the presence of BEG with Syva EMIT reagents and the Syva ETS Immunoanalyzer. The screening cutoff in urine for benzoylecgonine is 300 lag/L. GC/MS confirmation. A second sample was prepared for GC/MS analysis by suspending 0.5-0.75 g meconium in 5 mL methanol and vortex mixing for 5 min. Deuterated BEG was
262
added to the sample to a concentration of 1 p.g/g. The sample was centrifuged at 500 • g for 10 minutes. The methanolic supernatant was removed and evaporated to dryness at 40~ under a stream of air. Five mL of 0.1M phosphate buffer, pH 6.0, was used to resuspend the sample, which was then added to a Bond-Elut Certify solid phase extraction (SPE) column (preconditioned sequentially with methanol and 0.1M phosphate buffer [pH 6.0]). The column with adsorbed sample was sequentially washed with water, 0.1M HCI, and methanol. The cocaine and BEG were eluted with 2 mL methylene chloride-isopropyl alcohol (80:20) with 2% ammonium hydroxide. The eluent was dried at 40~ under a stream of air and resuspended in 0.5 mL methylene chloride, which was evaporated again (to remove any trace of water or methanol). The sample was suspended in 25 [xL BSTFA (with 1% TMCS) and heated at 60-70~ for 15 min to form a trimethylsilyl derivative of benzoylecgonine (12). Two I~L of this solution was injected into an HP GC/MS operating in the SIM mode.
Results and Discussion Immunoasny screen
The analytical sensitivity of the technique was determined by adding BEG to the negative control meconium. The analytical sensitivity of FPIA was found to be 0.6 lag BEG per gram fresh weight of meconium. The urine sensitivity is 300 tag/L. The method of Ostrea (8,9) employed an acid extraction. This method was attempted by us initially. However, analytical sensitivity was very poor. Positive results by FPIA (and GC/MS) were obtained only at high (5 mg/g and greater) BEG concentrations. The use of methanol for extraction proved to be the best solvent for optimizing benzoylecgonine recovery for FPIA. When compared with urine, the recovery from a spiked meconium sample was 61%. This led to a concentration cutoff of 0.6 lag/g.
GC/MS confirmation The chromatogram of a positive sample (Figure 1) demonstrated baseline resolution of cocaine and the trimethylsilyl derivative of benzoylecgonine with retention times of 5.91 and 6.77 min, respectively. Additionally, the chromatograms were clean and free of interferences. The sensitivity of the assay was determined by adding cocaine and BEG to negative meconium. The assay was found to be sensitive to less than 0.25 ktg/g cocaine and 0.5 lag/g BEG. Further dilution was not performed, given the sensitivity of the screen to 0.6 lag/g. The prechromatographic extraction efficiency was checked by adding the same amount of cocaine and BEG stock solution to blank meconium and to an empty test tube. The meconium was then subjected to methanolic extraction and solid phase extraction. The cocaine and BEG solution added to the clean test tube was evaporated to dryness at 40~ under a stream of air. Both samples were then reacted with BSTFA (1% TMCS) and analyzed by GC/MS. The extraction efficiency for cocaine was 99%, whereas the extraction efficiency for BEG was 30%. The linearity of the GC/MS assay for cocaine and BEG was found to be linear to 10 lag/g with regression coefficients, r 2, of 0.99 and 0.97, respectively. The within-run precision for 5 I.tg/g COC and BE was found to be 8.1% and 11.2% respectively. FPIA and GC/MS were used to analyze meconium from 59
Journal of AnalyticalToxicology,Vol. 16, July/August1992
samples. The 34 samples from cocaine-exposed infants were measured by FPIA, with 18 positive for BEG and cocaine for a sensitivity o f 52%. GC/MS analysis was performed on 27 samples, yielding an analytical sensitivity of 74% (20/27) for BEG. Cocaine was found in each sample positive for BEG. Cocaine was not found alone. Seven samples had insufficient meconium for GC/MS analysis. Infant urinalysis on 29 samples yielded an analytical sensitivity of 38% (11/29). The GC/MS assay is apparently more sensitive than the FPIA screen. The FPIA was, in turn, more sensitive than the infant BEG urinalysis. Further work is underway to study the efficacy of the use of meconium to determine fetal exposure to cocaine (13). Fluorescence polarization immunoassay, GC/MS confirmation, and maternal and infant hair analysis for cocaine will be compared with maternal interview. However, at this juncture, it can be said that the use of meconium may be useful for the determination of gestational exposure. The advantages of tiffs approach include the use of a nonradioactive immunoassay for the analysis of BEG in meconium, the use of GC/MS for confirmation, and the use of a technology (FPIA) that is available in many toxicology laboratories. Another theoretical advantage is that meconium begins to accumulate in the fetus at approximately 16 weeks gestation (14). Therefore, the "window" of drug exposure that can be detected by a toxicology screen is about 20 weeks, compared with a 3-5 day window of detection for a urine drug abuse screen.
4000 I 8 2 i 5aOO~ 3000 272 3/~3 2090 227 S '] " g I 5" ~ " 3r 2c10 t2~ 24QMI,z20 c~ 28R 3~m 32~I 34D 9s |/Oh m~'.p..~
~ |00Bm soQe
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Figure 1. Total ion chromatogramand selectiveion monitoringspectrum of cocaine(top) and benzoylecg0nine(bottom) of a sampleof an infant who wasexposedto cocainein utero.
Acknowledgments Fellowships from the Department of Laboratory Medicine for GDC and IBR are gratefully acknowledged. This research was funded in part by the Alcoholism and Drug Abuse Institute, Seattle, Washington, and the National Institute on Drug Abuse grant number R01 DA05365 to Ann P. Streissguth. We greatly appreciate both the technical assistance of the medical technologists at Harborview Medical Center and the cooperation of the nursing staff at the University of Washington Medical Center, Valley Medical Center, and Providence Medical Center.
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