DETERMINATION OF COCAINE AND ITS METABOLITES IN SPECIMENS OF NEONATAL AND MATERNAL ORIGIN By RUTH ELLEN WINECKER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996 dedicate this work to my husband, John. See, told you we would make I it TOGETHER! ACKNOWLEDGMENTS I would like to thank my doctoral committee. First and foremost, I would like to thank Roger L. Bertholf, Ph.D., for his guidance and enthusiasm during the past four and a half years! He introduced me to the world of clinical chemistry and toxicology with its mix of medicine and analytical chemistry, which I have found to be rewarding, exciting, and fulfilling. For this, I will be forever grateful. Thanks also go to Ian Tebbett, Ph.D., for allowing me to work on his cocaine project and without which this dissertation would not be possible. To Bruce A. Goldberger, Ph.D., and Catherine Hammett-Stabler, Ph.D., thanks for assuming the roles of "Honorary Gainesville Mentors;" you provided me with invaluable advice, you were always there when needed you, and your I guidance has been right on target! Thanks also go to Richard A. Yost, Ph.D., for keeping me on the analytical straight and narrow. My appreciation also goes to the following folks: Dr. Goldberger's lab group; in particular Jeri Ropero, Dr. Diana Garside, Abraham Tsadik, and Zafer Sabawi for all their help and encouragement; Dr. Tebbett's lab group, in particular, Diane Phillips and Siya, for teaching me HPLC. in My gratitude to Robert C. Hart, John's boss, for allowing John the time off from work to help me. Finally, I would like to thank the National Institutes of Health, and the Division of Sponsored Research at the University of Florida for funding my research. IV TABLE OF CONTENTS ACKNOWLEDGMENTS jjj ABSTRACT vii CHAPTERS INTRODUCTION 1. 1 Pharmacology, Metabolism, and the Addictive Nature of Cocaine 3 Maternal-Fetal Circulation and Fetal Exposure to Cocaine n Adverse Effects Associated with Cocaine Use 13 Scope of the Project 18 Scope of the Dissertation 21 2. UTILITY OF MATERNAL HAIR FOR DETERMINING PRENATAL COCAINE EXPOSURE 23 Introduction 23 Materials and Methods 30 Results 35 Discussion 52 QUANTITATIVE DETERMINATION OF COCAINE AND 3. ITS METABOLITES IN AMNIOTIC FLUID, COLOSTRUM, AND UMBILICAL CORD 59 Introduction 59 Materials and Methods 63 Results 67 Discussion 87 4. URINE VS. MECONIUM: MORE FUEL FOR THE FIRE 93 Introduction 93 Materials and Methods 96 Results 103 Discussion 127 5. IS THERE A "GOLD STANDARD" FOR DETERMINING PRENATAL COCAINE EXPOSURE 135 Introduction 135 Materials and Methods 137 Results and Discussion 137 CONCLUSIONS AND FUTURE WORK 6. 151 APPENDIX GC/MS CALIBRATION CURVES 155 I: APPENDIX HPLC CALIBRATION CURVES 177 II: REFERENCES 182 BIOGRAPHICAL SKETCH 193 VI Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DETERMINATION OF COCAINE AND ITS METABOLITES IN SPECIMENS OF NEONATAL AND MATERNAL ORIGIN By Ruth Ellen Winecker December, 1996 Chairperson: Dr. Roger L. Bertholf Major Department: Pathology The increased use of cocaine by women of childbearing 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 various specimens of maternal and neonatal origin was undertaken. Urine (n=75), meconium (n=74), maternal hair (n=66), umbilical cord tissue (n=70), amniotic fluid (n=32) and colostrum (n=21) were collected from pregnant subjects admitted to labor and delivery at Shands Hospital at the University of Florida (Gainesville, FL). Drug use status was ascertained in a confidential maternal interview. Subjects reporting cocaine use were designated as target subjects and those denying use as control subjects. VII Meconium and urine specimens were screened by immunoassay for the cocaine metabolite, benzoylecgonine. All specimens were subjected to solid- phase extraction and analyzed for cocaine and its metabolites by high performance liquid chromatography (HPLC) and/or gas chromatography/mass spectrometry (GC/MS). The percentage of positive specimens for each subject type is presented in Table A-1. Table A-1. Percentage of positive specimens in target and control subjects. Subject Hair Colostrum Amniotic Meconium Urine Cord Fluid Tissue Target (%) 73 60 54 50 48 43 Control (%) 12 11 9 9 2 Statistical analysis was undertaken to determine if any of these specimens is more likely to detect prenatal cocaine exposure. Results indicate that hair is more likely than umbilical cord tissue to detect prenatal cocaine exposure, but was equal in ability to all other specimens. Moreover, maternal hair may have a greater ability to detect prenatal cocaine exposure but, more studies, with larger numbers of subjects, are needed for results to attain statistical significance. In conclusion, this study has shown that no single specimen is ideal for the identification of cocaine exposed infants for the purposes of classification as exposed or non-exposed in health outcome research. This will continue to be a major stumbling block in studies investigating the adverse effects of prenatal cocaine exposure. VIII CHAPTER 1 INTRODUCTION Cocaine is the principal alkaloid obtained from leaves of the coca tree, Erythroxylon coca, and is the only known naturally occurring local anesthetic. Archaeologists and historians have chronicled the use of the coca tree for medicinal, religious, and stimulatory purposes at least 15 centuries.1 Unlike the ancient Incas, who chewed the coca leaves with ash achieving a slow absorption of cocaine, today's user is more likely to ingest more concentrated and highly addictive forms of the drug, such as cocaine powder or crack cocaine.1,2 In fact, modern forms of cocaine use produce a more intense "high" and a stronger 13 "craving" for repeated use. While cocaine was introduced to the United States as a general anesthetic and used in some wine and soft drink preparations during the early to mid 1800s, it became clear by 1900 that the use of cocaine could have serious adverse side effects, including a high potential for abuse. Today cocaine is classified as a Schedule II drug, recognized for its high abuse potential and limited medical usefulness.1 Despite increased regulation of its use and a highly publicized governmental "war on drugs," the abuse of cocaine, especially the preparation 1 known as crack, continues to increase.4,5 'Crack" is a freebase form of cocaine that can be smoked because of its stability at temperatures required for vaporization, and is sold in vials that contain one or more "rocks." Crack cocaine first appeared in the United States in 1986 and brought with it a significant increase in the average dose, resulting in an increase in cocaine related morbidity and mortality.6 The low price, ease of use, and availability have contributed to what many researchers characterize as an explosion of cocaine use in the last decade.3,7 The intense euphoria within one minute of smoking crack, followed by the swift inactivation and elimination of the drug from circulation, may contribute to its highly addictive nature and explain the trend away from casual use toward abuse. In fact, while the number of current users (current use is defined by the Substance Abuse and Mental Health Services Administration (SAMHSA) as use of the drug within the past thirty days) of cocaine has declined from 5.8 million in 1985 to 1.9 million in 1991, the number of daily users has increased from 246,000 to 336,000 over the same period.2,8 Importantly, drug usage surveys indicate that the heaviest users of cocaine are between 18 and 30 years of age, which in females, are peak childbearing years.9, 10, 11 The lifestyle of many of these young adult female cocaine abusers includes drug binges in "crack houses," where prostitution is 9,12 also prevalent. Naturally, this has resulted in an increasing number of infants being exposed to cocaine in utero. Estimates of the prevalence of cocaine