laboratory methods for detecting prenatal exposure to cocaine roger l. bertholf, ph.d. associate...

Laboratory Methods for Detecting Prenatal Exposure to Cocaine

Laboratory Methods for Detecting Prenatal Exposure to Cocaine

Roger L. Bertholf, Ph.D.Associate Professor of Pathology

Chief of Clinical Chemistry & Toxicology

University of Florida College of Medicine

Health Science Center/Jacksonville

Roger L. Bertholf, Ph.D.Associate Professor of Pathology

Chief of Clinical Chemistry & Toxicology

University of Florida College of Medicine

Health Science Center/Jacksonville

Why test babies for cocaine?Why test babies for cocaine?

• Prevalence of maternal cocaine use

• Effect of in utero cocaine exposure on fetal and postnatal development

• Medical/social/legal interventions

• Prevalence of maternal cocaine use

• Effect of in utero cocaine exposure on fetal and postnatal development

• Medical/social/legal interventions

Prevalence of maternal cocaine usePrevalence of maternal cocaine use

• 1998 Household Survey revealed that 1.8 million Americans use cocaine (0.8% of population >12 years old)– 2% of 18-25 year-olds; 1.2% of 26-35 year-olds– 0.5% of women are cocaine users

• Estimates of the prevalence of cocaine use during pregnancy range from less than 2% to more than 15%

• Reasons for variations– Geographic/socioeconomic/racial– Methods used to detect use

• 1998 Household Survey revealed that 1.8 million Americans use cocaine (0.8% of population >12 years old)– 2% of 18-25 year-olds; 1.2% of 26-35 year-olds– 0.5% of women are cocaine users

• Estimates of the prevalence of cocaine use during pregnancy range from less than 2% to more than 15%

• Reasons for variations– Geographic/socioeconomic/racial– Methods used to detect use

Is prenatal cocaine exposure harmful?Is prenatal cocaine exposure harmful?

• Extensive literature on clinical effects of prenatal cocaine exposure

• Confounding factors are almost always present– Poor prenatal care– Concomitant drug use (alcohol, nicotine)

• Reliable animal models do not exist– Sheep– Pig– Rodent

• Extensive literature on clinical effects of prenatal cocaine exposure

• Confounding factors are almost always present– Poor prenatal care– Concomitant drug use (alcohol, nicotine)

• Reliable animal models do not exist– Sheep– Pig– Rodent

Well documented physiological effects of cocaine

Well documented physiological effects of cocaine

• Vasoconstriction– Decreases uterine blood flow in sheep– Cerebral vasodilation and hypertension in newborn sheep and pigs

• Neurotransmitter deficiencies– ACTH and corticosterone response in rats– Dopamine transporter binding in mice

• Consistent observations in cocaine exposed neonates– Intrauterine growth retardation (LBW)– Prematurity– Complications/LOS

• Vasoconstriction– Decreases uterine blood flow in sheep– Cerebral vasodilation and hypertension in newborn sheep and pigs

• Neurotransmitter deficiencies– ACTH and corticosterone response in rats– Dopamine transporter binding in mice

• Consistent observations in cocaine exposed neonates– Intrauterine growth retardation (LBW)– Prematurity– Complications/LOS

Does cocaine cross the placenta?Does cocaine cross the placenta?

• Studies in pregnant sheep and rodents indicate that both cocaine and its metabolites cross the placenta– Fetal levels approximately 15% of maternal

• There is also evidence that the placenta has AChE activity– May have a protective effect

• Placental transfer of cocaine does not appear to be flow-limited• Cocaine can be measured in the amniotic fluid, but there is

disagreement over whether it originates solely from fetal urine

• Studies in pregnant sheep and rodents indicate that both cocaine and its metabolites cross the placenta– Fetal levels approximately 15% of maternal

• There is also evidence that the placenta has AChE activity– May have a protective effect

• Placental transfer of cocaine does not appear to be flow-limited• Cocaine can be measured in the amniotic fluid, but there is

disagreement over whether it originates solely from fetal urine

Other sources of cocaine exposureOther sources of cocaine exposure

• Breast Milk

• Passive inhalation

• Intentional/accidental ingestion

• Breast Milk

• Passive inhalation

• Intentional/accidental ingestion

Detecting prenatal cocaine exposure: Analytical considerations

Detecting prenatal cocaine exposure: Analytical considerations

• Cocaine metabolism

• Quantitative thresholds

• Choice of specimen

• Cocaine metabolism

• Quantitative thresholds

• Choice of specimen

Cocaine metabolismCocaine metabolism

N

H3C

O

O

CH3

O

O

N

H3C

O

O

CH3

OH

N

H3C

OH

O

O

O

HN

O

O

CH3

O

O

Ecgonine methyl ester Benzoylecgonine Norcocaine

- C6H5COO

- CH3

- CH3

CocaethyleneCocaethylene

N

H3C

O

O

CH3

O

O

CH3CH2OH

CH3OH

N

H3C

O

O

CH2CH3

O

O

Quantitative thresholdsQuantitative thresholds

• For immunochemical methods, quantitative thresholds are established by calibration– Pure compound or mixture of isomers– Semi-quantitative results for real specimens

• Purely quantitative results are only available using specific methods– GC/MS– HPLC– GC

• For immunochemical methods, quantitative thresholds are established by calibration– Pure compound or mixture of isomers– Semi-quantitative results for real specimens

• Purely quantitative results are only available using specific methods– GC/MS– HPLC– GC

Detecting prenatal cocaine exposure: Methods

Detecting prenatal cocaine exposure: Methods

• Immunochemical methods– RIA, ELISA– EMIT– FPIA– CEDIA– POC

• Spectrometric methods– GC/MS– HPLC– GC

• Immunochemical methods– RIA, ELISA– EMIT– FPIA– CEDIA– POC

• Spectrometric methods– GC/MS– HPLC– GC

Immunochemical methodsImmunochemical methods

• Most are calibrated to a threshold of 300 ng benzoylecgonine per mL of urine– Corresponds to the SAMHSA specifications

– May be adapted to other specimens

• Most are designed to measure benzoylecgonine in urine– Benzoylecgonine is not the principal metabolite in all

specimens

• Most are calibrated to a threshold of 300 ng benzoylecgonine per mL of urine– Corresponds to the SAMHSA specifications

– May be adapted to other specimens

• Most are designed to measure benzoylecgonine in urine– Benzoylecgonine is not the principal metabolite in all

specimens

Chromatographic methodsChromatographic methods

• Quantitative GC/MS analysis (by isotope dilution) is considered the reference method– Sensitivity <10 ng/mL– Requires extensive specimen preparation (extraction, derivatization,

isotopically-labeled internal standards)

• HPLC has similar sensitivity to GC/MS applications, but detection is less specific– Does not require preparation of volatile derivatives

• GC is robust and sensitive, but lacks detector specificity– Nitrogen/Phosphorus detectors are very sensitive but expensive

• Quantitative GC/MS analysis (by isotope dilution) is considered the reference method– Sensitivity <10 ng/mL– Requires extensive specimen preparation (extraction, derivatization,

isotopically-labeled internal standards)

• HPLC has similar sensitivity to GC/MS applications, but detection is less specific– Does not require preparation of volatile derivatives

• GC is robust and sensitive, but lacks detector specificity– Nitrogen/Phosphorus detectors are very sensitive but expensive

Detecting prenatal cocaine exposure:Specimens

Detecting prenatal cocaine exposure:Specimens

• Maternal specimens– Urine– Amniotic fluid– Hair

• Neonatal specimens– Urine– Meconium– Hair– Nails

• Maternal specimens– Urine– Amniotic fluid– Hair

• Neonatal specimens– Urine– Meconium– Hair– Nails

Cocaine in maternal specimensCocaine in maternal specimens

• Maternal urine is easy to collect, and measurements are reliable– Usually reflects exposure within the past 2-3 days

• Uncontaminated amniotic fluid is harder to obtain– More direct measure of fetal exposure (may be substitute for screening

fetal urine)– Pharmacokinetics of cocaine and metabolites in amniotic fluid have not

been established.

• Hair is the most difficult specimen to analyze, but shows promise as a clinical specimen– Incorporation of cocaine in hair has been widely documented

• Maternal urine is easy to collect, and measurements are reliable– Usually reflects exposure within the past 2-3 days

• Uncontaminated amniotic fluid is harder to obtain– More direct measure of fetal exposure (may be substitute for screening

fetal urine)– Pharmacokinetics of cocaine and metabolites in amniotic fluid have not

been established.

• Hair is the most difficult specimen to analyze, but shows promise as a clinical specimen– Incorporation of cocaine in hair has been widely documented

Cocaine in neonatal specimensCocaine in neonatal specimens

• Urine– Sometimes difficult to collect– Can be extracted from diapers

• Meconium– More difficult to collect uncontaminated specimens– Can also be extracted from diapers

• Hair– Shows promise, but methods are not widely available

• Nails– Most data on postmortem specimens

• Urine– Sometimes difficult to collect– Can be extracted from diapers

• Meconium– More difficult to collect uncontaminated specimens– Can also be extracted from diapers

• Hair– Shows promise, but methods are not widely available

• Nails– Most data on postmortem specimens

Urine drug testing in neonatesUrine drug testing in neonates

• Typically uses immunoassays configured for workplace drug testing– Positive threshold >300 ng/mL (benzoylecgonine)

• Most studies indicate that neonatal urine drug screening at conventional thresholds fails to detect up to half of cocaine-exposed infants

• Results of neonatal urine cocaine screening usually correlate with maternal urine

• Typically uses immunoassays configured for workplace drug testing– Positive threshold >300 ng/mL (benzoylecgonine)

• Most studies indicate that neonatal urine drug screening at conventional thresholds fails to detect up to half of cocaine-exposed infants

• Results of neonatal urine cocaine screening usually correlate with maternal urine

Use of lower screening thresholdsUse of lower screening thresholds

Distribution of urinary benzoylecgonine levels in neonates

<5010%

50-15015%

150-30025%

>30050%

Distribution of urinary benzoylecgonine levels in neonates

<5010%

50-15015%

150-30025%

>30050%

Urine collection from diapersUrine collection from diapers

• Several studies have documented the correlation between chemistry and microscopic laboratory results on bag-collected and diaper-extracted urine

• Cocaine, benzoylecgonine, and ecgonine methyl ester can be extracted from diapers with methanol.

• Several studies have documented the correlation between chemistry and microscopic laboratory results on bag-collected and diaper-extracted urine

• Cocaine, benzoylecgonine, and ecgonine methyl ester can be extracted from diapers with methanol.

Meconium drug testingMeconium drug testing

The fetus swallows drug-laced amniotic fluid

Drugs are deposited in the intestinal contents(or biliary excretion)

Meconium, passed in the first bowel movement, contains drugsthat have accumulated during the 2nd and 3rd trimesters

Is meconium testing better than urine?Is meconium testing better than urine?

• Several studies comparing urine and meconium results concluded that meconium testing was more sensitive– Analytical bias (RIA vs. EIA)– Cannot exclude the possibility of contamination

• Studies using urine and meconium methods with comparable sensitivities have reported equivocal results

• Several studies comparing urine and meconium results concluded that meconium testing was more sensitive– Analytical bias (RIA vs. EIA)– Cannot exclude the possibility of contamination

• Studies using urine and meconium methods with comparable sensitivities have reported equivocal results

Hair testingHair testing

• Animal studies have demonstrated a dose-related cocaine and benzoylecgonine accumulation in hair

• Cocaine and benzoylecgonine have been detected in hair specimens collected from cocaine abusing mothers and their infants

• Potentially the most sensitive method

• Animal studies have demonstrated a dose-related cocaine and benzoylecgonine accumulation in hair

• Cocaine and benzoylecgonine have been detected in hair specimens collected from cocaine abusing mothers and their infants

• Potentially the most sensitive method

Other specimensOther specimens

• Blood

• Amniotic fluid

• Breast milk

• Saliva

• Nails

• Blood

• Amniotic fluid

• Breast milk

• Saliva

• Nails

Distribution of cocaine metabolitesDistribution of cocaine metabolites

0

20

40

60

80

100

Urine Meconium Blood Hair AF

Cocaine Benzoylecgonine Ecgonine methyl ester

0

20

40

60

80

100

Urine Meconium Blood Hair AF

Cocaine Benzoylecgonine Ecgonine methyl ester

Which specimens are most stable?Which specimens are most stable?

0

20

40

60

80

100

% P

osit

ives

Meconium Urine Blood Hair

Percent positive results by specimen type and days since last use

<7 8 to 29 30 to 89 90 to 179 >180

0

20

40

60

80

100

% P

osit

ives

Meconium Urine Blood Hair

Percent positive results by specimen type and days since last use

<7 8 to 29 30 to 89 90 to 179 >180

Comparison of sensitivitiesComparison of sensitivities

01020304050607080

Per

cent

Pos

itiv

esPositive History Negative History

01020304050607080

Per

cent

Pos

itiv

esPositive History Negative History

ConclusionsConclusions

• Testing for prenatal cocaine exposure is widespread, but clinical indications are not well established

• None of the methods currently available are capable of measuring the magnitude or duration of prenatal cocaine exposure

• Urine screens must have greater analytical sensitivity to be useful for detecting prenatal cocaine exposure

• Neonatal hair testing appears to offer the best clinical sensitivity

• Testing for prenatal cocaine exposure is widespread, but clinical indications are not well established

• None of the methods currently available are capable of measuring the magnitude or duration of prenatal cocaine exposure

• Urine screens must have greater analytical sensitivity to be useful for detecting prenatal cocaine exposure

• Neonatal hair testing appears to offer the best clinical sensitivity